The Role of hsa-miR-548l Dysregulation as a Putative Modifier Factor for Glaucoma-Associated FOXC1 Mutations

Mutations of the FOXC1 transcription factor are involved in a variety of autosomal dominant ocular anterior segment defects, ranging from Axenfeld-Rieger malformations to isolated glaucoma in some patients. In this study we have evaluated the possible role of the c.*734A>T FOXC1 variant as a modifier factor of the activity of two FOXC1 mutations previously identified in families primarily affected by dominant glaucoma (haplotypes p.G447_G448insDG-c.*734A>T and p.I126S-c.*734A>T). Previous bioinformatic analyses indicated that the c.*734A>T variant is located in a potential target sequence for hsa-miR-548l. Co-expression of this miRNA with a reporter cDNA construct in which the wild-type 3'UTR sequence of FOXC1 was fused to the 3'-end of the firefly luciferase coding region, led to approximately 20% decreased luciferase activity compared to the controls, confirming the presence of a target sequence for hsa-miR-548l. In contrast, this miRNA did not show any effect on the luciferase activity associated with the mutant 3'UTR FOXC1 sequence, showing that it resulted in a loss-of-function of the has-miR-548l target sequence. In addition, functional evaluation of the two glaucoma-associated haplotypes revealed increased protein levels and transactivation, compared to the corresponding individual coding mutations (approximately 1.2-fold on average). These data support the role of hsa-miR-548l as a regulator of FOXC1 translation and provide evidence for the c.*734A>T variant as a modifier factor for the activity of coding glaucoma-associated FOXC1 mutations.