Corneal epithelium in keratoconus underexpresses active NRF2 and a subset of oxidative stress-related genes.

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

Identification of PITX3 mutations in individuals with various ocular developmental defects.

BACKGROUND: Congenital cataract displays large phenotypic (syndromic and isolated cataracts) and genetic heterogeneity. Mutations in several transcription factors involved in eye development, like PITX3, have been associated with congenital cataracts and anterior segment mesenchymal disorders. MATERIALS AND METHODS: Targeted sequencing of 187 genes involved in ocular development was performed in 96 patients with mainly anophthalmia and microphthalmia. Additionally, Sanger sequencing analysis of PITX3 was performed on a second cohort of 32 index cases with congenital cataract and Peters anomaly and/or sclereocornea. RESULTS: We described five families with four different PITX3 mutations, two of which were novel. In Family 1, the heterozygous recurrent c.640_656dup (p.Gly220Profs*95) mutation cosegregated with eye anomalies ranging from congenital cataract to Peters anomaly. In Family 2, the novel c.669del [p.(Leu225Trpfs*84)] mutation cosegregated with dominantly inherited eye anomalies ranging from posterior embryotoxon to congenital cataract in heterozygous carriers and congenital sclereocornea and cataract in a patient homozygous for this mutation. In Family 3, we identified the recurrent heterozygous c.640_656dup (p.Gly220Profs*95) mutation segregating with congenital cataract. In Family 4, the de novo c.582del [p.(Ile194Metfs*115)] mutation was identified in a patient with congenital cataract, microphthalmia, developmental delay and autism. In Family 5, the c.38G>A (p.Ser13Asn) mutation segregated dominantly in a family with Peters anomaly, which is a novel phenotype associated with the c.38G>A variant compared with the previously reported isolated congenital cataract. CONCLUSIONS: Our study unveils different phenotypes associated with known and novel mutations in PITX3, which will improve the genetic counselling of patients and their families.