Conclusions
A copy number variant (CNV) that confers risk for strabismus affects gene expression of several genes involved in neural development, highlighting that strabismus most likely results from abnormal neural development, and identifying several new genes and pathways for further research into the pathophysiology of strabismus.
Methods
Using CRISPR and bridging oligos, we introduced the duplication into the Kolf2.1J iPSC line. We differentiated the parent line and the line with the duplication into cortical neurons using a three-dimensional differentiation protocol, and performed bulk RNASeq on neural progenitors (day 14) and differentiated neurons (day 63).
Results
We successfully introduced the duplication into Kolf2.1J iPSCs by nucleofecting a bridging oligo for the newly formed junction along with cas9 ribonucleoparticles. We confirmed that the cells had a tandem duplication without inversion or deletion. The parent line and the line with the duplication both differentiated into neurons reliably. There were a total of 37 differentially expressed genes (DEGs) at day 63, 25 downregulated and 12 upregulated. There were 55 DEGs at day 14, 18 of which were also DEGs at day 63. The DEGs included a number of protocadherins, several genes involved in neuronal development, including SLITRK2, CSMD1, and VGF, and several genes of unknown function. Conclusions: A copy number variant (CNV) that confers risk for strabismus affects gene expression of several genes involved in neural development, highlighting that strabismus most likely results from abnormal neural development, and identifying several new genes and pathways for further research into the pathophysiology of strabismus.