Classification of Posterior Polymorphous Corneal Dystrophy as a Corneal Ectatic Disorder Following Confirmation of Associated Significant Corneal Steepening

Abstract

Importance

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 ZEB1 in keratocyte function.

Objective

To determine whether PPCD is characterized by significant corneal steepening.

Design

Cross-sectional study

Setting

University-based and private ophthalmology practices

Participants

Thirty-eight individuals (27 affected and 11 unaffected) from 23 families with PPCD

Interventions

Slit lamp examination and corneal topographic imaging were performed for individuals with PPCD and unaffected family members. Saliva or blood was collected from each individual for DNA isolation and ZEB1 sequencing. Corneal ZEB1 expression was measured using immunohistochemistry.

Main Outcome Measures

Percentage of individuals affected with PPCD and controls with average keratometry value >48.0D in each eye; mean keratometry value averaged for both eyes of individuals with PPCD and controls; correlation of ZEB1 mutation with keratometry value

Results

ZEB1 coding region mutations were identified in 7 of the 27 affected individuals. Ten of the 38 individuals (26.3%) had average keratometry values >48.0D OU: 10/27 (37.0%) individuals with PPCD (6/7 with ZEB1 mutations (85.7%) and 4/20 without ZEB1 mutations (20.0%)) and 0/11 unaffected individuals (p=0.037 for unaffected vs. affected; p=0.004 for PPCD with vs. without ZEB1 mutation). The mean keratometry value of each eye of affected individuals (48.2D) was significantly greater than unaffected family members (44.1D) (p value = 0.029). Affected individuals with ZEB1 mutations demonstrated a mean keratometry value of 53.3D, significantly greater than affected individuals without ZEB1 mutations (46.5D; p value = 0.004). 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.

INTRODUCTION

Posterior polymorphous corneal dystrophy (PPCD; MIM #122000) is an autosomal dominant corneal endothelial dystrophy characterized by well-described corneal endothelial abnormalities. Although the corneal endothelial dystrophies have traditionally been considered isolated disorders of the corneal endothelium, each has been associated with extraocular abnormalities: PPCD with abdominal hernias and Alport syndrome,^1–3 and both Fuchs endothelial corneal dystrophy (FECD; MIM #613267) and congenital hereditary endothelial dystrophy (CHED; MIM #217700) with hearing loss.^4–6 PPCD has also been associated with a number of other ocular abnormalities, including glaucoma, Terrien’s marginal degeneration and abnormalities of corneal curvature, including keratoconus.^7–20 While the association of PPCD with keratoconus was initially reported in the English-language literature almost 40 years ago, the subsequent 9 reports of this association published between 1989 and 2010 consisted of either individual case reports (4) or small case series (3, 3, 4, 5 and 7 subjects).^{7, 8, 10–15, 18, 20} Given the relative frequency of keratoconus in the general population, estimated to have an incidence of 1 in 2000, as well as its reported association with a variety of ocular and non-ocular disorders, the significance of the reported association with PPCD has been questioned.²¹ However, mutations in the visual system homeobox gene 1 (VSX1; MIM #605020) have been implicated as playing a pathogenic role in both PPCD and keratoconus, thus providing support to the contention that the reported association between the disorders is more than just coincidental.^22–30 In 2011, Raber and colleagues reported 18 patients from 10 families with PPCD who demonstrated steep corneal curvatures, with average keratometry values >48.0 D in each eye of 15 of the 18 patients.¹⁶ However, the authors acknowledge that the cohort that they reported included only individuals with PPCD and an average keratometry > 46.0 D and no clinical or topographic evidence of keratoconus. Thus, it is not possible to determine from their study what percentage of individuals with PPCD demonstrate abnormally steep corneal curvatures. Additionally, the authors did not screen the zinc finger E-box binding homeobox 1 gene (ZEB1; MIM 189909), previously known as the transcription factor 8 gene (TCF8), in which pathogenic mutations have been identified in approximately 1/3 of probands with PPCD. In fact, ZEB1 screening has been performed for only 4 individuals reported to date with PPCD associated with steep corneal curvatures.^{14, 20} Thus, it is not known whether abnormalities of corneal curvature are associated with PPCD that has been linked to chromosome 20 (the PPCD1 locus) or to PPCD associated with mutations in ZEB1 (located on chromosome 10, also known as the PPCD3 locus). Therefore, we performed corneal topographic imaging for all available affected probands and affected and unaffected family members from 45 families with PPCD. We also screened the ZEB1 coding and promoter regions in all 45 probands and determined segregation of identified presumed pathogenic variants in all available affected and unaffected family members.

MATERIALS AND METHODS

The authors followed the tenets of the Declaration of Helsinki in the treatment of the subjects reported herein. Study approval was obtained from the Institutional Review Board at The University of California, Los Angeles (UCLA IRB # 94-07-243-(14-33A), 02-10-092-(4,11), 10-001932).

Patient Identification/ZEB1 Coding and Promoter Region Screening

The diagnosis of PPCD was based on the presence of characteristic corneal endothelial changes in one or both eyes.¹ Additionally, the diagnoses of clinical and topographic keratoconus were based on previously published criteria.³¹ After informed consent was obtained, either a peripheral blood sample, saliva sample (Oragene saliva collection kits; DNA Genotek, Inc., Ontario, Canada) or buccal epithelial sample (Cyto-SoftTM Cytology Brush; Medical Packaging Corporation, Camarillo, CA) was collected as a source of genomic DNA. After genomic DNA was prepared from the buccal epithelial cells and peripheral blood leukocytes using the QIAamp DNA Blood Mini Kits and FlexiGene DNA (Qiagen, Valencia, CA), respectively, each of the nine exons of ZEB1 and the 1 Kb upstream of the initiation methionine were amplified using previously described primers and conditions.^{1, 32} After purification of the PCR products, DNA sequencing was performed on an ABI-3100 Genetic Analyzer (Applied Biosystems). The coding region nucleotide sequence (including the donor and acceptor splice sites) was read manually by comparison to the ZEB1 cDNA sequences (GenBank accession numbers NM_001128128.2 and NM_030751), while promoter region sequences were compared to the ZEB1 RefSeqGene sequence (GenBank accession number NG_017048.1).

Corneal Topographic Imaging

A recruitment letter regarding the performance of corneal topography was sent to all individuals enrolled into the investigators’ ongoing study on PPCD for whom corneal topographic imaging had not been previously performed. Individuals who expressed interest in participating were encouraged to come to the Jules Stein Eye Institute for topographic imaging. For patients who were unable to travel to the Jules Stein Eye Institute, prior corneal topographies were obtained, when available, or patients were encouraged to return to their local ophthalmologist for corneal topographic imaging. For patients examined at the Jules Stein Eye Institute, corneal topographic imaging was performed using a commercially available corneal topographer (Marco OPD-Scan III, Jacksonville, FL). After a single topographic image was obtained of each cornea, the average keratometry value was determined using the mean of the simulated K readings from the keratometric map. Eyes in which penetrating keratoplasty was performed prior to topographic imaging or in which other pathology, such as bullous keratopathy, prevented an accurate assessment of the corneal curvature were excluded.

Immunohistochemical detection of ZEB1 in the Corneal Stroma

A donor cornea obtained from a commercial eye bank was fixed in 4% PFA and subsequently placed in 30% sucrose for cryoprotection. Immunodetection of ZEB1 (ab87280; Abcam, Inc., Cambridge, MA) and CD34 (keratocyte marker; 3569; Cell Signaling Technology, Beverly, MA) was performed using a standard immunohistochemistry protocol. Briefly, sections were rehydrated in PBS + 0.3% TritonX100, washed 2× in PBS and blocked in PBS + 0.05% Tween-20 supplemented with 1% BSA and 10% normal serum. The sections were then incubated overnight with each primary antibody diluted 1:100 in blocking buffer, washed 1× in PBS + 0.05% Tween-20 followed by 2× in PBS. The sections were subsequently incubated with the secondary antibody (Alexa Fluor 488 or 594; Life Technologies, Carlsbad CA), diluted 1:500 in blocking buffer, washed 1× in PBS + 0.05% Tween-20 followed by 2× in PBS and mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA) aqueous mounting medium containing 40, 6-diamidino-2-phenylindole (DAPI). A negative control consisting of a species-specific normal IgG (Jackson ImmunoResearch, West Grove, PA) was used at the same concentration as each primary antibody. Fluorescence imaging was performed on an epifluorescence Zeiss microscope (Axio Imager.A2, Carl Zeiss, Oberkochen, Germany).

Statistical Analyses

Fisher’s exact test was used to compare the percentage of affected and unaffected individuals with PPCD with average keratometry values greater than 48.0 D in each eye and the percentage of affected individuals with and without ZEB1 mutations with average keratometry values greater than 48.0 D in each eye. The student t-test was used to determine the significance of the difference between the mean keratometry value of eyes of affected individuals and unaffected family members as well as for eyes of affected individuals with and without ZEB1 mutations. P values less than 0.05 were considered significant.

RESULTS

Corneal Topographic and Slit Lamp Imaging

Corneal topographic imaging was performed for 38 individuals (27 affected and 11 unaffected) from 23 of the 45 families with PPCD recruited to date. Ten of the 38 subjects (26.3%) for whom corneal topographic imaging was performed demonstrated average keratometry values greater than 48.0 D in each eye, including a significantly greater percentage of individuals with PPCD (37.0%; 10/27) compared with unaffected individuals (0%; 0/11) (Fisher’s exact test: p=0.037). Corneal topographic imaging was performed for affected family members of two of these 10 subjects, demonstrating average keratometry values greater than 48.0 D in each eye of these individuals as well. The mean keratometry value of each eye of affected individuals measured 48.2 D (SD = 5.8 D), which was significantly greater than the mean keratometry value of 44.1 D (SD = 2.2 D) for unaffected family members (t-test p value = 0.029).

Slit lamp examination of the 10 individuals with PPCD who demonstrated average keratometry values greater than 48.0 D in each eye revealed characteristic clinical features of keratoconus in both eyes of two individuals (Table 1). In both of these patients, corneal topographic imaging demonstrated central corneal steepening, consistent with keratoconus. Additionally, imaging of the posterior corneal surface in one patient using slit scanning topography (Orbscan, Bausch & Lomb, Rochester, New York) demonstrated significant elevation of the posterior corneal profile compared to a best fit sphere (OD 0.172 mm; OS 0.078 mm), the apex of which corresponded in location to the thinnest portion of the cornea in each eye (OD 354 microns; OS 403 microns). A third patient demonstrated inferior nasal steeping on corneal topographic imaging, but did not demonstrate any clinical features of keratoconus on slit lamp biomicroscopy (Figures 1 and 2).

Table 1.

Clinical features of patients with PPCD and steep corneal curvature (> 48.0 D OU)

Patient	Sex	Eye	Simulated Keratometry			Keratoconus		ZEB1 Mutation
			Steep K	Flat K	Average K	Clinical Features	Topographic Features
1	M	OD	50.50	49.50	50.00	No	No	p.(Gln12*)
		OS	50.75	48.25	49.50	No	No
2*	M	OD	52.08	48.70	50.39	No	Yes (Inferonasal steepening)	p.(Gln884fs)
		OS	49.34	47.87	48.61	No	Yes (Inferonasal steepening)
3	M	OD	51.50	54.00	52.75	No	No	No
		OS	51.25	52.25	51.75	No	No
4	M	OD	51.25	55.75	53.50	No	No	p.(Gln214*)
		OS	55.25	55.72	55.49	No	No
5	M	OD	55.00	56.00	55.50	No	No	No
		OS	53.50	53.50	53.50	No	No
6	F	OD	57.00	57.00	57.00	No	No	p.(Glu495fs)
		OS	55.50	58.00	56.75	No	No
7**	F	OD	56.20	57.98	57.09	No	No	p.(Arg325*)
		OS	58.02	59.32	58.67	No	No
8**	F	OD	58.46	61.26	59.86	No	No	p.(Arg325*)
		OS	56.02	59.21	57.62	No	No
9	F	OD	58.60	56.80	57.70	Yes (conical corneal deformation; central and interior corneal thining; s/p hydrops)	Yes (Central corneal steepening; anterior protrusion of posterior corneal profile)	No
		OS	55.40	53.50	54.45	Yes (conical corneal deformation; central and interior corneal thining; s/p hydrops)	Yes (Central corneal steepening; anterior protrusion of posterior corneal profile)
10	F	OD	59.31	56.72	58.02	Yes (conical corneal deformation)	Yes (Central corneal steepening)	No
		OS	64.90	59.52	62.21	Yes (conical corneal deformation)	Yes (Central corneal steepening)

^*Individual depicted in Figures 1 and 2;

^**Individuals are related (7 is the mother of 8)

Figure 1.

Slit lamp photomicrographs of individual with PPCD demonstrating confluent areas of grey-white opacification at the level of Descemet membrane (right). Scattered endothelial vesicles are seen with retroillumination against the red reflex (left). Screening of ZEB1 revealed the novel mutation p.(Gln884Argfs*37).

Figure 2.

Corneal topographic imaging of the affected individual shown in Figure 1 demonstrating inferior temporal steepening in each cornea, with average keratometry values measuring 50.39 D in the right eye and 48.61 D in the left eye. No clinical features of keratoconus were noted on slit lamp biomicroscopic imaging of either cornea.

While corneal topographic imaging was not performed An insufficient number of individuals were recruited from each family in which an individual with PPCD and steep corneal curvature was identified to confirm that all affected relatives with PPCD had steep corneas as well. However, in both families in which two affected individuals underwent corneal topographic imaging, both individuals had steep corneas. Additionally, in the family in which an unaffected individual underwent corneal topographic imaging, the individual did not demonstrate a steep cornea in either eye.

ZEB1 Coding and Promoter Region Screening

Screening of the ZEB1 coding region demonstrated nonsense mutations in 7 of the 27 (26%) affected individuals, including 6 of the 23 (26%) probands. Screening of the ZEB1 promoter region in each affected individual did not reveal any presumed pathogenic sequence variants.³² A significantly greater percentage of affected individuals with a ZEB1 mutation (85.7%; 6/7) demonstrated average keratometry values greater than 48.0 D in each eye compared with affected individuals without a ZEB1 mutation (20.0%; 4/20) (Fisher’s exact test: p =0.004). Additionally, the mean keratometry value of each eye of affected individuals with a ZEB1 mutation (53.3 D; SD = 4.8 D) was significantly greater than the mean keratometry value of affected individuals without a ZEB1 mutation (46.5 D; SD = 5.0 D) (t-test: p value = 0.004).

Clinical Course and Management

The mean age of the 10 individuals with PPCD and average keratometry values > 48.0 D in each eye at the time of the initial slit lamp examination and corneal topographic imaging was 37.7 years (range, 12–66 years). Three of the individuals were less than 30 years old when first examined, but serial topographies are not available for any of the three patients to evaluate for progressive corneal steepening. Additionally, two of the three underwent penetrating keratoplasty in either one or both eyes, thus not permitting detection of changes in the native corneal curvature. Overall, seven of the 10 (70%) affected individuals with steep keratometry values underwent corneal transplantation in one or both eyes, a significantly higher percentage than the 15.9% (10/63) of affected individuals with either unknown keratometry or average keratometry values < 48.0D in each eye who required corneal transplantation (p = 0.0009). In six of the seven individuals, corneal transplantation was performed for visually significant corneal edema, while in the seventh, the indication was corneal ectasia and central corneal scarring.

Immunohistochemical detection of ZEB1 in the Corneal Stroma

To determine whether ZEB1 is expressed in the corneal stroma, fluorescence immunodetection of keratocytes in an eye bank cornea was performed using an antibody to CD34, a type-I transmembrane glycophosphoprotein that is typically present in stromal fibroblasts (Figure 3). Antibodies directed against ZEB1 demonstrated that the protein is expressed by the stromal keratocytes, as well as by the corneal epithelial and endothelial cells. In each cell type, expression was localized to the nucleus, identified with the DAPI stain, as can clearly be seen in the merged images (Figure 3). Isotype and secondary only controls showed either no or low and diffuse background staining (data not shown).

Figure 3.

Immunodetection of ZEB1 in normal donor cornea. A. A cross-section of the cornea probed with anti-ZEB1 (Red, 594nm) and anti-CD34 (Green, 488nm) demonstrates ZEB1 expression in the epithelium, stroma and endothelium. Nuclei are stained with 40, 6-diamidino-2-phenylindole (DAPI) (10× objective). B. Higher magnification views of the indicated sections of the cornea demonstrate that ZEB1 is present in the nuclei of epithelial cells, stromal keratocytes and endothelial cells (100× oil objective).

DISCUSSION

While traditionally considered an isolated disorder of the corneal endothelium, PPCD has now been shown to be associated with significantly steeper mean keratometry values than unaffected individuals, as previous investigators have indicated in smaller, uncontrolled series. We defined abnormally steep corneal curvature as a mean keratometry value of >48.0 D in each eye, as this value is approximately three standard deviations (SD) above the mean keratometry value of 43.97 D described in normal individuals (SD = 1.54 D).^{16, 33} Thirty seven percent of individuals with PPCD and 86% of individuals with PPCD3 demonstrated corneal curvatures that were > 48.0 D in both eyes, as compared to none of the unaffected individuals. In addition, the mean keratometry value averaged for both eyes of individuals with PPCD (48.2 D) is approximately 3 SDs above the population mean and for individuals with PPCD3, the mean keratometry value (53.3 D) is 6 SDs above the population mean. Therefore, PPCD should be considered a corneal ectatic disorder, along with keratoconus, keratoglobus and pellucid marginal degeneration. Although the corneal ectasias are characterized by corneal stromal thinning, as corneal transplantation was performed for visually significant stromal edema in the majority of individuals with steep keratometry values, we are not able to correlate decreased corneal stromal thickness with steep corneal curvature. Additionally, as we had not performed corneal topographic imaging in patients with PPCD until recently, we do not have serial topographies in affected individuals to assess for progression. However, we plan to do so going forward to determine whether the abnormally steep corneal curvature associated with PPCD is progressive throughout life, or whether it stabilizes at a particular age in affected individuals, as is typically the case with the other corneal ectatic disorders.

An argument may be made that PPCD itself is not a corneal ectatic disorder, but instead presents in association with corneal steepening as it shares a common genetic basis with keratoconus. Supporters of this argument would point to the publications reporting mutations in VSX1 purported to play a pathogenic role in both PPCD and keratoconus.^22–30 However, just as many publications have been published either not substantiating or refuting a role for VSX1 in both PPCD and keratoconus.^{27, 31, 34–44} Additionally, multiple genome-wide linkage and association studies for keratoconus have not identified involvement of either the PPCD1 locus on chromosome 20, containing VSX1, or the PPCD3 locus on chromosome 10, containing ZEB1.^{39, 45–55} Given this, and the association of PPCD with corneal steeping in the absence of clinical features of keratoconus described in this report as well as by Raber and others, significant evidence exists to suggest that PPCD is associated with corneal steepening independent of an association with keratoconus (Table 2).^{7, 11, 13, 16, 20}

Table 2.

Previous reports of PPCD associated with abnormalities of corneal curvature

Author	Year of Publication	Subjects	Subjects with Average K value > 48 D OU	Subjects with Average K value > 50 D OU	Subjects Diagnosed with Keratoconus	ZEB1 Screening
Gasset12	1974	2	2	2	2	No
Weissman18	1989	4	3	2	4	No
Bechara7	1991	1	Not mentioned	Not mentioned	0*	No
Blair8	1992	1	0	0	1	No
Driver11	1994	3	2	2	2	No
John13	1998	5	4	2	0	No
Mazotta15	2008	1	0	0	1	No
Cremona10	2009	7	≥1**	≥1**	7	No
Lam14	2010	1	0	0	1	Yes (negative)
Liskova20	2010	3	0†	0†	0††	Yes (all positive)
Raber16	2011	18	15†	9†	0	No

^*Topographic and histopathologic features but no clinical features of keratoconus;

^**keratometry values provided for only 1 of the 7 patients;

^†One subject had undergone corneal transplantation in one eye, and pre-operative keratometry was not available;

^††One subject demonstrated topographic features but no clinical features of keratoconus

Clarification of the association of PPCD with steep corneal curvature will require further elucidation of the genetic factors that influence corneal curvature as well as the role that ZEB1 plays in both corneal endothelial and stromal development and function. To date, we have examined and collected DNA from 73 affected individuals with PPCD. Seventeen of these 73 individuals (23.3%) have required corneal transplantation, which includes 9 of the 26 individuals (34.6%) with ZEB1 mutations. The fact that this percentage is twice that of affected individuals without ZEB1 mutations (17.0%; 8/47) is suggestive that ZEB1 haploinsufficiency results in more several endothelial dysfunction than the yet to be identified protein dysfunction associated with non-ZEB1 PPCD, such as PPCD1. Similarly, the significantly greater mean keratometry value of affected individuals with a ZEB1 mutation compared to affected individuals without a ZEB1 mutation is additional evidence of a more severe clinical phenotype associated with PPCD3, and indicates an association between more severe endothelial dysfunction and more pronounced corneal steepening.

We acknowledge several limitations of this study, one of which is the use of the need for corneal transplantation as an indicator of the degree of endothelial dysfunction. As we have indicated that a significantly greater percentage of patients with average keratometry values > 48.0 D in each eye required corneal transplantation than those with average keratometry values < 48.0 D, this suggests that the difference is due the presence of corneal ectasia as the indication for transplantation in the former group. However, corneal transplantation was performed for visually significant corneal edema, not corneal ectasia, in all but one of these patients. The suggestion that the degree of endothelial dysfunction is greater in eyes with more pronounced corneal steepening is an association that will require more detailed clinical characterization in a larger number of affected individuals to definitively demonstrate. Another limitation of this study is the fact that keratometry and ZEB1 screening data were available and analyzed for 37% (27 of 73) of all recruited affected individuals from just over one-half (23 of 45) of the families recruited to date. Obviously, individuals with a more severely affected phenotype may be more likely to participate in research studies than those with more mild manifestations of a disease, a selection bias that may affect the study findings.

While PPCD is clearly a corneal endothelial dystrophy, we believe that this and the previous reports listed in Table 2 are sufficient to consider PPCD to be a corneal ectatic disorder as well. As truncating mutations in ZEB1 account for approximately 1/3 of PPCD, and the genetic basis of the other 2/3 remains to be elucidated, an understanding of the genetic basis of the endothelial dysfunction and corneal steepening that characterize PPCD begins with an analysis of ZEB1 expression and function. We have previously demonstrated ZEB1 expression in the corneal endothelium and present evidence of its expression in the corneal stroma in this report.⁵⁶ In PPCD3, we have demonstrated decreased expression of ZEB1 and increased expression of collagen, type IV, alpha 3 (COL4A3; MIM 120070) in the corneal endothelium, leading to the proposed pathogenesis underlying the endothelial cell abnormalities observed in affected individuals and in Zeb1-heterozygous and -null mice.⁵⁷ We are currently investigating whether ZEB1 and COL4A3 also demonstrate inversely related corneal stromal expression levels in PPCD3 and whether interaction of ZEB1 with other proteins expressed in the corneal stroma that contain E2-box motifs (to which ZEB1 binds) and/or are the product of genes implicated in the determination of corneal curvature (such as FRAP1 and PDGFRA) may be involved in the pathogenesis of the significant corneal steepening.^{58, 59}