Description
Background
Expansion of an intronic CTG repeat within the TCF4 gene (termed CTG18.1) is the most common genetic risk factor for Fuchs endothelial corneal dystrophy (FECD), an age-related corneal-specific eye disease. PureTarget is an amplification-free targeted long-read sequencing approach that enables simultaneous assessment of TCF4 repeat size, sequence composition and 5mC methylation status.
Purpose: To determine whether DNA methylation status is altered between expanded and non-expanded alleles in affected corneal endothelial cells (CECs).
Methods
Ultra-high molecular weight DNA from eight CEC samples derived from patients with monoallelic expansions was investigated with PureTarget. HiFi reads were aligned to hg38, phased into non-expanded and expanded alleles using TRGT, and CpG methylation positions were identified with Fiberseq. Expanded reads with at least 5% CCG content were classified as interruption-containing reads using BBDuk, and methylation at reference CpG sites was quantified using CpGtools and assessed for differential methylation with MethBat.
Methylation was then assessed using two complementary pipelines: within-repeat methylation was summarised by binning per-read CpG methylation positions into 1 kb windows along each molecule, while methylation of the repeat-flanking regions was quantified at reference CpG sites near the repeat.
Results
Within the CTG repeat tract, interrupted expanded reads displayed the greatest levels of methylation and variability, exceeding 300 methylated CpGs per 1 kb bin, while non-expanded reads generally remained below ~50 methylated CpGs per bin. Expanded reads without interruptions showed intermediate levels of methylation.
At reference CpG sites upstream of CTG18.1, non-expanded reads maintained low baseline methylation (<5%). In contrast, interrupted expanded reads reached >60% methylation, compared with ~25% across expanded reads without interruptions, defining a nearby differentially methylated region.
Conclusions
Methylation status at this TCF4 locus is shaped not simply by repeat length, but by repeat sequence composition, with CCG-interrupted expanded alleles forming a distinctly hypermethylated subgroup in CECs. This suggests that repeat interruptions may underlie tissue-specific epigenetic changes relevant to FECD pathogenesis.
Lay Abstract
Fuchs endothelial corneal dystrophy (FECD) is an eye disease that affects the cornea, the clear front surface of the eye. In many patients, FECD is linked to an elongated DNA repeat in the TCF4 gene. Using a DNA sequencing method called PureTarget, we studied this repeat in corneal cells, the cell type most directly affected in FECD. This approach allowed us to examine DNA methylation, which is a chemical modification on DNA that can affect how genes behave.
We analysed eight corneal samples from patients carrying the expanded repeat. Non-expanded DNA molecules, expanded molecules, and expanded molecules containing sequence interruptions were compared by examining methylation both within the repeat itself and in the surrounding DNA region.
Interestingly, we found that expanded molecules with interruptions showed the highest methylation levels. Expanded molecules without interruptions showed intermediate levels, while non-expanded molecules showed low methylation. This pattern was seen both within the repeat and in the nearby region.
These findings suggest that in FECD, methylation at the TCF4 repeat is influenced not only by repeat length, but also by the sequence pattern within the repeat. This may therefore help explain how repeat length, structure and methylation contributes to the disease in the cornea.
| Lay Title | Studying DNA modifications in Fuchs endothelial corneal dystrophy |
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| Role | Research Assistant |