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.