Abstract
Purpose :
Usher syndrome (Usher) is the leading inherited cause of deaf-blindness, with 2.5% of cases attributed to mutations in the USH1C gene. Transgenic mice expressing the human USH1C c.216A mutation develop retinitis pigmentosa (RP), deafness, and vestibular dysfunction similar to Acadian Usher patients. Due to low expression of Ush1c in the retina, previous methods have been unable to quantify mRNA transcripts, identify isoform variants, or measure splice deletions created by the 216A mutation. We tested the hypothesis that Next Generation Sequencing (NGS) RNA-Seq could be used to measure these factors in Usher mice and wild type littermates. We also developed an NGS method to calculate percentage splice correction in mice treated with antisense oligonucleotides (ASOs) targeting the 216A mutation.
Methods :
NGS RNA-Seq was used to measure Ush1c variant expression and differential gene expression in the retinal transcriptome of Usher mice and wild type littermates over a 1-year time-course. An NGS method was used to measure splice correction in ASO-treated Usher mice on Nextera XT DNA libraries made from retinal cDNA enriched for Ush1c by nested PCR. A visual cliff behavioral assay was then used to correlate Ush1c expression data with visual perception in mice.
Results :
Higher levels of Ush1c mRNA were detected in Usher retinas compared to wild type littermates at all ages. Variants a, b, and c were detected at different ages in both groups; however, nearly all variants were abnormally spliced in Usher retinas. ASO treatment significantly increased the levels of full-length and decreased the levels of mutant Ush1c transcripts in Usher retinas. Differentially expressed genes in pathways related to cilia function/transport, ER stress and the unfolded protein response, oxidative stress, and apoptosis were detected between Usher and normal retinas. Visual cliff behavioral analysis showed that Usher mice have reduced visual discrimination at all ages tested compared to normal littermates.
Conclusions :
NGS is a highly sensitive method to identify the effects of the 216A mutation on Ush1c splice variants, calculate splice correction with treatment, and measure transcriptome expression in the retina, which can be correlated with visual behavior in mice. These results will improve our understanding of vision loss in Usher and guide the development of genetic strategies for long-term vision correction in RP.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.