Transcriptional characterization of the nonsense allele of FOXE3 responsible for Peters anomaly

Abstract

Purpose: We previously identified a homozygous nonsense FOXE3 mutation (p.C240X) in a Pakistani family responsible for Peters anomaly and subsequently found that the nonsense mutation does not affect the localization of FOXE3, a transcription factor to the nucleus. We propose that the causative effect lies in the DNA-binding activity of the mutated FOXE3 protein. To test our hypothesis, we examined the differential expression of mRNAs targeted by wild type and mutant FOXE3 in human embryonic kidney (HEK) 293T cells.

Methods: The wild-type and mutant (p.C240X) FOXE3 alleles were cloned in a commercially available expression vector (pT-Rex™-DEST30). Both wild type and mutant constructs were transfected in quadruplets in HEK293T cells, whereas non-transfected HEK293T cells were used to control the background. Cells were harvested 24 hours post transfection and immediately subjected to total RNA isolation, which was used to prepare whole transcriptome next-generation sequencing libraries. The libraries were subjected to massive parallel sequencing on the Illumina HiSeq 2000 genome analyzer. In parallel, the wild-type, mutant and control cells lysates were digested and peptides were labeled using 8-plex iTRAQ reagents and were subjected to LC-MS/MS for proteome sequencing. The data thus generated by HiSeq 2000 and LC-MS/MS were analyzed for differentially expressed genes.

Results: Two lanes of HiSeq 2000 genome analyzer generated approximately 3 billion reads for wild-type, mutant and control samples. The sequencing reads were analyzed by DESeq software, using raw read counts and negative-binomial distributions to measure the differential gene expression. DESeq analysis was performed by comparing wild-type and control samples, followed by wild-type and mutant samples with selection for differentially expressed genes with false discovery rate (FDR) of <0.05 and minimum 1x log-fold change. The initial analyses of transcriptome and proteome data revealed 75 genes that were significantly up- or down-regulated in response to mutant protein expression in HEK293T cells.

Conclusions: These results suggest that nonsense allele (p.C240X) affects the transcriptional profile of FOXE3 transcription factor protein. We are currently examining the role of the candidate genes in anterior ocular segment dysgenesis through in vivo zebrafish complementation and physiologically relevant assays.

Keywords: 421 anterior segment • 539 genetics • 638 pathology: human