Abstracts

Rationale: MicroRNAs (miRNAs) have a significant role in health and disease. Circulating miRNAs in body fluids are currently being investigated as biomarkers in many diseases. There is a critical need for novel biomarkers for diagnosis and to predict disease progression and/or response to treatment in epilepsy. Profiling miRNA expression changes following seizures can be explored as potential biomarkers. However, expression changes may vary in different experimental models of epilepsy, where seizures are induced. Further the diurnal and  temporal variation in miRNA expression in epilepsy is poorly understood. Our objective is to understand blood miRNA expression changes in relation to the time of seizure occurrence in dogs with naturally occurring epilepsy. This will provide us information regarding optimum sample collection times in patients with epilepsy for biomarker analysis. Methods: Blood samples were collected in PAXgene blood RNA tubes (Qiagen, CA) from five dogs maintained at the University of Minnesota Veterinary Medical Center. Samples were collected twice from each animal as baseline, when they were not having active seizures. When seizures occurred, we collected samples after the episode, at different time points that were clinically feasible. Total RNA was extracted using PAXgene blood miRNA kit (Qiagen, CA) and small RNA sequencing performed on an Illumina HiSeq 2000 (24 samples per lane, ~ 10 million reads per sample) platform. Differentially expressed (DE) miRNAs were determine between groups, using Edge R package. For data visualization and comparisons between groups, we performed Principal Component Analysis and clustering in R package. Network and pathway analyses were performed using Ingenuity Pathway Analysis (IPA) software. Results: We identified ~180 unique canine miRNAs with > 5x coverage in whole blood collected from these dogs. Our preliminary analyses showed miRNA expression varies with breed and the time of day. However, only 6 miRNAs displayed statistically significant differences between the 2 time-points. Currently, we have data from 2 dogs who had seizures. Analysis of samples collected after a seizure episode showed different profiles for these 2 dogs that are consistent with differences in seizure frequency and duration. We observed a total of 18 and 25 unique DE miRNAs (> 2-fold; p < 0.05) in each dog with 7 overlapping miRNAs (cfa-miR-9, -19b, -19a, -144, -127, -122, -1343). Many of these expression changes were observed within 24hrs after seizure, while few miRNAs continued to show changes after several days. Predictive analysis of miRNA function showed oxidative stress, cell proliferation and epigenetic regulation to be altered in both dogs. Conclusions: We have developed a method to isolate and characterize circulating miRNAs from dogs with epilepsy that can be adopted in a clinical setting. Our data suggests that miRNA expression profile changes with time of sample collection. Further studies are needed in more canine samples, possibly comparing dogs with chronic epilepsy with new onset seizures to understand the potential utility of circulating miRNA expression as biomarkers. Funding: American Kennel Club Canine Health Foundation, Inc.