Yijing Liang

Antisense oligonucleotides (ASOs) are a versatile therapeutic modality for inborn genetic diseases. ASOs can induce skipping of “dispensable” exons containing disease-causing variants to rescue protein amount and function, but this approach has been studied for only a small number of genes. We developed a high-throughput in silico tool for assessing exon dispensability and designing exon-skipping ASO sequences. Parameters were optimized using known dispensable and in-frame indispensable exons. Across 72,644 exons of 5,057 disease genes, we identified thousands of new targets for exon-skipping ASOs (3.4% of exons with most stringent filters, 24.5% with less stringent filters) that collectively include 0.97%-15.6% of disease-causing variants in large-scale databases. To facilitate recognition of DNA variants potentially amenable to exon skipping as a therapeutic strategy, we established the HAWK-EYE database as a repository of exon dispensability predictions and corresponding in silico -optimized ASO sequences, available as an open-access web application ( https://hawk-eye.research.sickkids.ca/ ). To illustrate translational utility, we experimentally validated a subset of the in silico -optimized ASO sequences that were generated for all Dispensable exons in the HAWK-EYE database, and showed that skipping a Dispensable exon in SOX5 preserves protein function using in vivo and in vitro assays. This scalable platform approach to exon-skipping ASOs will accelerate identification and testing of amenable genetic variants.

Integrin heterodimers containing an Integrin alpha V subunit are essential for development and play critical roles in cell adhesion and signaling. We identified biallelic variants in the gene coding for Integrin alpha V (ITGAV) in three independent families (two patients and four fetuses) that either caused abnormal mRNA and the loss of functional protein or caused mistargeting of the integrin. This led to eye and brain abnormalities, inflammatory bowel disease, immune dysregulation, and other developmental issues. Mechanistically, the reduction of functional Integrin αV resulted in the dysregulation of several pathways including TGF-β–dependent signaling and αVβ3-regulated immune signaling. These effects were confirmed using immunostaining, RNA sequencing, and functional studies in patient-derived cells. The genetic deletion of itgav in zebrafish recapitulated patient phenotypes including retinal and brain defects and the loss of microglia in early development as well as colitis in juvenile zebrafish with reduced SMAD3 expression and transcriptional regulation. Taken together, the ITGAV variants identified in this report caused a previously unknown human disease characterized by brain and developmental defects in the case of complete loss-of-function and atopy, neurodevelopmental defects, and colitis in cases of incomplete loss-of-function.

Congenital heart disease (CHD) is the most common congenital anomaly. Non-canonical splice-disrupting variants are not routinely evaluated by clinical tests. Algorithms including SpliceAI predict such variants, but are not specific to cardiac-expressed genes. Whole genome (WGS) (n=1083) and myocardial RNA-Sequencing (RNA-Seq) (n=114) of CHD cases was used to identify splice-disrupting variants. Using features of variants confirmed to affect splicing in myocardial RNA, we trained a machine learning model that outperformed SpliceAI for predicting cardiac-specific splice-disrupting variants (AUC 0.92 vs 0.66), and was independently validated in 43 cardiomyopathy probands (AUC 0.88 vs 0.64). Application of this model to 971 CHD WGS samples identified 9% patients with splice-disrupting variants in CHD genes. Forty-one% of predicted splice-disrupting variants were deeply intronic. The burden of variants in CHD genes was higher in cases compared with 2,570 controls. Our model improved genetic yield by identifying splice-disrupting variants that are not evaluated by routine tests.