Identification of differentially expressed genes in keratoconus epithelium analyzed on microarrays.
OBJECTIVE
The present study was conducted to investigate differential gene expression in individual samples derived from fresh-frozen human keratoconus and normal corneal epithelium, using gene microarrays.


METHODS
Total RNA was extracted (11 keratoconus and 8 normal samples), labeled, and hybridized to microarrays (GeneChip; Affymetrix, Inc., Santa Clara, CA). GeneChip data were validated by verifying the expression profiles of 10 genes by real-time PCR and by recalculation using dChip software (Wong Laboratory, Department of Biostatistics, Harvard School of Public Health, Boston, MA). Furthermore, 3 of the 10 encoded proteins were stained by immunohistochemistry.


RESULTS
In comparison with normal cornea, the expression of 471 of the 5600 genes on the microarrays was changed in the keratoconus samples. This number was reduced to 47 with increased expression and 9 with decreased expression when more stringent selection parameters were applied. These genes are believed to be involved in keratoconus. Two of the candidate genes, lysyl oxidase and tissue inhibitor of metalloproteinase 3, are known to be involved in other eye diseases. Expression profiles were reproduced with the software dChip (Wong Laboratory) and real-time PCR. Increases in keratin 6 and 13 were also detected at the protein level.


CONCLUSIONS
Keratoconus epithelium appears to be characterized by massive changes of the cytoskeleton, reduced extracellular matrix remodeling, altered transmembrane signaling, and modified cell-to-cell and cell-to-matrix interactions. Validation of gene expression with dChip analysis and real-time PCR indicates GeneChip to be a valid technique for investigation of epithelium from single dissected corneal samples. Association between alterations at RNA and protein levels was observed for some of the tested candidates.
Nielsen
Kim
K
Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark.
Birkenkamp-Demtröder
Karin
K
Ehlers
Niels
N
Orntoft
Torben Falck
TF
D000328:Adult; D016039:Corneal Transplantation; D003598:Cytoskeletal Proteins / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D007640:Keratoconus / Q000235:genetics* / Q000378:metabolism / Q000601:surgery; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D012313:RNA