Identification and Characterization of SLC4A11 Mutations in Fuchs Endothelial Corneal Dystrophy (FECD)

Abstract

Purpose:: The endothelial (posterior) corneal dystrophies, which result from primary endothelial dysfunction, include Fuchs endothelial dystrophy (FECD), posterior polymorphism dystrophy (PPCD) and congenital hereditary endothelial dystrophy (CHED). As they share common features of disease it is possible that a proportion of them could be clinical manifestations of different mutations of the same gene. Accordingly, mutations in COL8A2 gene which encodes the alpha 2 (VIII) collagen chain have been identified in both familial and sporadic FECD as well as in a family with PPCD. The aim of our work was to determine whether mutations in the SLC4A11 gene, recently implicated in recessive CHED may also play a pathogenic role in the development of the more common Fuchs corneal endothelial dystrophy.

Methods:: Exons 1-19 of SLC4A11 gene was PCR amplified in 90 FECD cases (64 Chinese and 25 Indian) and then subject to bi-directional sequencing. Eighty Chinese and 30 Indian samples were used as normal age matched controls. Wild type and mutant cDNA constructs were transfected in to HEK293 cells and protein extracts used for immunoblot analysis and cell surface processing assays. Confocal immunolocalization were also performed using established protocols.

Results:: Four heterozygous mutations absent in ethnically matched controls were identified in this screen of 89 FECD patients. These were three missense mutations (E399K, G709E and T754M) and one deletion mutation (99-100delTC). In addition, several silent mutations and conservative amino acid substitutions present in both FECD patients and controls were also identified. Missense mutations involved amino acid residues showing high interspecies conservation indicating that mutations at these sites would be deleterious. Accordingly, immunoblot analysis, biochemical assay of cell surface localization and confocal immunolocalization showed that missense mutants were defective in localization to the cell surface.

Conclusions:: SLC4A11 may therefore play a role in the pathogenesis of FECD through haploinsufficiency.

Keywords: gene screening • mutations • cornea: basic science