RAD21 involved in corneal stroma development by regulating expression of the cadherin and cell adhesion pathway

Abstract

Purpose : Sclerocornea is a congenital anomaly of the eye. The cornea of the patients blends with sclera, resulting in no clear boundary that can cause a range of corneal opacity from peripheral to total sclerocornea. We recently identified a RAD21 variant R450C from a peripheral sclerocornea pedigree. Overexpression of this mutated variant mRNA in Xenopus laevis embryos disrupted the organization of corneal stroma fibrils. To understand the potential mechanisms of RAD21 in leading to corneal stroma defects, RNAseq analysis was performed on cells isolated from the peripheral sclerocornea family members.

Methods : Whole blood was collected from sclerocornea pedigree members and the peripheral blood mononuclear cell layer was isolated and transformed with B98-5 Epstein-Barr virus. Cells were cultured with Iscove’s Modified Dulbecco’s Medium supplemented with 20% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 ^○C in a 95% air-5% CO₂ incubator. Total RNA was extracted and the cDNA library was constructed by using the Illumina TruSeq RNA sample preparation kit v2. RNAseq was conducted on Illumina HiSeq 2500. The fold change between the RNA abundance from the affected and unaffected members was calculated as the two “reads per kilobase per million mapped reads” (RPKM) values of two affected members divided by the two RPKM values of the two unaffected members. Gene ontology enrichment analysis was conducted by using DAVID and Enrichr.

Results : The expression of the cell adhesion genes were upregulated 30 folds in cells carrying the heterozygous RAD21 variant compared to the cells isolated from the unaffected family members (p=1.6 x 10^-30), suggesting this RAD21 variant affects cell migration. In addition, genes related to the mitochondrial respiratory chain and MHC class II were downregulated 6 and 3.5 folds respectively in cells carrying the heterozygous RAD21 variant compared to the cells isolated from the unaffected family members (p=3.9 x 10^-4 and p=1.4 x 10^-7 respectively).

Conclusions : Our results suggest RAD21 plays a crucial role in corneal stroma development by regulating the cadherin and cell adhesion pathway, as well as the mitochondrial respiratory chain and MHC class II pathways

This is a 2020 ARVO Annual Meeting abstract.