Identification and Characterisation of a Novel Missense Homeodomain Mutation in ZEB1 Resulting in Keratoconus

Abstract

Purpose: : Mutations in zinc finger E-box binding homeobox 1 (ZEB1; MIM*189909) have been reported in posterior polymorphous corneal dystrophy (PPCD) and Fuch’s endothelial corneal dystrophy (FECD). Although PPCD and keratoconus (KTCN) involve different layers of the eye, PPCD has been associated with KTCN in several reports and mutations in VSX1 have been reported in both conditions. The purpose of this study was to screen ZEB1 for mutations in KTCN patients.

Methods: : The patient cohort consisted of 70 patients with sporadic and familial KTCN of Northern European ethnicity. All patients had a full ophthalmic examination and the diagnosis of keratoconus was made on the basis of clinical examination, a history of penetrating keratoplasty for keratoconus and corneal topography. Mutational analysis of ZEB1 was performed by direct cycle sequencing of all 9 exons. One hundred unrelated individuals (200 chromosomes) without ocular disease from the Northern Irish population were used as normal controls. An anterior corneal lamellar was available for one patient with a novel ZEB1 homeodomain mutation and corneal keratocytes were cultured. RT-qPCR was used to analyse the expression levels of ZEB1 transcriptional targets.

Results: : A novel heterozygous missense mutation, c.1920G>T, in exon 7 of ZEB1 which was detected in a patient familial KTCN and was not seen in 200 normal chromosomes. This nucleotide change results in a nonsynonymous substitution of a glycine with hisitdine (p.Gln640His), a highly conserved residue in the homeodomain of ZEB1. The mutation segregated within the family. RT-qPCR was performed on cultured keratocytes from the proband to investigate the downstream consequences of the homeodomain ZEB1 mutation on ZEB1 transcriptional activation. From 18 screened transcriptional targets, three genes were highly down-regulated and five genes were moderately down-regulated, including members of the collagen IV group.

Conclusions: : Mutations in ZEB1 have not previously been associated with keratoconus. The functional consequences of this mutation on potential ZEB1 transcriptional targets provides significant insight into keratoconus pathogenesis.

Keywords: cornea: stroma and keratocytes • cornea: epithelium • cornea: endothelium