Marked Reduction of ADH1B Gene Expression in Keratoconus Fibroblasts

Abstract

Purpose:: To identify genes that are abnormally expressed in the keratocytes of human corneas with keratoconus (KC).

Methods:: 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.

Results:: 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).

Conclusions:: Decreased expression of ADH1B in corneal keratocytes (fibroblasts) represents a strong marker of KC and may contribute to its pathogenesis.

Keywords: keratoconus • gene microarray • cornea: stroma and keratocytes