Chromosome 4 Duplication Associated with Strabismus Leads to Gene Expression Changes in iPSC-Derived Cortical Neurons.

Background/objectives: Strabismus is the most common ocular disorder of childhood. Three rare, recurrent genetic duplications have been associated with both esotropia and exotropia, but the mechanisms by which they contribute to strabismus are unknown. This work aims to investigate the mechanisms of the smallest of the three, a 23 kb duplication on chromosome 4 (hg38|4:25,554,985-25,578,843).

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 , , and , 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.