Integrated analysis of microRNA expression, UTR binding sites, and human variation in ocular tissues. T. Gaasterland1,2, A. N. Dubinsky3, L. E. Edsall2,4, T. S. Mondala5, P. Ordoukhanian5, S. R. Head5 1) Institute for Genomic Medicine, Univ California San Diego, La Jolla, CA; 2) Scripps Institution of Oceanography, Univ California San Diego, La Jolla, CA; 3) Pediatrics Department, School of Medicine, Univ California San Diego, La Jolla, CA; 4) University Program in Genetics and Genomics, Duke University, Durham, NC; 5) Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA.

   We present a platform to evaluate and rank genome variants in untranslated regions (UTR) of mRNA transcripts and their potential relevance to eye diseases through disruption of microRNA binding. We evaluate microRNA and mRNA expression in human eye tissues to identify UTR variants with the potential to disrupt microRNA regulatory activity and contribute to risk of, or progression in, glaucomatous optic nerve degeneration, a blinding eye disease affecting over 60 million people worldwide. We overlaid eye tissue gene expression patterns for microRNA and mRNA, microRNA binding sites in UTRs, and UTR variants detected through exome sequencing. We sequenced ~500 human exomes with capture probes that include UTR targets (Nimblegen SeqCap EZ Exome). 90,358 variant sites located in UTR regions were observed in at least one exome. Total RNA was extracted from optic nerve, optic disc with lamina cribrosa, retina, trabecular meshwork, ciliary body, and choroid from human donor eyes and subjected to high-throughput sequencing (Illumina, Hiseq 2000; Nugen) to measure microRNA and mRNA expression levels. MicroRNA reads were matched against the 2,555 unique, known human microRNAs (, v20), counted, and normalized. mRNA reads were mapped to the reference human genome, counted per gene, and normalized (, hg19; bowtie; cuffdiff). MicroRNAs and mRNAs were stratified by high, medium, and low expression and ranked within tissue by expression level. The first 8-9 bases of a microRNA can guide its binding to an mRNA. Generally, these seed sites start at base 2 of the microRNA and match their mRNA targets in reverse-complement. With our tool, ZoomMiR, we tabulated and scored all seed sites for the known human microRNAs in 124,315 5-prime UTR and 194,503 3-prime UTR sequences from all mRNA transcript isoforms annotated in hg19. Exome variants, microRNA and mRNA eye-tissue expression levels, and microRNA seed sites were merged based on genomic chromosome positions to identify all UTR variants within or near microRNA seed sites and detected through exome sequencing. Known variants received dbSNP identifiers and population frequencies from the 1000 Genomes and Exome Sequencing Project sites (; Together, these data provide a tool to evaluate the impact of UTR variants with alternate alleles over-represented in patients with disease compared to general or control populations.

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