A substantial proportion of patients with posterior polymorphous corneal dystrophy (PPCD) lack a molecular diagnosis. We evaluated 14 unrelated probands who had a clinical diagnosis of PPCD who were previously determined to be negative for mutations in ZEB1 by direct sequencing. A combination of techniques was used including whole-exome sequencing (WES), single-nucleotide polymorphism (SNP) array copy number variation (CNV) analysis, quantitative real-time PCR, and long-range PCR. Segregation of potentially pathogenic changes with disease was confirmed, where possible, in family members. A putative run of homozygosity on chromosome 10 was identified by WES in a three-generation PPCD family, suggestive of a heterozygous deletion. SNP array genotyping followed by long-range PCR and direct sequencing to define the breakpoints confirmed the presence of a large deletion that encompassed multiple genes, including ZEB1. Identification of a heterozygous deletion spanning ZEB1 prompted us to further investigate potential CNVs at this locus in the remaining probands, leading to detection of two additional heterozygous ZEB1 gene deletions. This study demonstrates that ZEB1 mutations account for a larger proportion of PPCD than previously estimated, and supports the hypothesis that haploinsufficiency of ZEB1 is the underlying molecular mechanism of disease for PPCD3.

Department of Ophthalmology 1st Faculty of Medicine Charles University Prague and General University Hospital Prague Czech Republic

Department of Ophthalmology New Zealand National Eye Centre Faculty of Medical and Health Sciences University of Auckland Auckland New Zealand

Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine University Hospital Motol Charles University Prague Prague Czech Republic

Eye Department Greenlane Clinical Centre Auckland District Health Board Auckland New Zealand

Gennet Centre for Fetal Medicine and Reproductive Genetics Prague Czech Republic

Institute of Inherited Metabolic Disorders 1st Faculty of Medicine Charles University Prague and General University Hospital Prague Czech Republic

Moorfields Eye Hospital London UK

UCL Genetics Institute London UK

UCL Institute of Ophthalmology London UK

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