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Keratoconus (KC) was a non-inflammatory progressive thinning disorder of the cornea. As the cornea thins, it leads to progressive mixed myopic and irregular astigmatism. KC was commonly reported as an isolated disorder, yet there are published papers on its association with other known genetic disorders such as Down syndrome, Leber congenital amaurosis, and mitral valve prolapse (Gullen 1963; Krachmer, 1984; Sharif, 1992; Rabinowitz, 1998). The age of onset was at puberty (Li et al., 2007) and the condition tends to be progressive until the third or fourth decade of life. It usually arrests at this stage, but may adopt a variable pattern of progression (Rabinowitz, 1998).
In humans, the paraoxonase (PON) gene family has three members, consisting of PON1, PON2 and PON3 which were aligned next to each other on the long arm of chromosome 7q21.3-22.1 (La Du et al., 1999). There were two forms of PON1: (1) a membrane-associated tissue form and also (2) in a form of high-density lipoprotein (HDL) which circulates in the blood plasma (Mackness et al., 1996). PON1 was a serum esterase that exhibits a wide substrate specificity, which was 27 kb in size, and contains nine exons. More than 192 single-nucleotide polymorphisms were known for PON, including its promoter and coding regions (SeattleSNPs: http://pga.gs.washington.edu/). PON1 was synthesized primarily in the liver, and to a lesser extent in the brain, heart, kidney, lungs and small intestine (Mackness et al., 1996). The main function of paraoxonase was to inhibit low density lipoprotein (LDL) oxidation by destroying the biologically active phospholipids in oxidatively modified LDL (Ausejo et al., 1999; Jaouad et al., 2003).
Among human populations such as blacks and whites, Asians and Europeans, the frequency of the PON1 allele fluctuates significantly. It has been observed that the 192R and 55L alleles were more common in blacks than whites and paraoxonase activity was higher in blacks than whites. However, race remained a predictor of paraoxonase activity even after 192Q/R and 55L/M polymorphisms were accounted for (Troughton et al., 2008). The two PON1 55L/M and 192Q/R coding region polymorphisms perform different antiorganophosphate and antioxidant hydrolyzing abilities as both encode two different PON1 isoforms. In addition to the PON1 55L/M and 192Q/R coding region polymorphisms, there have been five reported polymorphisms in the PON1 regulatory region. Each of these five polymorphisms was associated with different effects on PON1 activity (Leviev et al., 2000; Brophy et al., 2001; Suehiro et al., 2000). The 192Q/R and 55L/M polymorphisms have been the focus of many population genetic studies as both polymorphisms affects the PON1 activity. However, the second exonic polymorphism of the PON1 gene which occurs at amino acid position 55L/M has less effect on PON1 activity compared to that of polymorphism 192Q/R. Thus, the emphasis in this study was the 192Q/R polymorphism. The PON1 192Q/R polymorphism involved the base pair substitution of CAA to CGA in exon 5. This results in the substitution of amino acid glutamine with arginine at position 192 and introduces the restriction site of AlwI.
Each disease manifests a discrete free radical damage profile. The role of free radicals shows prominently in the pathogenesis of KC as the formation of nitrotyrosine and immunoreactive malonaldehyde (MDA) increases in KC corneas (Buddi et al., 2002; Shoham et al., 1998). As such, oxidative stress appears to play a more important role in KC. In addition, abnormal expression of several major antioxidant enzymes has also been reported in KC corneas (Shoham et al., 1998). Hence, free radical damage and oxidative stress appear to be correlated to the pathogenesis of KC.
The association of the PON1 192Q/R polymorphism with many other diseases apart from KC has also been studied, for example coronary artery disease, cancer, diabetes mellitus Type 2, Alzheimer's disease and Parkinson's disease (Goswami et al., 2009). The reported results show that paraoxonase play a certain role in those diseases.
Until now, the aetiology and pathogenesis of KC were still remaining unclear. Moreover, studies on the association between PON1 192Q/R polymorphism with KC are lacking. Hence, this study aims to evaluate the association between PON1 192Q/R polymorphism with KC by performing polymerase chain reaction (PCR) and restriction enzyme digestion on the extracted DNA from blood specimens of KC patients.
OBJECTIVES OF THE STUDY
To examine the allele and genotype frequencies of PON1 192Q/R polymorphism in KC patients and normal controls.
To examine the significance of association of the PON1 192Q/R polymorphism with KC.
To provide data on genetic polymorphisms of KC patients for possible prognosis and diagnosis of KC.
MATERIALS AND METHOD
A total of 9 KC propositi and 2 forme fruste KC were recruited from Ophir Eye Clinic and Surgery in Klang and Tun Hussein Onn National Eye Hospital (THONEH) in Petaling Jaya. 56 normal controls who have no known clinical signs of KC were recruited from the family members and relatives of propositi, as well as other patients who were ascertained at Ophir Eye Clinic and Surgery and team members of the study. The research protocol was approved by the Ethics Committee of the University of Malaya. Written informed consent forms were obtained from all participants. All participants were interviewed using a questionnaire that includes information on demography, medical history and family history.
Of these subjects, 9 KC patients, 2 forme fruste KC patients and 56 normal controls were examined using the autorefractormeter to determine the spectacle power, followed by blood taking, then using the keratometer to obtain the corneal topography, the Snellen's chart to perform the visual acuity test and lastly the Haag-Streit Slit Lamp to carry out biomicroscopy test.. The diagnosis of KC was based on clinical examination and was performed by Dr. Jenny Parameshvara Deva, a consultant ophthalmologist and refractive surgeon. KC patients were those who had one or more of the following clinical signs with no other pathology in one eye: distinct stromal thinning, Vogt striae, or a Fleischer ring detected by slit-lamp examination; obvious scissoring of the red reflex or the Charleaux oil droplet sign as identified by retinoscopy. The blood samples were obtained by venipuncture and were drawn into EDTA vacutainer tubes. The blood samples were stored in a -20°C freezer prior to DNA extraction.
DNA was isolated from the peripheral blood samples using the conventional phenol-chloroform method. Firstly, DNA was extracted from the blood samples using 10% w/v sodium dodecyl sulphate (SDS) and 10mg/mL Proteinase K after being washed with 10/10mM Tris-EDTA buffer pH 8.0 for at least three times. The extracted DNA was then purified by using phenol-chloroform. Lastly, the DNA was precipitated with 4M sodium chloride (NaCl) and ethanol.
The concentration and the purification of the purified DNA were determined by using the following formula:
The DNA samples were then diluted to a concentration of approximately 0.4µg/µl which was equivalent to 400ng of DNA.
Polymerase Chain Reaction (PCR)
DNA amplification was carried out to amplify a 99-bp PCR product containing a native AlwI restriction enzyme at codon 192. DNA amplification was performed using GoTaq® Green Master Mix. The master mix contains GreenTaq Reaction Buffer pH8.5, 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP and 3mM magnesium chloride (MgCl2). The components of PCR and the primers that were used were shown as follows:
GoTaq® Green Master Mix
100µM forward primer
100µM reverse primer
DNA template (400ng)
Forward: 5'TAT TGT TGC TGT GGG ACC TGA3'
Reverse : 5'CAC GCT AAA CCC AAA TAC ATC TC3'
The amplified DNA samples were electrophoresed in 1.0 TBE buffer at a voltage of 5V/cm on a 1.5% w/v agarose gel containing ethidium bromide against a 20-bp ladder. The ladder was used to determine the DNA quality and to estimate the DNA quantity as well. The agarose gel was viewed under ultraviolet (UV) light and an image of the agarose gel were captured using polaroid.
Restriction Fragments Length Polymorphism (RFLP)
PON 1 genotyping was performed by restriction digestion using the restriction endonuclease AlwI where the amplicons were digested into fragments. The 192Q/R polymorphism was determined by restriction enzyme digestion analysis of PCR products amplified from genomic DNA.
Agarose Gel Electrophoresis (AGE)
The resulting fragments of the digested PCR products were separated by electrophoresis using a 3% w/v of agarose gel in 1.0 TBE buffer at a voltage of 5V/cm until the ethidium bromide (blue dye) reaches about 75% down the gel. The DNA marker that was used was the 20-bp DNA ladder. The agarose gel was viewed under ultraviolet (UV) light to determine the genotypes. An image of the agarose gel was captured using polaroid.
The data obtained from the study was analyzed using SPSS software version 14. The distribution of allele and genotype frequencies between KC cases and normal controls were evaluated using Genepop version 4.0. The association between KC and genotypes were then evaluated using Chi-square (χ2) test, Fisher's test and Microsoft Excel.
Polymerase Chain Reaction (PCR)
Using a forward and reverse primer 192 for amplification of the PON1 192 gene, a 99 bp PCR product was amplified. All amplified DNA were electrophoresed by 1.5% agarose gel before digestion to ensure that the 99 bp band was amplified.
Restriction Fragments Length Polymorphism (RFLP)
Amplified DNA were genotyped by RFLP using restriction endonuclease AlwI. The presence of the AlwI allows restiriction enzyme digestion of the 99 bp amplified DNA into 2 fragments: the 63 bp and 36 bp bands. Agarose gel electrophoresis was used to separate the digested fragments. In the presence of the mutant-type nucleotide CGA (RR), two bands of 63-bp and 36-bp bands were seen on complete digestion occurred; in the presence of the wild-type nucleotide CAA (QQ), only 99-bp band was seen as no digestion occurred; in the presence of both mutant- and wild-type nucleotides (QR), three bands of 99, 63 and 36-bp bands were observed. The results were indicated in Figure 1:
(mutant + wild type)
Figure 1: Genotypes of PON1 polymorphism as determined by RFLP.
3.1) Demographic Distribution
A total of 67 subjects were recruited in this study. There were 9 KC patients (13.4%), 2 forme fruste KC patients (3.0%), 20 myopic patients (29.8%) and 36 normal subjects (53.7%). The age range of the recruited subjects was 5 to 68 years old. The mean ages for the respective groups were: 24.33 + 7.84 (KC patients), 16.0 + 2.83 (Forme fruste KC patients), 29.75 + 13.63 (Myopic patients) and 34.14 + 17.51 (Normal subjects). In the sample population, the male to female ratio was 36:31 with the percentage of 53.7% of males and 46.3% of females.
In the pooled sample population, the Malay (41.2%) and Indian (44.8%)were equally distributed while the Chinese (13.4%) were lesser compared to the other two ethnics.
3.2) Genotype and Allele Frequencies
There were three genotypes in PON1 192Q/R polymorphism which were QQ, QR and RR genotypes. In the sample population, the genotype frequencies were 0.1791 (QQ), 0.5522 (QR) and 0.2687 (RR). The allele frequencies for Q allele and R allele were 0.4552 and 0.5448 respectively while the frequencies for Q carrier and R carrier were 0.7317 and 0.821 respectively.
3.3) Association between KC and genotypes
In this study, the genotypic distribution of 192Q/R polymorphism indicates no significant difference with KC patients in Malaysia and suggests that there may possibly be no relation between PON1 192Q/R polymorphism and the risk of an individual to KC. The χ2 value for 192Q/R polymorphism was 0.689, and the p-value was more than 0.05. From the analysis, the 192Q/R genotypes show the highest frequency in both of the KC patients and non-KC subjects.
3.4) Association between KC and alleles
The allelic distribution of 192Q/R polymorphism also shows no significant difference with KC patients in Malaysia. This suggests that there may be no relation between PON1 192Q/R polymorphism and the risk of an individual to KC. The χ2 value for 192Q/R polymorphism was 0.282, and the p-value was more than 0.05. The allele frequency between Q allele and R allele show not much difference between KC patients and non-KC subjects.
3.5) Association between KC and carriers
In this study, the carrier distribution of 192Q/R polymorphism indicates no significant difference with KC patients in Malaysia and suggests that there may possibly be no relation between PON1 192Q/R polymorphism and the risk of an individual to KC. The χ2 value for 192Q/R polymorphism was 0.270 and the p-value was more than 0.05. From the analysis, the Q and R carriers were equally distributed in both of the KC patients and non-KC subjects.
3.6) Association between Ethnics and Genotypes
Comparison of genotypes between Malays and Indians showed no significant difference. The χ2 value obtained was 5.818 (p=0.055). Chinese subjects were ignored in the analysis as there were only 9 Chinese subjects being recruitmed and no Chinese with the QQ genotype was recruited in this study.
There are few papers published on PON1 polymorphisms in other ocular diseases such as aged-related macular degeneration (. However, there are no published papers reported on PON1 polymorphism with KC. This study on the association of PON1 and KC may be the first to carry out. In the present study, there was no significant evidence that 192Q/R was associated with KC risk as our data did not detect statistical significance in the distribution of allele and genotype frequencies of 192Q/R between KC patients and non-KC subjects.
The limitation of this study was that the KC sample size was too small to obtain a significant association between PON1192Q/R polymorphism and KC. KC patients recruited in this study were from two selected areas: THONEH in Petaling Jaya and Ophir Eye Clinic and Surgery in Klang. Future work has been discussed to collect KC data from all states in Malaysia.