Altered expression of CLC, DSG3, EMP3, S100A2, and SLPI in corneal epithelium from keratoconus patients.
OBJECTIVE
This investigation was designed to determine whether the five genes, CLC, DSG3, EMP3, S100A2 and SLPI, are differentially expressed in keratoconus, as indicated from another study.


METHODS
Gene expression was monitored using quantitative real-time PCR on 14 keratoconus samples and 16 controls, and normalized to GAPDH and B2M. The DSG3, S100A2, and SLPI proteins were quantified by Western blotting, and the cellular localization was determined by immunohistochemistry. One of the genes, CLC, was reduced in gene expression and its four exons were sequenced.


RESULTS
The five genes were all differentially expressed in keratoconus (P < 0.04) and so were at least three of the encoded proteins (P = 0.009). DSG3 was expressed in association with the cell membrane of the basal and suprabasal epithelial cells, and S100A2 was expressed in the nucleus and cytoplasm, often as intracellular granules. Two SNPs (rs374185 and rs384138) were observed in the CLC gene, each with an allele frequency of 68%. No other mutations were detected.


CONCLUSIONS
The five genes, and three of the encoded proteins, were shown differentially expressed between a group of keratoconus patients and a reference group using different techniques. These alterations, in combination with earlier findings, strongly demonstrate the genes to be involved in the corneal disease. We suggest the unambiguously expressed DSG3 protein to be used as a marker for keratoconus.
Nielsen
Kim
K
Department of Ophthalmology, Aarhus University Hospital, Denmark.
Heegaard
Steffen
S
Vorum
Henrik
H
Birkenkamp-Demtröder
Karin
K
Ehlers
Niels
N
Orntoft
Torben Falck
TF
D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D002630:Chemotactic Factors / Q000235:genetics* / Q000378:metabolism; D051184:Desmoglein 3; D019573:Epithelium, Corneal / Q000378:metabolism*; D015870:Gene Expression; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007640:Keratoconus / Q000235:genetics* / Q000378:metabolism; D008245:Lysophospholipase; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D009418:S100 Proteins / Q000235:genetics* / Q000378:metabolism; D053481:Secretory Leukocyte Peptidase Inhibitor; D017422:Sequence Analysis, DNA
D015415:Biomarkers; D015820:Cadherins; D002630:Chemotactic Factors; C071894:DSG3 protein, human; D051184:Desmoglein 3; C485157:EMP3 protein, human; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D012333:RNA, Messenger; D009418:S100 Proteins; C098997:S100A2 protein, human; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D008245:Lysophospholipase; C011711:lysolecithin acylhydrolase