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V. V. Mootha, J. Kanoff, J. Shankardas, P. Prasher, V. K. Mootha, S. D. Dimitrijevich; Marked Reduction of ADH1B Gene Expression in Keratoconus Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1846.
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© ARVO (1962-2015); The Authors (2016-present)
To identify genes that are abnormally expressed in the keratocytes of human corneas with keratoconus (KC).
Keratocytes (fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using serum containing medium. RNA was isolated from 5 KC fibroblast cultures and 6 eye bank donor fibroblast cultures. The generated cDNA was analyzed using the Affymetrix U 133 Plus 2.0 microarrays. Select differentially expressed genes were further validated by immunohistochemistry with commercially available mouse monoclonal antibodies. Stromal keratocyte staining of archived keratoconus pathology specimens were graded using a 0 to 3 scale and compared to archived whole globes with normal corneas as well as Fuchs’ endothelial corneal dystrophy specimens.
Microarray data analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B gene) in the keratoconus fibroblasts (P=.03). Immunohistochemistry using a monoclonal mouse IgG against purified human liver alcohol dehydrogenase revealed a mean staining intensity of 0.1 in the stromal keratocytes in the KC group compared to 1.4 in the normal cornea group (P=.004). In a separate group of immunohistochemistry experiments, there was a mean intensity of 0.6 in the stromal keratocytes of the KC group compared to 1.65 in the Fuchs’ group (P=.004).
Decreased expression of ADH1B in corneal keratocytes (fibroblasts) represents a strong marker of KC and may contribute to its pathogenesis.
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