Strabismus can be both visually and socially debilitating and its underlying pathophysiological mechanisms remain poorly understood. Current treatments often do not restore full visual function and do not address the underlying pathology. Strabismus has a clear hereditary component, but precise genetic mechanisms have not been defined. We recently identified three rare, recurrent genetic duplications that increase risk of esotropia. Each of these duplications includes a long non-coding RNA (lncRNA), which are often involved in chromatin remodeling and regulation of gene expression. Duplications can also affect gene expression by insertion of regulatory elements in new locations or disruption of the 3D chromatin structure. We therefore hypothesize that regulation of gene expression is an important mechanism underlying strabismus. This is bolstered by the findings that known environmental risk factors for strabismus, including prematurity, maternal smoking, and low birth weight, affect epigenetic regulation through changes in methylation. This proposal aims to (1) define the consequences of these duplications on gene expression, chromatin structure, and neuronal morphology and function, (2) evaluate esotropic and exotropic patients for single nucleotide variants (SNVs) or small insertions or deletions (indels) in the genes and regulatory regions included in the duplications or affected by the duplications, and (3) identify additional genetic causes of strabismus through whole genome sequencing of large strabismus families. The precise breakpoints and insertion points of the duplications will be determined by long-read whole genome sequencing, then each duplication will be introduced into induced pluripotent stem cells (iPSCs) through CRISMERE (a variant of CRISPR/cas9). Gene expression, enhancer activity, and chromatin conformation will be compared between stem cells, neuroprogenitors, and differentiated neurons with and without each duplication. The effects of each duplication on neuronal morphology and function will be assessed. Fluidigm multiplexing and next-generation sequencing will allow cost-effective screening of our large strabismus cohort for SNVs and indels in the coding and regulatory regions of the genes included in the duplications, as well as genes whose expression is altered by the duplications. Variants identified in multiple individuals and predicted to be damaging bioinformatically will be evaluated with in vitro functional studies. Additional families with multiple members with strabismus will be enrolled, and coding, non-coding, and structural variants will be identified through whole genome sequencing. Variants will be prioritized based on linkage, bioinformatic predictions, and population frequency. In addition, the epigenetic and 3D interactome maps from neuroprogenitors and neurons will be used to prioritize variants. Functional studies will be done on high priority identified variants. This work, by identifying genes and signaling pathways that contribute to development of strabismus, will provide insights into strabismus pathogenesis, which will allow development of new strabismus treatments or preventative interventions based on the underlying pathophysiology.

Strabismus (misaligned eyes) can be both visually and socially debilitating. We recently identified three genetic duplications (areas with an extra copy of the DNA) that increase the risk of strabismus, and this project aims to understand the mechanism by which those duplications increase risk. In the long term, this work will provide fundamental knowledge about how and why strabismus develops and pave the way for the development of new treatments that address its underlying cause.