Supervisors info:
Χρήστος Κρούπης, Επίκουρος Καθηγητής Κλινικής Βιοχημείας-Μοριακής Διαγνωστικής, Ιατρική Σχολή Πανεπιστημίου Αθηνών
Summary:
Introduction: Fuchs endothelial corneal dystrophy is a slowly progressive, bilateral but asymmetric, posterior corneal dystrophy. It is characterized by reduced density of corneal endothelial cells, thickening of Descemet’s membrane and deposition of extracellular material in the form of guttae. As the endothelial cells regulate corneal hydration and maintain its transparency, their loss may eventually progress to corneal stromal edema, epithelial bullae and vision loss. For the end-stage disease corneal transplant surgery or keratoplasty represents the only definitive treatment. The prevalence of FECD varies markedly across the world.
FECD is a genetically heterogeneous disease. There are two forms defined by the age of onset. Early-onset FECD is rare and the more common late-onset FECD which can either be familial or sporadic, with onset typically after the age of 50 years. Early-onset FECD is associated with COL8A2 mutations. Furthermore, for the late-onset FECD has been identified a polymorphism g.97923C>A dbSNPrs613872, in the intron 3 of the transcription factor 4 (TCF4) which encodes a member of the E-protein family (E2-2) and increases FECD risk by a factor of 30 for persons homozygotes.
Methods: The purpose of the present study was to develop real-time PCR method and melting curve analysis in order to genotype the rs613872 polymorphism in TCF4 gene and to implement it on a sample of 22 Greek FECD patents (Krachmer scale ≥2) and 58 healthy controls, age and sex matched. DNA was isolated from blood samples, after signed informed consent, ophalmological evaluation and clinical data collection. The same samples were screened using DNA Sequencing method in order to detect the g.31753T>G L450W rs8035192 and g.31767C>A Q455K rs8035191 mutations in a COL8A2 genomic region. For the statistical analysis of the results, SPSS and SNPStats software packages were used.
Results: A rabit and reliable assay was developed for SNP rs613872 in TCF4 gene, using hybridization probes in the complementary stand and a melting Curve in the LightCycler (Roche) instrument. Analytical validation was performed (efficiency E=1.89, Cq between run precision CV≤1.2%, Tm T-allele 57.20 0C CV=1.7% and G-allele 63.75 0C CV=1.25%, ΔTm=7.39). The genotyping results were 100% concordant with the reference method of DNA Sequencing. A statistically significant association was found for SNPs rs613872 in TCF4 gene and late-onset FECD with OR 4.82 (CI 1.98-11.73) with the log-additive model and a significant increase of the G allele from 17.24% in healthy to 47.73% in patients. No statistically significant association was found between the G risk allele the age and the sex in our population. DNA Sequencing methodology was applied for screening of L450W and Q455K mutations in COL8A2 gene, but they were not detected in our population. However a novel polymorphism NM_005202.3:c1491G>A→p.A497T in one sample was identified.
Conclusions: The rs613872 SNP in TCF4 gene was confirmed to be strongly and statistically associated with late-onset FECD in the Greek population. Also it was confirmed that the L450W and Q455K mutations in COL8A2 gene were not associated with late-onset FECD.
Keywords:
Corneal Dystrophy, Fuchs, TCF4, rs613872, COL8A2
