Tissue-Specific Dynamics of TCF4 Triplet Repeat Instability Revealed by Optical Genome Mapping

Here, we demonstrate the utility of optical genome mapping (OGM) to interrogate the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1) and gain novel insights into the tissue-specific nature of the disease. Genomic DNA (gDNA) samples derived from peripheral blood leukocytes and primary corneal endothelial cells (CECs) were analysed by OGM. Concurrently, all samples were genotyped by standard PCR-based methods to classify their expansion status. Individuals with one or more CTG18.1-expanded alleles ([≥]50 CTG repeats) detected in their leukocyte-derived gDNA were classified as expansion-positive. A customised bioinformatics pipeline was developed to perform CTG18.1-targeted OGM analysis. All linearised gDNA molecules containing labels flanking CTG18.1 were extracted, corrected for the repeats on the reference human genome and sized. Analysis of paired bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin, but displayed significantly higher levels of instability within the diseased corneal endothelium. Clusters of CTG18.1 molecules of approximately 1,800-11,900 repeats, beyond the ranges observed in individual-matched leukocyte samples, were detected in all CEC gDNA samples from expansion-positive cases. In conclusion, OGM is a powerful method to analyse the somatically unstable CTG18.1 locus. More generally, this work exemplifies the broader utility of OGM in exploring somatically unstable short tandem repeat loci. Furthermore, this study has highlighted the extreme levels of tissue-specific CTG18.1 somatic instability occurring within the diseased corneal endothelium, which we hypothesise plays a pivotal role in driving downstream pathogenic mechanisms of CTG18.1-mediated FECD.