Characterization of Usher Syndrome Cell line Genotypes and Elucidation of Novel MicroRNA Biomarkers using MicroRNA Microarray and Droplet Digital PCR

Abstract Background:Usher syndrome (USH) is an inherited disorder characterized by sensorineural hearing loss (SNHL), retinitis pigmentosa (RP)-related vision loss, and vestibular dysfunction. USH presents itself as three distinct clinical types 1, 2 and 3, with no biomarker for early detection. This study aimed to explore novel microRNA (miRNA) biomarkers for USH by comparing miRNA expression patterns in cell lines derived from USH patients and control subjects. Methods:Lymphocytes from USH patients and healthy individuals were isolated and transformed into stable cell lines using Epstein-Barr virus (EBV). DNA from these cell lines was sequenced using a targeted panel to identify gene variants associated with USH types 1, 2, and 3. Microarray analysis was performed on RNA from both USH and control cell lines using NanoString miRNA microarray technology. Dysregulated miRNAs identified by the microarray were validated using droplet digital PCR technology. Results: DNA sequencing revealed that two USH patients had USH type 1 with gene variants in USH1B (MYO7A) and USH1D (CDH23), while the other two patients were classified as USH type 2 (USH2A) and USH type 3 (CLRN-1), respectively. The NanoString miRNA microarray detected 92 differentially expressed miRNAs in USH cell lines compared to controls. Significantly altered miRNAs exhibited at least a twofold increase or decrease with a p value below 0.05. Among these miRNAs, 20 were specific to USH1, 14 to USH2, and 5 to USH3. Three miRNAs that are known as miRNA-183-family which are crucial for inner ear and retina development have been significantly down regulated as compared to control cells. Subsequently, droplet digital PCR assays confirmed the dysregulation of twelve most prominent miRNAs in USH cell lines. Conclusion:This study identifies several miRNAs with differential expression in USH patients and their potential utility as biomarkers for Usher syndrome.