Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2

Abstract

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

DOMINO: Using Machine Learning to Predict Genes Associated with Dominant Disorders

“…As a ''validation set,'' we used 99 genes with Mendelian mutations (26 AD genes and 73 AR genes) that we ex- tracted from papers published from January 2016 to March 2017 in The American Journal of Human Genetics and in Nature Genetics, to mimic the discovery of newly-reported genes and confirm the absence of a potential bias toward well-studied and annotated genes, composing the bulk of the training set (Table S4). For the validation set, DOMINO predicted AD association with an AUC of 0.920 ( Figures 1D and 1E) and specificity and sensitivity of 88.5% and 78.1% at J max , respectively (Table S4, Figures 2D and 2E) 22 while KLHL24 has a start-loss DNA change resulting in the use of a downstream alternative initiation site. 23 The mechanisms of pathogenesis for SAMD9L are also rather unusual for a Mendelian condition and are characterized by particular chromosomal rearrangements.…”

An N‐Ethyl‐N‐Nitrosourea (ENU) Mutagenized Mouse Model for Autosomal Dominant Nonsyndromic Kyphoscoliosis Due to Vertebral Fusion

“…These include triphalangeal thumb/preaxial polydactyly (autosomal dominant with variable penetrance), due to mutations in the ZPA regulatory sequence, a long‐range cis‐acting regulator of Sonic Hedgehog ( SHH ) gene expression67, 68, 69; and autosomal recessive isolated pancreatic agenesis due to noncoding mutations downstream of pancreas‐specific transcription factor 1a ( PTF1A ) 70. Such mutations are frequently large deletions or duplications, but may also consist of single‐nucleotide mutations, similar to those most frequently induced by ENU, as found in isolated pancreatic agenesis, and recently described in the promoter region of ovo‐like zinc finger 2 ( OVOL2 ) in families with autosomal‐dominant corneal endothelial dystrophies 71. Mutations upstream of PTF1A were found to reduce the expression of PTF1A , whereas mutations in the OVOL2 promoter were able to induce OVOL2 expression, likely leading to aberrant ectopic OVOL2 expression in the developing cornea 70, 71.…”

“…Either of these two molecular mechanisms could account for the dominant disease presentation in Hvf mice, eg, through a dosage effect due to a reduction in the level of a critical transcript, or through ectopic induction of a transcript, and either of these may be time and/or spatially specific. Future work to identify the Hvf causative mutation will focus on analyses of the six intergenic and three intronic noncoding variants in transcriptional assays, such as luciferase assays, similarly to those undertaken for PTF1A and OVOL2‐associated noncoding variants 70, 71. To determine the specific role of the variants in somitogenesis, these assays may need to be undertaken in a somite cell‐line, such as cells derived from pluripotent stem cells,72 or in bone‐specific cells such as chondrocytes.…”

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

“…[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.…”

“…[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.…”