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A rare disease is defined as those that affect fewer than 1 in 2,000 individuals. There are about 7,000 rare diseases, which more than 90% of them have no treatment and 50-75% affects children. RNA splicing is the process by which introns are removed, and exons joined together in pre-mRNA processing. Genetic variants within introns can influence RNA splicing, leading to disease through the loss of protein expression and/or function. However, the molecular consequences of deep intronic variants are challenging to predict. We have developed a massively parallel minigene splicing assay that can experimentally detect variant-induced splicing aberrations. The assay consists of a GFP minigene reporter containing a short human intron. Centered within the intron of the reporter gene is a cloning cassette that can be used to insert intronic sequences of interest. We generated a library of 12,400 sequences corresponding to 620 rare genetic variants detected within introns of undiagnosed rare disease patients enrolled in the Genomic Answers for Kids program. We introduced the library into HEK293T cells and evaluated splicing outcomes using a high-throughput sequencing-based strategy. We found that 2.5% of variants profiled gave rise to novel splice acceptor sites and 7% of variants profiled gave rise to novel splice donor sites. We are currently in the process of validating these splicing defects in patient-derived cell models. The approach we describe here has the potential to significantly improve the ability to detect pathogenic splice-disrupting variants in rare disease genomes.

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High-throughput identification of deep intronic splice-disrupting variants using a massively parallel minigene splicing assay