Molecular Studies of Tcf4 Gene and Correlation with Late‐Onset Fuchs Endothelial Corneal Dystrophy in the Greek Population: A Novel Cost‐Effective Diagnostic Algorithm

Background/Aim: Late-onset Fuchs’ endothelial corneal dystrophy (FECD) is a hereditary, progressive, bilateral and irreversible disorder that is characterized by thickening of Descemet's membrane, microscopic collagenous protuberances known as guttae and accelerated loss of corneal endothelial cells. Patients initially complain of blurred vision and as the disease progresses, painful epithelial edema develops. Untreated cases of FECD often result in blindness and then, the only treatment is corneal transplantation. DNA polymorphisms in many genes have been implicated, among them TCF4 on chromosome 18q encoding a transcription factor protein E2-2, which is involved in regulating cellular growth and differentiation in the cornea. In our previous published study, we confirmed the association of an intronic TCF4 SNP (rs613872) with the disease in our population. The purpose of this present study is to investigate further into another intronic point of interest in the same gene, the CTG18.1 trinucleotide repeat expansion. Methods: DNA was isolated from EDTA blood from a well-ascertained group of 36 Greek patients with FECD (Krachmer scale ≥2) and 58 healthy individuals, age- and sex-matched after obtaining their informed consent. STR-PCR and triplet-repeat primed PCR (TP-PCR) were performed and followed gel electrophoresis and fragment analysis in an ABI SeqStudio genetic analyzer. Our real-time qPCR genotyping method was used for the SNP in the LightCycler (Roche). Statistical analysis of both genetic results was performed with SPSS and SNPStats. Results: The TCF4 expansion method was validated adequately and a cost-effective diagnostic algorithm is proposed. An expanded allele was defined as having over 40 trinucleotide repeats. 20 out of 36 patients (56%) possessed at least one expanded allele compared to only 3 out of 58 healthy controls [5%, odds ratio=19.85 (95% C.I.=5.23-75.29)]. The frequency of TCF4 risk G allele was increased to 40% in patients with FECD compared to 17% in healthy subjects [odds ratio=3.59 (95% C.I.= 1.68-7.69)]. Conclusion: The need for an international effort for TCF4 triplet repeat method standardization is stressed along with the provision of suitable reference materials. Since 58.4% of the FECD patients possessed a range of 2-4 risk alleles in both TCF4 loci, we conclude that TCF4 is strongly and statistically associated with late-onset FECD in the Greek population. The missing heritability could be attributed to other genes that deserve further attention in the future.