Classification of posterior polymorphous corneal dystrophy as a corneal ectatic disorder following confirmation of associated significant corneal steepening.

IMPORTANCE: The identification of steep corneal curvatures in a significant percentage of patients with posterior polymorphous corneal dystrophy (PPCD) confirms this previously reported association and suggests a role for the ZEB1 protein in keratocyte function. OBJECTIVE: To determine whether PPCD is characterized by significant corneal steepening. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study at university-based and private ophthalmology practices of 38 individuals (27 affected and 11 unaffected) from 23 families with PPCD. EXPOSURE: Slitlamp examination and corneal topographic imaging were performed for individuals with PPCD and unaffected family members. Saliva or blood samples were obtained from each individual for DNA isolation and ZEB1 sequencing. Corneal ZEB1 expression was measured using immunohistochemistry. MAIN OUTCOMES AND MEASURES: Percentage of individuals affected with PPCD and controls with an average keratometric value greater than 48.0 diopters (D) in each eye; the mean keratometric value averaged for both eyes of individuals with PPCD and controls; and the correlation of ZEB1 mutation with keratometric value. RESULTS: ZEB1 coding region mutations were identified in 7 of the 27 affected individuals. Ten of the 38 individuals (26.3%) had average keratometric values greater than 48.0 D OU: 10 of 27 individuals with PPCD (37.0%; 6 of 7 individuals with ZEB1 mutations [85.7%] and 4 of 20 individuals without ZEB1 mutations [20.0%]) and 0 of 11 unaffected individuals (P = .04 for unaffected vs affected individuals; P = .004 for individuals with PPCD with vs without ZEB1 mutation). The mean keratometric value of each eye of affected individuals (48.2 D) was significantly greater than that of each eye of unaffected family members (44.1 D) (P = .03). Affected individuals with ZEB1 mutations demonstrated a mean keratometric value of 53.3 D, which was significantly greater than that of affected individuals without ZEB1 mutations (46.5 D; P = .004). Fluorescence immunohistochemistry demonstrated ZEB1 expression in keratocyte nuclei. CONCLUSIONS AND RELEVANCE: Abnormally steep corneal curvatures are identified in 37% of all individuals with PPCD and 86% of affected individuals with PPCD secondary to ZEB1 mutations. ZEB1 is present in keratocyte nuclei, suggesting a role for ZEB1 in keratocyte function. Therefore, ZEB1 may play a role in both corneal stromal and endothelial development and function, and PPCD should be considered both an endothelial dystrophy and an ectatic disorder.

Analysis of the role of ZEB1 in the pathogenesis of posterior polymorphous corneal dystrophy.

PURPOSE: To determine how nonsense mutations in the transcription factor ZEB1 lead to the development of posterior polymorphous corneal dystrophy type 3 (PPCD3). METHODS: Whole-cell extracts were obtained from cultured human corneal epithelial cells (HCEpCs) as a source of ZEB1 protein. DNA-binding assays were performed using the whole-cell extract and oligonucleotide probes consisting of the two conserved E2-box motifs and surrounding nucleotides upstream of COL4A3. ZEB1 and COL4A3 mRNA expression in primary human corneal endothelial cells (HCEnCs) was assayed in both PPCD3 and control corneas by RT-PCR. Immunohistochemistry was used to localize ZEB1 and COL4A3 expression in normal human cornea. RESULTS: Electromobility shift assays (EMSAs) and competition EMSAs demonstrated binding of protein(s) in the cultured HCEpCs to the E2-box motifs in the probes. The supershift EMSA confirmed that ZEB1, demonstrated to be present in the whole-cell extracts, binds to both the proximal and distal E2-box motifs in the COL4A3 promoter region. Both COL4A3 and ZEB1 are expressed in normal HCEnCs, although in PPCD3, ZEB1 expression is decreased and COL4A3 expression is increased compared with levels of both genes in healthy control corneas. CONCLUSIONS: Inversely related HCEnC expression levels of ZEB1 and COL4A3 in PPCD3 indicate that ZEB1-mediated alterations in COL4A3 expression are most likely associated with the pathogenesis of this corneal endothelial dystrophy. However, the demonstration of COL4A3 expression in healthy adult primary HCEnCs suggests that PPCD3 is more likely to involve an alteration in the timing and/or degree of COL4A3 expression than to result from the dichotomous change implied by the previously proposed ectopic expression model.

The utility of next-generation sequencing in the evaluation of the posterior polymorphous corneal dystrophy 1 locus.

PURPOSE: To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1) using next-generation sequencing (NGS) of the common PPCD1 support interval, in which Sanger sequencing failed to identify a pathogenic mutation. METHODS: Enrichment of the portion of chromosome 20 containing the common PPCD1 interval was performed on DNA extracted from an affected and an unaffected member of a family previously linked to the PPCD1 locus. NGS using the Roche 454 Titanium platform was performed, followed by computational analysis using NextGENe Software. RESULTS: NGS of the selectively enriched chromosomal 20 region between markers D20S48 and D20S190 produced over 400,000 DNA sequence reads with an average of 350 bases for each of the two DNA samples. Alignment of the DNA sequence reads with the reference sequence from the National Center of Biotechnology Information (NCBI) resulted in over 119 million matched bases per sample. Approximately 68,000 DNA sequence variants were identified in the common PPCD1 support interval in the affected individual, which was approximately twice the number of sequence variants identified in the unaffected individual. In both individuals, approximately 0.5% of the identified variants mapped to the 13 known and 16 predicted genes in the PPCD1 support interval, including 16 of the 17 (94%) variants previously identified by Sanger sequencing in the 13 known genes. In both individuals, the variant not identified by NGS was located in a region of inadequate coverage. CONCLUSIONS: NGS identified all of the exonic sequence variants that were previously identified by Sanger sequencing in known genes in adequately covered regions of the common PPCD1 interval, although the pathogenic variant is yet to be discovered. Given adequate coverage of a selectively enriched chromosomal region of interest, NGS represents a useful technique to screen for sequence variants in candidate gene loci that has multiple advantages over previously employed techniques for mutation discovery.