Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4.

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.

Active transforming growth factor-ß2 in the aqueous humor of posterior polymorphous corneal dystrophy patients.

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal proliferation of corneal endothelial cells. It was shown that TGF-ß2 present in aqueous humor (AH) could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to determine whether the levels of this protein are changed in AH of PPCD patients. METHODS: We determined the concentrations of active TGF-ß2 in the AH of 29 PPCD patients (42 samples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients were divided based on either the molecular genetic cause of their disease as PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples). RESULTS: The level of active TGF-ß2 in the AH of all PPCD patients (mean ± SD; 386.98 ± 114.88 pg/ml) in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001). Compared to the control group, a significantly higher level of active TGF-ß2 was found in the PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-ß2 were not significantly affected by gender, age, secondary glaucoma or by the progression of dystrophy when one or repeated PK were performed. CONCLUSION: The levels of active TGF-ß2 in the AH of PPCD patients are significantly higher than control values, and thus the increased levels of TGF-ß2 could be a consequence of the PPCD phenotype and can be considered as another feature characterizing this disease.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.

Correction of moderate to high hyperopia with an implantable collamer lens: medium-term results.

PURPOSE: To evaluate the medium-term results of phakic posterior chamber implantable collamer lens implantation to correct moderate and high hyperopia. METHODS: In this retrospective study, patients were treated for hyperopia with the Visian Implantable Collamer Lens (ICH model V3; STAAR Surgical AG, Nidau, Switzerland). Examined parameters were manifest refraction spherical equivalent, uncorrected distance visual acuity, corrected distance visual acuity, vault, anterior chamber depth, anterior chamber angle width, endothelial cell density, intraocular pressure, patient satisfaction, and complications. RESULTS: The mean age of 15 patients (28 eyes) was 28 years (range: 18 to 36 years), with a mean follow-up period of 3.6 years (range: 3 to 6 years). The mean manifest refraction spherical equivalent decreased from +6.30 ± 1.42 diopters (D) (range: +4.25 to +8.50 D) preoperatively to -0.37 ± 0.56 D (range: -1.25 to +1.00 D) at 3 years postoperatively. The mean uncorrected distance visual acuity improved from 0.77 ± 0.38 logMAR (range: 0.16 to 1.30 logMAR) to 0.20 ± 0.17 logMAR (range: 0.00 to 0.48 logMAR) at the 3-year follow-up. Postoperatively, 62% of eyes gained one line of corrected distance visual acuity or remained unchanged. The mean vault reduced from 367.1 ± 253.6 µm (range: 70.0 to 1,190.0 µm) at 1 month postoperatively to 283.6 ± 210.0 µm (range: 75.0 to 915.0 µm) at the last follow-up visit (P = .005). The mean preoperative anterior chamber depth and anterior chamber angle width also decreased at the last follow-up visit (P = .037 and < .0001, respectively). The mean endothelial cell loss was 4.91% (P = .089). No serious complications occurred. Thirteen (87%) patients were satisfied with the outcomes and no patient was dissatisfied. CONCLUSIONS: Implantation of a posterior chamber implantable collamer lens is a safe, effective, predictable, and stable method for the correction of moderate and high hyperopia in highly selected patients. No case of cataract or anterior subcapsular opacities formation was recorded in relation to the decrease of vault over the studied period and low vault in some eyes.

The application of autologous serum eye drops in severe dry eye patients; subjective and objective parameters before and after treatment.

AIM: To assess the impact of autologous serum (AS) eye drops on the ocular surface of patients with bilateral severe dry eye and to draw a comparison between the clinical and laboratory examinations and the degree of subjective symptoms before and after serum treatment. MATERIALS AND METHODS: A three-month prospective study was conducted on 17 patients with severe dry eye. AS eye drops were applied a maximum of 12 times a day together with regular therapy. Dry eye status was evaluated by clinical examination (visual acuity, Schirmer test, tear film breakup time, vital staining, tear film debris and meniscus), conjunctival impression cytology (epithelial and goblet cell density, snake-like chromatin, HLA-DR-positive and apoptotic cells) and subjectively by the patients. RESULTS: The application of AS eye drops led to a significant improvement in the Schirmer test (p < 0.01) and tear film debris (p < 0.05). The densities of goblet (p < 0.0001) and epithelial cells (p < 0.05) were significantly increased, indicating a decrease of squamous metaplasia after AS treatment. A significant decrease (p < 0.05) was found in the number of apoptotic, HLA-DR-positive and snake-like chromatin cells on the ocular surface. A significant improvement was found in all evaluated subjective symptoms. Altogether, the clinical results were improved in 77%, the laboratory results in 75% and the subjective feelings in 63% of the eyes. CONCLUSIONS: We found that three-month AS treatment led especially to the improvement of ocular surface dryness and damage of the epithelium. The improvement of dry eye after AS treatment correlated well with the clinical, laboratory and subjective findings. From the patients' subjective point of view, the positive effect of AS decreased with time, but still persisted up to three months after the end of therapy.

High prevalence of posterior polymorphous corneal dystrophy in the Czech Republic; linkage disequilibrium mapping and dating an ancestral mutation.

Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1-12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64-133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene, ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.

Recurrence of posterior polymorphous corneal dystrophy is caused by the overgrowth of the original diseased host endothelium.

Posterior polymorphous corneal dystrophy (PPCD) is a rare, bilateral autosomal dominant disorder affecting primarily the corneal endothelium and descemet membrane (DM). The aim of this study was to establish the origin of abnormal endothelium in a patient with PPCD exhibiting cornea graft failure after keratoplasty surgery. A sex-mismatched graft obtained from a patient with PPCD who underwent repeat penetrating keratoplasty and the patient's original cornea were investigated. Combined fluorescent immunohistochemistry for cytokeratin (CK) 19 (a marker of aberrant PPCD endothelium) with fluorescence in situ hybridization (FISH) of the sex chromosomes were used in order to characterize the cells on the posterior graft surface. The pathological endothelium of the failed PPCD cornea revealed strong positivity for CK19 using fluorescent immunohistochemistry. In all the CK19-positive cells, both X and Y chromosomes were simultaneously detected using FISH. The results clearly showed the original cells of the patient (XY), within 3.5 years, almost totally overgrown the posterior corneal surface of the graft (XX). Moreover, an abnormal posterior collagenous layer populated by fibroblast-like cells was observed between DM and the endothelium in the failed graft, but its exact origin could not be established due to the low number of cells. Simultaneous detection of CK19 using fluorescent immunohistochemistry together with the detection of gonosomes using FISH was performed for the first time in the cornea and allowed us to prove that the recurrence of PPCD was caused by pathological abnormal proliferation and migration of recipient cells into donor graft.

Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model.

Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0±1.8 days and xenografts in 6.5±1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4+ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.

Variable ocular phenotypes of posterior polymorphous corneal dystrophy caused by mutations in the ZEB1 gene.

PURPOSE: To describe the ocular features of 6 Czech and British patients with posterior polymorphous corneal dystrophy (PPCD) caused by mutations in the zinc finger E-box binding homeobox 1 gene (ZEB1). METHODS: Case note review of 4 individuals with p.E776fs mutation, one with p.Y719X and one with p.F375fs mutation within the ZEB1 gene. RESULTS: Five individuals exhibited endothelial and Descemet membrane changes consistent with the diagnosis of PPCD. We concluded that one 70-year-old female who had a normal endothelium at both slit lamp and non-contact specular microscopy was a case of non-penetrance. The onset of disease was as early as 3 months after birth. One patient had irregular astigmatism with inferior corneal steepening on videokeratography, but without corneal thinning or other signs of keratoconus. Two others had corneal steepening >49D but with regular astigmatism. Three individuals underwent penetrating keratoplasty (PK) in 1 eye, with one patient treated for secondary glaucoma prior to the PK. CONCLUSIONS: The phenotype associated with changes in the ZEB1 gene exhibits variable expression and incomplete penetrance and seems to have a low risk for secondary glaucoma or the need for keratoplasty compared to PPCD linked to 20p11.2. There is insufficient data for phenotype correlations with PPCD caused by other genes.

Matrix metalloproteinases in recurrent corneal melting associated with primary Sjörgen's syndrome.

PURPOSE: To investigate the contribution of matrix metalloproteinases (MMPs) to recurrent corneal melting in keratoconjunctivitis sicca associated with primary Sjörgen's syndrome (pSS). METHODS: One native melted cornea and ten melted corneal grafts from two patients with severe pSS were used. The presence of MMPs (1, 2, 3, 7, 8, 9, and 13) was detected using indirect enzyme immunohistochemistry. The active forms of MMP 2 and 9 and MMP 3 and 7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP 1 were measured using an activity assay. Eleven unaffected corneas served as controls. RESULTS: The average values of the staining intensity revealed very intense MMP 1, intense MMP 2, 7, and 9 and moderate MMP 3 and 8 positivity, in the corneal epithelium of melted corneas. Intense MMP 1 and 9 staining, moderate MMP 2, 3, and 8 staining, and weak MMP 7 staining were found in the anterior stroma. The posterior stroma revealed intense MMP 1, moderate MMP 3 and 9, and weak MMP 2, 7, and 8 positivity. Immunostaining of the endothelium was moderate for MMP 9 and weak for MMP 1, 2, 3, 7, and 8. MMP 13 was negative in all but four melted specimens, where weak-to-moderate staining was found in the epithelium and stroma. Control corneas revealed moderate MMP 1 and 2 and weak MMP 8 staining in the epithelium, weak MMP 2 staining in the anterior stroma, and weak MMP 1 and 8 staining in the endothelium. Significantly elevated MMP 1 activity and extremely elevated MMP 9 activity were found in most of the tested pathological specimens, compared to healthy controls, where no activity of the two enzymes was present. Markedly elevated MMP 2 activity was found in 2 of 11 specimens, compared to normal tissue. The inactive form of MMP 3 was detected in half of the tested specimens, and inactive MMP 7 in all melted corneas. Active MMP 3 and 7 were found in one melted sample. Neither of these MMPs was found in any of the control specimens. CONCLUSIONS: The increased expression and elevated activity of a wide range of MMPs in melted cornea samples from two patients diagnosed with pSS confirm that these enzymes contribute to the tissue destruction, leading to serious consequences such as corneal perforation and loss of vision.

A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient.

PURPOSE: To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse. METHODS: Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro. RESULTS: Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer. CONCLUSIONS: These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12(-)p63(+) population being closer to the primitive limbal SC.

Phenotype associated with the H626P mutation and other changes in the TGFBI gene in Czech families.

AIMS: To evaluate mutations in the transforming-growth-factor-beta-induced (TGFBI) gene in patients of Czech origin with autosomal dominant corneal dystrophies. METHODS: The coding sequence of the TGFBI gene was analysed in 22 affected Czech individuals from 7 apparently unrelated families. Comparison of phenotype to genotype was performed. RESULTS: A H626P mutation, previously only described in a family with a variant of lattice corneal dystrophy (LCD), was detected in one family with superficial geographic corneal opacities. Light microscopy of 2 samples obtained following either a prior superficial keratectomy or keratoplasty showed amyloid but no fuchsinophilic deposits. In a family with LCD type I, an R124C mutation was identified. The R124L mutation was shown to be causative of Reis-Bucklers corneal dystrophy in 2 families. A family with Thiel-Behnke corneal dystrophy exhibited an R555Q mutation. In 2 families with granular corneal dystrophy type I, the typical R555W change was identified. CONCLUSION: The phenotype of the family with the H626P mutation differed from the phenotype previously reported for this change.

Effect of suturing technique on corneal xenograft survival.

PURPOSE: Understanding xenograft rejection is crucial for the potential introduction of xenotransplantation into clinical practice. Small-animal models play an essential role in this context and substantially contribute to our knowledge about mechanisms of xenograft rejection. METHODS: Rat-to-mouse corneal xenografts were performed by using 2 suturing techniques. Sutures were left either as long or as short as possible to limit the extent of a nonspecific inflammatory response. Cyclosporine A (CsA), monoclonal antibody anti-T cells, and a specific inhibitor of inducible NO synthase (alone or in a combination with CsA) were tested as immunosuppressants. RESULTS: Grafts with long sutures were rejected in 7.3 +/- 1.2 days, whereas those with short sutures were rejected after 11.8 +/- 1.0 days (P < 0.001). Similarly, long sutures induced more pronounced corneal neovascularization (P < 0.001). Although groups of recipients with long sutures all tested immunosuppressants significantly (P < 0.01-0.001) prolonged corneal graft survival, none of them showed a comparable efficacy in groups of recipients with short sutures. CONCLUSIONS: This study showed that suturing technique significantly affects the outcome of corneal concordant xenograft transplantation, influences the effectiveness of immunosuppressive regimens, and therefore must be taken into account when evaluating their efficacy.

Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei.

OBJECTIVE: To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity. STUDY DESIGN: Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC-negative; and group 3, KCS SLC-positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted. RESULTS: The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75 +/- 8.09 MNi/1,000 cells) as well as SLC-negative cells (4.0 +/- 3.83 MNi/1,000 cells) of group 3. CONCLUSION: We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlates with impaired values in clinical test as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity.

A new model of corneal transplantation in the miniature pig: efficacy of immunosuppressive treatment.

Corneal allograft rejection is frequently studied in small rodent or rabbit models. To study mechanisms of rejection in a model that more closely mimics transplantation in humans, we performed orthotopic corneal transplantation in the miniature pig using a 7-mm diameter donor graft. Four groups of recipients were studied: 1) untreated naive, 2) untreated vascularized (high risk), 3) high-risk grafts treated by topical application of prednisolone, or 4) high-risk grafts treated with a combined systemic immunosuppression regime of oral prednisone, cyclosporine A, and mycophenolate mofetil. Both the clinical features and histological assessment of corneal graft rejection showed close similarities to graft rejection in humans. Interestingly, preliminary results indicated that topical steroid treatment was superior to systemic immunosuppression in significantly promoting graft survival. Thus, corneal transplantation in the pig represents an animal model most closely resembling corneal grafting in humans, and offers possibilities for testing various clinically applicable immunosuppressive treatments.

Novel mutations in the ZEB1 gene identified in Czech and British patients with posterior polymorphous corneal dystrophy.

We describe the search for mutations in six unrelated Czech and four unrelated British families with posterior polymorphous corneal dystrophy (PPCD); a relatively rare eye disorder. Coding exons and intron/exon boundaries of all three genes (VSX1, COL8A2, and ZEB1/TCF8) previously reported to be implicated in the pathogenesis of this disorder were screened by DNA sequencing. Four novel pathogenic mutations were identified in four families; two deletions, one nonsense, and one duplication within exon 7 in the ZEB1 gene located at 10p11.2. We also genotyped the Czech patients to test for a founder haplotype and lack of disease segregation with the 20p11.2 locus we previously described. Although a systematic clinical examination was not performed, our investigation does not support an association between ZEB1 changes and self reported non-ocular anomalies. In the remaining six families no disease causing mutations were identified thereby indicating that as yet unidentified gene(s) are likely to be responsible for PPCD.

Immunohistochemical characterization of cytokeratins in the abnormal corneal endothelium of posterior polymorphous corneal dystrophy patients.

Posterior polymorphous corneal dystrophy (PPCD) is a hereditary bilateral disorder affecting Descemet's membrane and the endothelium. The aim of the present study was to determine the spectrum of cytokeratin (CK) expression in cells on the posterior surface of the cornea in PPCD patients. Ten corneal buttons and one specimen of the trabecular meshwork (TM) from PPCD patients who underwent graft or glaucoma surgery were used, as well as six corneal buttons and two TM specimens obtained from healthy donors as controls. Cryosections were fixed and indirect immunofluorescent staining was performed using antibodies directed against a wide spectrum of cytokeratins (CKs). The number of positive cells and the intensity of the staining were assessed using fluorescent microscopy. All 10 PPCD corneal specimens had areas of endothelium displaying typical endothelial morphology as well as areas consisting of layers two to six cells thick with both flat endothelial-like cells and polygonal cells with round nuclei and a large cytoplasm. Both of these morphologically distinct cell types showed strong immunostaining for CK7, CK19, CK8 and CK18, while weaker positive signals were observed for CK1, CK3/12, CK4, CK5/6, CK10, CK10/13, CK14, CK16 and CK17. PPCD endothelium was completely negative for CK2e, CK9, CK15, and CK20. Focal positivity was detected in PPCD TM for CK4, CK7 and CK19. CK8 and CK18 were the only CKs expressed in control endothelium. PPCD and control epithelium displayed similar staining patterns. The distinct positivity for CK3/12, CK4, CK5/6, CK10/13, CK14, CK16 and CK17 was observed in aberrant PPCD endothelium for the first time. We demonstrate that the abnormal endothelium of PPCD patients expresses a mixture of CKs, with CK7 and CK19 predominating. In terms of CK composition, the aberrant PPCD endothelium shares features of both simple and squamous stratified epithelium with a proliferative capacity. The wide spectrum of CK expression is most probably not indicative of the transformation of endothelial cells to a distinct epithelial phenotype, but more likely reflects the modified differentiation of metaplastic epithelium.

Mucolipidosis IV: report of a case with ocular restricted phenotype caused by leaky splice mutation.

PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.