Lower Fractions of TCF4 Transcripts Spanning over the CTG18.1 Trinucleotide Repeat in Human Corneal Endothelium

Abstract

Fuchs’ endothelial corneal dystrophy (FECD) is a bilateral disease of the cornea caused by gradual loss of corneal endothelial cells. Late-onset FECD is strongly associated with the CTG18.1 trinucleotide repeat expansion in the Transcription Factor 4 gene (TCF4), which forms RNA nuclear foci in corneal endothelial cells. To date, 46 RefSeq transcripts of TCF4 are annotated by the National Center of Biotechnology information (NCBI), however the effect of the CTG18.1 expansion on expression of alternative TCF4 transcripts is not completely understood. To investigate this, we used droplet digital PCR for quantification of TCF4 transcripts spanning over the CTG18.1 and transcripts with transcription start sites immediately downstream of the CTG18.1. TCF4 expression was analysed in corneal endothelium and in whole blood of FECD patients with and without CTG18.1 expansion, in non-FECD controls without CTG18.1 expansion, and in five additional control tissues. Subtle changes in transcription levels in groups of TCF4 transcripts were detected. In corneal endothelium, we found a lower fraction of transcripts spanning over the CTG18.1 tract compared to all other tissues investigated.

Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease

“…Alongside RNA toxicity, repeat-associated non-AUG (RAN) dependant translation of repetitive (CUG) n and (CAG) n transcripts 12 , dysregulation of TCF4 itself has also been hypothesized to contribute to the underlying pathology of CTG18.1 Exp+ mediated FECD, but exactly how this non-coding repeat expansion drives dysregulation remains elusive 13,14 . This is largely because the gene comprises >90 alternative spatially and temporally expressed isoforms [13][14][15] . Such diversity is reflected in the varied functional roles TCF4 plays in human development, alongside the diverse range of human disease in addition to FECD associated with TCF4 variants.…”

“…However, a comprehensive understanding of the transcriptome-wide consequences of this process and its relevance to FECD pathophysiology and personalized therapies has yet to be realized 6,10,11 . Alongside RNA toxicity, repeat-associated non-AUG (RAN) dependant translation of repetitive (CUG) n and (CAG) n transcripts 12 , dysregulation of TCF4 itself has also been hypothesized to contribute to the underlying pathology of CTG18.1 Exp+ mediated FECD, but exactly how this non-coding repeat expansion drives dysregulation remains elusive 13,14 . This is largely because the gene comprises >90 alternative spatially and temporally expressed isoforms 13–15 .…”

“…dysregulation has yet to be associated with CTG18.1 expansions in corneal endothelial cells (CECs) (14)(15)(16). Such diversity is reflected in the varied functional roles TCF4 plays in human development, alongside the range of human disease associated with TCF4 variants in addition to FECD.…”

“…However, a comprehensive understanding of consequences and role of both the spliceopathy and downstream effects of the TCF4 dysregulation have yet to be characterised in parallel in the same model system. Furthermore, because TCF4 comprises >90 alternative spatially and temporally expressed isoforms, deciphering transcript-specific level dysregulation has proven to be extremely challenging and a consistent pattern of TCF4 dysregulation has yet to be associated with CTG18.1 expansions in CECs (12)(13)(14). Such diversity is reflected in the varied functional roles TCF4 plays in human development, alongside the range of human disease associated with TCF4 variants in addition to FECD.…”

Alternative Splicing in Human Physiology and Disease

“…This gene is characterized by the production of 46 known transcripts. The expression levels of these transcripts harboring CTG18.1 expansion were investigated in patients with Fuchs endothelial corneal dystrophy by Westin et al, concluding with some preliminary data regarding the effect of this trinucleotide repeat on the expression of TCF4 transcripts, which require further investigation [ 11 ].…”