Non-coding mutations identified as corneal endothelial dystrophy cause

Understanding the genetic cause for PPCD is becoming increasingly important and relevant in all medical fields, including ophthalmology.

Researchers studying families affected by posterior polymorphous corneal dystrophy 4 (PPCD4) have identified new mutations causing the rare autosomal-dominant corneal endothelial dystrophy. The discovery and related investigations that are described in a recent publication [Liskova P, et al. Am J Hum Genet. 2018;102:447–459.] provide insight into the molecular basis of PPCD and have implications for both clinical care and genetic researchers, said Alison Hardcastle, PhD, and Petra Liskova, MD, PhD.

Studying individuals from a Czech family and using linkage analysis, the investigators identified a single locus mapping to chromosome 8q22.3–q24.12. Through whole-genome sequencing, a unique variant (c.20+544G>T) was found in the locus that was within intron 1 of GRHL2.

Targeted screening of GRHL2 regulatory regions in individuals from unrelated, previously unsolved PPCD4-affected families led to the discovery of two additional unique genetic variants in the intronic regulatory region of GRHL2. In vitro tests showed that the three PPCD4 variants induced increased expression of GRHL2.

Histological evaluation and immunostaining studies of corneal tissue from a PPCD4-affected eye and unaffected controls showed that endothelial cells in the diseased eyes inappropriately expressed GRHL2 as well as epithelial markers (E-cadherin and cytokeratin 7), indicating that the cells had transitioned from an endothelial to an epithelial-like cell type.

Dr. Hardcastle and Dr. Liskova explained that the mutations discovered in patients with PPCD4 and those found previously in PPCD1- and PPCD3-affected families all involve genes that are key regulators of pathways involved in epithelial to mesenchymal transition/mesenchymal to epithelial transition (EMT/MET) pathways.

“An imbalance in transcription factors involved in the EMT/MET pathways appears to be a convergent disease mechanism leading to dysfunction of the endothelial barrier and the development of PPCD,” said Dr. Hardcastle, professor of molecular genetics, UCL Institute of Ophthalmology, University College London, London. “The mutations cause either an increase or decrease in expression of the transcription factors that causes the cells to transition to an epithelial-like cell state.”

Clinical relevance

Dr. Hardcastle and Dr. Liskova noted that elucidation of the genetic causes for PPCD has several clinical ramifications.

“Traditionally, disorders leading to corneal opacity have been treated surgically, and knowledge of the underlying molecular causes has not been considered essential for patient management,” said Dr. Liskova, associate professor, Departments of Ophthalmology and Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University, Prague, Czech Republic. “However, understanding the genetic cause is becoming increasingly important and relevant in all medical fields, including ophthalmology.”

Dr. Liskova noted that in some patients, PPCD is phenotypically indistinguishable from congenital hereditary endothelial dystrophy.

“The ability to identify disease-causing mutations would facilitate an accurate diagnosis. Because congenital hereditary endothelial dystrophy may be associated with hearing loss, it is recommended that affected children undergo regular evaluations of auditory function. This type of follow-up is not needed for children with PPCD,” said Dr. Liskova.

There are also implications for counseling of patients with PPCD.

Dr. Liskova explained, “Patients with both Mendelian disorders and complex disorders seek genetic counseling and testing. By dissecting the genetic cause of PPCD we can provide more accurate predictions of disease progression, associated risks, such as secondary glaucoma development, and if a corneal transplant may be necessary as disease progresses,” she said.

Dr. Liskova added, “Knowledge of the underlying genetic cause is fundamental to understanding disease mechanisms and facilitates genetic testing to determine if individuals are at risk of developing disease. Ophthalmologists have an essential role in informing patients and their families about genetic testing.”

Obtaining a clearer understanding of pathogenic mechanisms for disease development also opens up opportunities for identifying targeted therapies, and as an accessible tissue, the cornea is well suited for genetic treatment.

“Switching off inappropriately expressed genes could be considered as a possible therapeutic option for corneal dystrophies,” said Dr. Liskova.

“We have recently demonstrated the potential utility of a gene targeted therapy for Fuchs corneal endothelial dystrophy. We found that treating patient cells with a targeted antisense oligonucleotide reversed toxic effects caused by a mutation in TCF4. In the future, a similar type of gene-directed therapy could be applicable for PPCD.” said Dr. Hardcastle.

The discovery of the genetic cause of PPCD4 was possible because of the use of whole-genome sequencing (WGS).

“Whole-genome sequencing enabled interrogation of the entire genome rather than just the protein coding portion that is more typically and routinely investigated,” said Dr. Liskova.

Disclosures:

Petra Liskova, MD, PhD
E: petra.liskova@lf1.cuni.cz

Alison Hardcastle, PhD
E: a.hardcastle@ucl.ac.uk

Dr. Liskova and Dr. Hardcastle have no relevant disclosures