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

Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study

<p>Abstract</p> <p>Background</p> <p>The observation of cytokeratins (CK's) in mass spectrometry based studies raises the question of whether the identified CK is a true endogenous protein from the sample or simply represents a contaminant. This issue is especially important in proteomic studies of the corneal epithelium where several CK's have previously been reported to mark the stages of differentiation from corneal epithelial stem cell to the differentiated cell.</p> <p>Methods</p> <p>Here we describe a method to distinguish very likely endogenous from uncertain endogenous CK's in a mass spectrometry based proteomic study. In this study the CK identifications from 102 human corneal samples were compared with the number of human CK identifications found in 102 murine thymic lymphoma samples.</p> <p>Results</p> <p>It was anticipated that the CK's that were identified with a frequency of <5%, <it>i.e. </it>in less than one spot for every 20 spots analysed, are very likely to be endogenous and thereby represent a 'biologically significant' identification. CK's observed with a frequency >5% are uncertain endogenous since they may represent true endogenous CK's but the probability of contamination is high and therefore needs careful consideration. This was confirmed by comparison with a study of mouse samples where all identified human CK's are contaminants.</p> <p>Conclusions</p> <p>CK's 3, 4, 7, 8, 11, 12, 13, 15, 17, 18, 19, 20 and 23 are very likely to be endogenous proteins if identified in a corneal study, whilst CK's 1, 2e, 5, 6A, 9, 10, 14 and 16 may be endogenous although some are likely to be contaminants in a proteomic study. Further immunohistochemical analysis and a search of the current literature largely supported the distinction.</p

The spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva

The aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions

The OV-TL 12/30 Clone of Anti-cytokeratin 7 Antibody as a New Marker of Corneal Conjunctivalization in Patients with Limbal Stem Cell Deficiency

Compared with cytokeratin (CK)19, CK7 is the more reliable marker for distinguishing between the corneal and conjunctival epithelium, particularly in patients with limbal stem cell deficiency

Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas

The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process