Abstract
Abstract: :
Purpose: To evaluate the mitochondrial DNA (mtDNA) isolated from keratoconus corneas for rearrangements and/or deletions. Methods: Total DNA was isolated from normal human corneas (n=9, range 29 to 73, mean 43.6 yrs) and keratoconus corneas (n=10, range 12 to 41, mean 31.1 yrs). Frozen, pulverized corneas were homogenized in 100mM NaCl, 25mM Na2EDTA, 10mM Tris–HCl, pH 8.0, and then digested with Proteinase K (15ug/ml), 0.5% SDS, ribonuclease A (5µg/ml) followed by phenol extraction and ammonium acetate/ethanol precipitation. Long Extension–Polymerase Chain Reaction (LX–PCR) was carried out with 50ng of extracted DNA, mitochondrial specific primers, Premix D, and Failsafe Enzyme Mix in a total volume of 50 µl. Amplified products were separated by electrophoresis on a 0.8% agarose gel and stained with ethidium bromide. Some samples were also digested with HindIII, Apa I, EcoR I, and Pst I. The mtDNA to nuclear DNA (nDNA) ratio was measured by real–time PCR by assessing amplification of mtDNA sequences compared to that of r18S. Results: The LX–PCR products for normal and keratoconus corneas were 16kb, the expected size for mtDNA. Restriction digests of the LX–PCR products indicated keratoconus corneas (5/10) displayed unusual fragmentation patterns compared to normal corneas (2/9). The mtDNA to nDNA ratios for both normal and keratoconus corneas were similar to each other. Conclusions: The mtDNA for keratoconus corneas had increased frequency of rearrangements. There was not a common deletion/mutation pattern in the keratoconus corneas. These rearrangements/mutations of the keratoconus mtDNA will require further analysis to identify the specific alterations and potential affects on mitochondrial functions.
Keywords: keratoconus • mitochondria • oxidation/oxidative or free radical damage