Accumulation of mitochondrial DNA damage in keratoconus corneas.
OBJECTIVE
To determine whether keratoconus (KC) corneas have more mitochondrial (mt)DNA damage than do normal corneas.


METHODS
Thirty-three normal corneas and 34 KC corneas were studied. Immunohistochemistry for mitochondria-encoded cytochrome c oxidase (complex IV) subunit 1 (CO-Iota) and porins was performed. Total DNA was isolated and mtDNA genome amplified by either long-extension-polymerase chain reaction (LX-PCR) or short-extension-PCR (SX-PCR). LX-PCR mtDNA was digested with restriction enzymes to confirm full-length mtDNA amplicon. SX-PCR mtDNA was probed by Southern blot analysis. The T414G mutation was analyzed by peptide nucleic acid directed clamping PCR. Real-time PCR measured the ratio of mtDNA to nuclear (n)DNA.


RESULTS
KC corneas had decreased CO-Iota in areas of corneal thinning. LX-PCR mtDNA digested with restriction enzymes showed expected size bands except for PstI, which showed two additional bands in some KC corneas (2/18). By both LX-PCR (7.4 +/- 3.8 vs. 4.3 +/- 2.7, P < 0.04) and SX-PCR (5.5 +/- 0.55 vs. 2.4 +/- 2.0, P < 0.006), KC corneas had an increased number of smaller-sized bands (representing mtDNA deletions/mutations) compared with normal corneas. Southern blot analysis of SX-PCR products confirmed their mtDNA origin. The T414G mutation was not detected in either KC or normal corneas. KC corneas showed a trend of lower mtDNA-to-nDNA ratio (26%, P < 0.7) than did normal corneas.


CONCLUSIONS
KC corneas exhibit more mtDNA damage than do normal corneas. The previously reported increased oxidative stress and altered integrity of mtDNA may be related to each other and may be important in KC pathogenesis.
Atilano
Shari R
SR
Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California, USA.
Coskun
Pinar
P
Chwa
Marilyn
M
Jordan
Nicole
N
Reddy
Vinitha
V
Le
Khoi
K
Wallace
Douglas C
DC
Kenney
M Cristina
MC
D000293:Adolescent; D000328:Adult; D000368:Aged; D015139:Blotting, Southern; D002648:Child; D003315:Cornea / Q000378:metabolism*; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007640:Keratoconus / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D018272:Porins / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism
D004272:DNA, Mitochondrial; D018272:Porins; D026361:Reactive Nitrogen Species; D003576:Electron Transport Complex IV