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

Abstract

The predominant cause of Fuchs endothelial corneal dystrophy (FECD) is a CTG repeat expansion (termed CTG18.1) situated within an intron of the transcription factor encoding gene,TCF4. Here we use a primary FECD case-derived corneal endothelial cell (CEC) system to enhance our understanding of multiple pathogenic processes underlying CTG18.1-mediated FECD. We define differential gene expression and alternative splice events using long- and short-read RNA-seq datasets generated from 15 biologically independent primary CEC lines, comprehensively characterizing aberrant splicing-related aspects of the disease. Further targeted analysis of these data, alongside a complementary spatial transcriptomics approach, reveals a unique and distinctive pattern ofTCF4-specific dysregulation that underpins expansion-positive FECD and isolates downstream consequences of this shift as an important pathogenic component of disease. To explore whetherTCF4dysregulation, irrespective of CTG18.1 expansions, could cause FECD we also interrogated exome data generated from a large (n=141) genetically refined cohort of CTG18.1 expansion-negative FECD cases. We identify four rare and predicted deleterious (minor allele frequency <0.005; CADD>15)TCF4variants all encompassed by only a small fraction (10/93) of totalTCF4transcripts suggesting that, in rare instances, disruption of a specific subset of TCF4 isoforms may also confer CEC-specific disease independent of CTG18.1 expansions. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity andTCF4isoform-specific dysregulation, underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

Tissue-Specific Dynamics ofTCF4Triplet Repeat Instability Revealed by Optical Genome Mapping

“…A strong correlation between CTG18.1 expansion length in peripheral blood leukocytes and molecular hallmarks of pathology in the corneal endothelium, including the presence of nuclear CUG-specific RNA foci and isoform-specific patterns of TCF4 downregulation, is well documented (Zarouchlioti et al 2018;Hu et al 2018;Powers et al 2022;Bhattacharyya et al 2024). Our study has shown that the mean repeat length in mono-allelic expanded CEC lines is 4.4 to 17.0-fold longer than in leukocyte gDNA samples.…”

“…To overcome this limitation and acquire enough cells for the OGM protocol (approximately 1 million cells per sample), we used primary CEC cultures generated from endothelial keratoplasty specimens removed following planned corneal surgery to analyse CEC-specific gDNA. We have previously demonstrated that these primary CEC cultures robustly maintain the biomarkers of CTG18.1-mediated disease and generate a pure corneal endothelial cell model that enables the investigation of FECD in a disease-relevant cell context (Zarouchlioti et al 2018;Bhattacharyya et al 2024). We acquired paired biosamples (corneal endothelial specimens and peripheral blood leukocytes) from 9 FECD patients to directly compare instability levels between the affected and unaffected cell types.…”