Confirmation of the OVOL2 Promoter Mutation c.-307T>C in Posterior Polymorphous Corneal Dystrophy 1.

Doug D Chung,Anthony J Aldave,Katherine M Gee,Aleck E Cervantes,Elise Heon,Marina Zakharevich,Evelyn M Hanser,Edwin M Stone,Ricardo F Frausto,Andreas R Janecke

doi:10.1371/journal.pone.0169215

Abstract

PurposeTo identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without ZEB1 coding region mutations.MethodsThe promoter, 5’ UTR, and coding regions of OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity.ResultsOVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type.ConclusionsPreviously identified as the cause of PPCD1, the OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.

Highlights

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder characterized by the presence of characteristic corneal endothelial opacities as well as corneal steepening
Array CGH in the PPCD1 and PPCD3 loci excluded the presence of copy number variation (CNV) involving either ovolike zinc finger 2 (OVOL2) or zinc finger E-box binding homeobox 1 gene (ZEB1), respectively
Identified as the cause of PPCD1, the OVOL2 promoter variant c.-307T>C was identified in the original family that established the PPCD1 locus

Summary

Introduction

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder characterized by the presence of characteristic corneal endothelial opacities as well as corneal steepening. [24] Davidson and colleagues recently reported the identification of c.-307T>C and two other novel OVOL2 promoter region variants (c.-274T>G and c.-370T>C) in two British and 16 Czech PPCD families, which include two of the four families that have been previously mapped to the PPCD1 locus.[25] In addition, another OVOL2 promoter region variant (c.339_361dup) was identified in a family originally reported with autosomal dominant congenital hereditary endothelial dystrophy (CHED) mapped to the CHED1 locus, which overlaps the PPCD1 locus.[25] OVOL2 is a transcription factor that has been shown to downregulate ZEB1 expression, thereby suppressing EMT and driving mesenchymal-to-epithelial transition (MET) instead.[26, 27] Reporter activity assays using OVOL2 promoter constructs each containing one of the four identified variants demonstrated increased in vitro reporter gene expression compared to the wild type promoter in HEK293 cells.[25] Davidson and colleagues proposed that PPCD-associated OVOL2 promoter variants cause increased OVOL2 expression and the encoded protein subsequently represses ZEB1 expression, leading to a PPCD phenotype similar to PPCD3, in which ZEB1 truncating mutations likely result in ZEB1 haploinsufficiency.[4, 12]

Methods

Results

Conclusion