T35 - Microrna Regulation Of Candidate Genes For Tourette Syndrome

Abstract

Background Tourette Syndrome (TS) is a neurodevelopmental disorder that presents early in childhood and is marked by the appearance of multiple involuntary motor tics and at least one vocal tic. It presents high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). Despite a strong genetic contribution, the molecular mechanisms behind TS are still uncertain, although multiple lines of evidence suggest involvement of specific candidate genes and corresponding epigenetic mechanisms via miRNA regulators. Methods To date only a few genetic findings have been replicated in TS. Among these the nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene has been recently suggested as candidate susceptibility gene. CHRNA7 is known to regulate a wide variety of developmental and secretory functions, however, the mechanism of its transcriptional regulation is still unclear [1]. Another recent promising finding in TS genetic research is Netrin 4 (NTN4), which belongs to a family of extracellular proteins that direct axon outgrowth and guidance [2]. Several microRNAs have already been associated with neuropsychiatric disorders, but most importantly a few miRNA have also been associated with Tourette Syndrome [3,4].Our goal was to investigate the role of selected candidate genes in a case-control setup along with functional validation involving miRNA regulation in the possible involvement in TS pathogenesis through analysis of 3’UTR regions. Results An OpenArray platform (TaqMan® OpenArray® Genotyping System) was used for the case-control analysis of TS patients (N=564) and healthy controls. Starting from a list of candidate genes previously indicated as possible risk factors for TS, we screened for presence of SNPs in the 3’UTR regulatory regions. This analysis (data retrieved by mirSNP and polymiRTS databases) led to the identification of 32 SNPs which were predicted to change the seed sequence of in silico proposed miRNAs. To proof this concept, we performed functional validation study using a luciferase assay reporter containing 3’ UTR regions of CHRNA7 and NTN4 transfected with their predicted miRNA. Specifically, miRNA-106b and miRNA-198b were identified as the most potent miRNA candidates for regulation of CHRNA7 and NTN4 gene expression. SKNF1 and HEK human cell lines were co-transfected using lipofectamine with the luciferase reporter-3’ UTR construct of CHRNA7 and NTN4 (SwitchGear Genomics) and the corresponding putative miRNAs (miR-106b, miR-198b) along with non-targeting control miRNAs (miR-196b, miR-641b) in the functional validation studies. Discussion In order to increase our understanding of the underlying genetic and epigenetic mechanisms of TS, we aimed to study the possible miRNA regulation processes in TS-related genes, which would help not only to better understand the full genetic architecture of this disorder but also to determine how miRNAs contribute to the complexity of gene regulation in the development of disease. OpenArray analysis identified significant differences among the selected candidates genes (LHX6, iMMP2L) confirming the implication of those genes in TS etiology. In the luciferase assays we characterized the regulatory effect of the predicted miRNAs on the expression candidate genes in a concentration-dependent manner, which showed up to 5-fold change in the relative gene expression levels in case of CHRNA7 and NTN4 thereby proving the concept of our study.