Spatial transcriptomics reveal isoform-specific patterns of TCF4 dysregulation in CTG18.1 expansion positive FECD case-derived corneal endothelial cells

Abstract

Purpose : Expansion of an intronic CTG repeat element, termed CTG18.1, is the most common genetic risk factor for Fuchs endothelial corneal dystrophy (FECD). Patients with CTG18.1 expansions (≥50 repeats) display corneal endothelial-specific disease, though TCF4 is ubiquitously expressed. TCF4 comprises >90 alternative spatially and temporally expressed isoforms and is inconsistently downregulated in expansion-mediated FECD (Exp+). Deciphering the tissue specificity of this disease and characterizing transcript-specific dysregulation has proven challenging given the transcriptomic complexity of TCF4. Here, we aim to determine if CTG18.1 expansions induce isoform-specific patterns of TCF4 dysregulation in patient-derived corneal endothelial cells (CECs).

Methods : Primary patient-derived CEC cultures of Exp+ (n=3), expansion negative (Exp-, n=3) and unaffected controls (n=3) were established and incubated with two sets of RNAScope probes. One probe set visualizing total TCF4 transcripts, and another designed to target 5’ exons present in a subset of longer isoforms (~35/93), which flank the repeat-containing intron. Collectively, these probes enable us to visualise the relative expression of the repeat encompassing transcripts when compared to total TCF4 expression.

Results : Our custom spatial transcriptomic approach showed that, on average, Exp+ CECs had a significantly smaller proportion of the longer repeat flanking transcripts (16.33% ± 4.53) compared to control (36% ± 6.9, p=0.0145) and Exp- (30.9% ± 5.17, p=0.0213) CECs. This suggests that the presence of the expanded repeat alone consistently skews TCF4 transcript ratios away from longer, repeat-containing, isoforms. This subset of longer isoforms typically contain all three of activation domains of TCF4 as compared to one or two in the shorter isoforms. Long isoforms are known to have a magnitude more transcription factor activity and may have altered dimerization.

Conclusions : A subset of TCF4 transcripts is significantly downregulated in the presence of CTG18.1 expansions in affected patient-derived CECs. We propose a compensatory mechanism where shorter TCF4 transcripts with transcription start site downstream of the repeat may be upregulated in response. This shift towards shorter transcripts would result in functional downregulation and may, in part, explain the tissue-specificity of FECD.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.