Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy.

Purpose: To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression. Methods: Next-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause). Results: Expression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes. Conclusions: Identification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.

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

PURPOSE: To 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. METHODS: The 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. RESULTS: OVOL2 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. CONCLUSIONS: Previously 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.

Active extravasation of gadolinium-based contrast agent into the subdural space following lumbar puncture.

A 38year-old male presented with cauda equina syndrome following multiple lumbar puncture attempts. Lumbar spine magnetic resonance imaging (MRI) showed a subdural hematoma and an area of apparent contrast enhancement in the spinal canal on sagittal post-contrast images. Axial post-contrast images obtained seven minutes later demonstrated an increase in size and change in shape of the region of apparent contrast enhancement, indicating active extravasation of the contrast agent. This is the first reported case of active extravasation of gadolinium-based contrast agent in the spine.