Abstract
Purpose :
Strabismus is one of the most common ocular disorders of childhood. Genetic risk plays a role in the development of strabismus, but specific mechanisms have not been defined. We identified three genetic duplications that increase the risk of both esotropia and exotropia. This study aims to identify how one of those duplications (a 23kb duplication on chromosome 4) affects neurodevelopment.
Methods :
The chromosome 4 duplication (hg38|4:25,554,985-25,578,843) was introduced into an established human induced pluripotent stem cell (iPSC) line using CRISPR. Control and duplication lines were differentiated (N=4) into cortical neurons. Cells were examined for changes in gene expression using RNAseq as stem cells (Day 0), neuroprogenitors (Day 14), and differentiated neurons (Day 63). Neurons were analyzed at Day 77 with immunohistochemistry for neurite outgrowth (MAP2 staining) and Day 84 for synapse formation (PSD95 and Synapsin staining). RNASeq data were analyzed to identify differentially expressed genes and significantly enriched KEGG pathways and gene ontology (GO) biological processes terms. Immune labeling experiments were imaged in the ImageXpress Micro Confocal High-Content Imaging System, measuring and analysis was assessed by using MetaXpress. Data were analyzed using unpaired student’s t-test and two-way ANOVA.
Results :
Stem cells, neuroprogenitors, and differentiated neurons from both the duplication and control lines expressed appropriate markers, indicating neurodifferentiation. MAP2 immunostaining of differentiated neurons showed a decreased number of differentiated cells with the duplication, but no difference in neurite length. PSD95 and Synapsin immunostaining of differentiated neurons showed a significant reduction in the number of synaptic puncta in the cells with the duplication. RNAseq revealed 10 differentially expressed genes (DEGs) at Day 63 and 13 at Day 14. Seven DEGs overlap between Day 14 and Day 63 (ZNF736, ALG10B, FPGT, PCDHGB4, TTC34, ANKRD30BL, LINC01139). Enrichment analyses showed a downregulation in pathways and biological processes related to synaptic membrane adhesion and presynapse assembly in the cells with the duplication compared with wildtype.
Conclusions :
This duplication on chromosome 4 may contribute to the development of strabismus by changing gene expression and synapse formation during neuronal development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.