Keratoconus (KC) is an eye disorder which is characterized by progressive thinning of cornea and conical shape of cornea. KC patients will usually have irregular astigmatism, shortsightedness (myopia) and progressive vision impairment. The prevalence of KC in general population is approximately one in 2000 (Rabinowitz, 1998). According to a study conducted in University of Malaya Medical Centre (UMMC), KC has 0.3% prevalence in the urban population (Reddy et al., 2008). KC can strike any age group and usually goes undetected. Early or sub-clinical stages of KC (forme fruste KC) can only be detected by using topography (measurement of the curvature of cornea), and pachymetry (measurement of the thickness of cornea). As KC is progressive, early treatment is crucial in order to prevent further progression.
To date, the exact cause of KC is unknown. According to Rabinowitz (1998), KC can be categorized into three types based on their pathogenesis: KC which is associated with rare genetic diseases (for example, atopic keratoconjunctivitis, Fuch's corneal dystrophy and congenital cataracts); KC which is associated with clinical settings (such as contact lens wearing, Down syndrome and eye rubbing); and KC without any association, which is the most common cause.
Keratoconus patients are subjected to wearing contact lenses to correct their vision. A special type of contact lens which is known as rigid gas permeable (RGP) lens is prescribed to patients (Rabinowitz, 1998). The RGP lens helps to correct the abnormal conical shape of the cornea, thus forming a clearer image. Furthermore, collagen crosslinking (CXL) treatment is indicated for patients when their corneal collagens are weakening progressively. In the CXL procedure, the ophthalmologist surgeon will drip riboflavin drop by drop into the patient's eye every two minutes for thirty minutes, and crosslinking is activated by using ultraviolet light (Trattler and Rubinfeld, 2009). Another approach for the treatment of advanced cases of KC is corneal transplant. The center of the patient's cornea is removed and the donor's cornea tissue is sewn back into patient's cornea. However, recurrent KC may occur in 15 to 20 years after the transplantation (Colin and Velou, 2002).
Oxidative stress is the imbalance of oxidants and antioxidants, resulting in cell or membrane damage. One of the previous studies had shown distinct staining of malondialdehyde (MDA) and nitrotyrosine (NT) in KC cornea which means that KC patients were under oxidative stress (Buddi et al., 2002). Paraoxonase 1 (PON1) is an antioxidant enzyme that metabolizes lipid peroxides, prevents oxidation of low density lipoprotein (LDL) and hydrolyzes xenobiotic compounds, for instance, organophosphates in the insecticide. The catalytic ability of PON1 can be affected by polymorphism at position 55 (L55M) and 192 (Q192R). In determination of PON1 status, both the polymorphism of PON1and PON1 level in plasma have to be taken into consideration (Costa and Furlong, 2002). Previous studies have reported that PON1 activity was decreased in the serum of patients with age-related macular degeneration (AMD) (Ates et al., 2009). To the best of our knowledge, there has been no research on PON1 activity and polymorphism in KC patients in the literature.
As the cornea is tissue-specific, corneal sample should be used to investigate the corneal-expressed gene. By understanding the gene expression of KC, we shall be able to know more about the pathogenesis of KC. In future, we can look for more approaches of treatment in terms of gene therapy. The fresh corneal samples can be obtained from patients who are undergoing corneal transplant. Gene-expression profiles can be examined by expressed sequence tag (EST) analysis of complementary DNA (cDNA) libraries (Wistow, 2002). ESTs are short DNA sequences that complement to cDNA which had been used in previous studies in genetic profiling of KC patients (Wistow, 2002; Rabinowitz, 2005).
Visual System Homeobox 1 (VSX1) is a homeodomain transcriptors factor which is encoded by a homeobox gene (Semina et al., 2000). VSX1 is suggested that involved in the ocular development and thus is believed that VSX1 expression plays a role in KC (Heon et al., 2002). However, controversy arose when a study revealed that VSX1 gene expression do not have any association with KC (Aldave et al., 2006). It is believed that different population will show different expression of VSX1 gene, thus this study will be interesting as this is a preliminary study of gene expression in KC patients.
Aquaporins (AQP) are known as channels that regulated the movement of water through cell plasma membranes. Eleven AQPs (AQP0-10) had been categorized into two types of proteins which are water-permeable and water-and-glycerol-permeable (Agre and Kozono, 2003). Among those AQPs, AQP1 and AQP5 are present in mammalian corneal epithelium and corneal endothelium, respectively. In this study, expression of AQP5 gene will be studied as controversy had also been arisen on the association between AQP5 and KC (Rabinowitz, 2005; Garfias et al., 2008).
Superoxide dismutase is an enzyme binds with copper and zinc ions (CuZn-SOD). It is a known antioxidant enzyme that protects the biological sites in body from oxidative damages by scavenging superoxide free radicals. Previous study proved markedly decreased CuZn-SOD level in KC patients (Behndig et al., 2001). In this study, the corneal expression of SOD1 gene will be studied.
A hypothesis about the degradative pathway which might be contributed in the development of KC had been proven by elevated levels of degradative enzymes (such as acid phosphatase and acid esterase) and decreased levels of protease inhibitors (example, proteinase inhibitor and α1-microglobulin) (Whitelock et al., 1997). Controversy arose when Zhou et al. (1998) discovered that not all of those enzymes and inhibitors showed changes that mentioned above. In this study, the expression of degradative enzymes and protease inhibitors in KC patients will be examined.
OBJECTIVES
To investigate gene expression of keratoconic and non-keratoconic corneas.
To examine the activities of degradative enzymes and protease inhibitors in KC patients.
To assess the oxidative stress of KC patients by evaluating the PON1 status.
MATERIALS AND METHODS
Corneal Samples
Corneal Sample Collection
KC patients and non-KC patients who will undergo corneal transplantation will be recruited as subjects. The fresh KC cornea samples which will otherwise be discarded will be collected at the time of corneal transplantation. The corneal tissue will be preserved in the appropriate media and stored at -80ËšC before being subjected to further procedures.
cDNA Library Construction and Sequencing
The collected corneal buttons will be pooled. The total RNA will be extracted by using a commercialized kit. Poly(A)+ RNA will be prepared to be used for cDNA synthesis (Simms, 1995). The cDNA will be fractionated and cloned in a vector to generate a cDNA library. The cDNA will be sequenced and analyzed.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Another few collected cornea samples will be stabilized. The total RNA will be isolated by using commercialized kit. The corneal tissue will be homogenized in solution and washed by adding into a mini silicone column (Rabinowitz et al., 2005). Next, the concentration of total RNA will be measured by spectrophotometer. Reverse transcriptase reactions will be performed.
Polymerase Chain Reaction (PCR)
The cDNA obtained from RT-PCR will be used as template to perform PCR for VSX1, SOD1, AQP5, acid phoaphatase and α-1 proteinase inhibitor.
Blood Samples
Blood Sample Collection
KC patients and non-KC patients will be recruited at the Ophir Eye Clinic and Surgery in Klang, Selangor. Questionnaires regarding the information and conditions of subjects will be filled up. Ophthalmic examination will be conducted by ophthalmologist. The tests included autorefraction test, refraction test (determination of visual acuity and prescription for eyeglasses or contact lenses), topography and pachymetry. Venous blood will be collected for EDTA tubes (6 ml) and heparinized tubes (6 ml).
PON1 activity measurement (Richter et al., 2004)
The blood in heparinized tubes will be spun down using centrifuge to get the plasma. The plasma will be diluted and added with paraoxon and diazoxon separately. Next, the absorbance will be measured each 15 seconds for 2 minutes by using UV spectrophotometer at 405nm (for paraoxon) and 270nm (for diazoxon).
Genotyping of PON1 Polymorphism (Richter et al., 2004)
DNA will be extracted from blood in the EDTA tubes. The DNA will be amplified by PCR and the products will be run on 1.5% (w/v) agarose gel. The DNA is then digested and digested products will be run on 3% (w/v) agarose gel.
Data Analysis
Initial rates of the substrate hydrolysis will be calculated (Richter et al., 2004). Graph of the rates of diazoxon hydrolysis versus the rates of paraoxon hydrolysis will be plotted. With that, the subjects of this study will be separated into three types of phenotypes: PON1192Q/Q, PON1192Q/R and PON1192Q/Q.
EXPECTED OUTCOMES
The cDNA libraries will be constructed and gene expression of cornea of KC patients will be studied.
The expression of degradative enzymes and protease inhibitors will be examined.
The association of polymorphism and activity of PON1 with KC patients will be obtained.
