Purpose: To 1) screen for mutations in the GRHL2 gene in PPCD probands in whom mutations in the ZEB1 and OVOL2 genes were not identified; and 2) determine whether GRHL2 expression is modulated by PPCD4-associated GRHL2 mutations in corneal endothelial cells using in vitro promoter activity assays.
Methods: Fifty-nine patients with PPCD were recruited from the Stein Eye Institute, Los Angeles, CA. Patients were examined via slit lamp biomicroscopic imaging to confirm the diagnosis of PPCD, and genomic DNA was purified from the patients' peripheral blood leukocytes or buccal epithelial cells. As part of a previous study, 37 of 59 PPCD probands were revealed to not harbor any mutations in the ZEB1 or OVOL2 genes and, therefore, were genetically unresolved. Of those 37 genetically unresolved PPCD patients, 24 had sufficient DNA quality and quantity for performing additional screening. The GRHL2 regulatory region containing the 5' UTR, exon 1, and the first 650 base pairs of intron 1 (2,728-bp total) was amplified using PCR. Sanger sequencing was performed on the purified PCR products, and sequencing results were screened for any novel or rare (MAF < 0.01) mutations. To generate the reporter vector for the in vitro promoter activity assay, the GRHL2 regulatory region was amplified from control genomic DNA and cloned into pCR4-TOPO vector for sub-cloning into a promoter-less luciferase reporter vector.
Results: Sanger sequencing screening of the GRHL2 regulatory region was negative for any novel or rare mutations in the 24 genetically unresolved PPCD patients. In an effort to generate the in vitro luciferase reporter vector, the GRHL2 regulatory region was successfully amplified from the control genomic DNA using PCR, but TOPO cloning did not yield any positive clones containing the entire regulatory region.
Conclusion: The negative screening result in the 24 unresolved PPCD patients likely reflects the genetically heterogeneous nature of PPCD. Future work will focus on optimizing experimental conditions to successfully clone the GRHL2 regulatory region into the luciferase reporter vector to verify the modulatory effects of PPCD4-associated GRHL2 mutations, as well as identifying novel pathological mutations in the genetically unresolved PPCD patients.