An Overview Of Keratoconus Biology Essay
Keratoconus ( KC ) was a non-inflammatory progressive thinning upset of the cornea. As the cornea thins, it leads to progressive assorted myopic and irregular astigmia. KC was normally reported as an stray upset, yet there are published documents on its association with other known familial upsets such as Down syndrome, Leber inborn amaurosis, and mitral valve prolapsus ( Gullen 1963 ; Krachmer, 1984 ; Sharif, 1992 ; Rabinowitz, 1998 ) . The age of oncoming was at pubescence ( Li et al. , 2007 ) and the status tends to be progressive until the 3rd or 4th decennary of life. It normally arrests at this phase, but may follow a variable form of patterned advance ( Rabinowitz, 1998 ) .
In worlds, the paraoxonase ( PON ) cistron household has three members, dwelling of PON1, PON2 and PON3 which were aligned following to each other on the long arm of chromosome 7q21.3-22.1 ( La Du et al. , 1999 ) . There were two signifiers of PON1: ( 1 ) a membrane-associated tissue signifier and besides ( 2 ) in a signifier of high-density lipoprotein ( HDL ) which circulates in the blood plasma ( Mackness et al. , 1996 ) .
PON1 was a serum esterase that exhibits a broad substrate specificity, which was 27 kilobit in size, and contains nine coding DNAs. More than 192 single-nucleotide polymorphisms were known for PON, including its booster and cryptography parts ( SeattleSNPs: hypertext transfer protocol: //pga.gs.washington.edu/ ) . PON1 was synthesized chiefly in the liver, and to a lesser extent in the encephalon, bosom, kidney, lungs and little bowel ( Mackness et al. , 1996 ) .
The chief map of paraoxonase was to suppress low denseness lipoprotein ( LDL ) oxidization by destructing the biologically active phospholipids in oxidatively modified LDL ( Ausejo et al. , 1999 ; Jaouad et al. , 2003 ) .Among human populations such as inkinesss and Whites, Asians and Europeans, the frequence of the PON1 allelomorph fluctuates significantly. It has been observed that the 192R and 55L allelomorphs were more common in inkinesss than Whites and paraoxonase activity was higher in inkinesss than Whites.
However, race remained a forecaster 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 part polymorphisms perform different antiorganophosphate and antioxidant hydrolyzing abilities as both encode two different PON1 isoforms. In add-on to the PON1 55L/M and 192Q/R coding part polymorphisms, there have been five reported polymorphisms in the PON1 regulative part. 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 focal point of many population familial surveies as both polymorphisms affects the PON1 activity. However, the 2nd exonic polymorphism of the PON1 cistron which occurs at amino acerb place 55L/M has less consequence on PON1 activity compared to that of polymorphism 192Q/R. Therefore, the accent in this survey was the 192Q/R polymorphism. The PON1 192Q/R polymorphism involved the base brace permutation of CAA to CGA in exon 5. This consequences in the permutation of amino acid glutamine with arginine at place 192 and introduces the limitation site of AlwI.Each disease manifests a distinct free extremist harm profile.
The function of free groups shows conspicuously 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 emphasis appears to play a more of import function in KC. In add-on, unnatural look of several major antioxidant enzymes has besides been reported in KC corneas ( Shoham et al.
, 1998 ) . Hence, free extremist harm and oxidative emphasis 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 besides been studied, for illustration coronary arteria disease, malignant neoplastic disease, diabetes mellitus Type 2, Alzheimer ‘s disease and Parkinson ‘s disease ( Goswami et al. , 2009 ) . The reported consequences show that paraoxonase play a certain function in those diseases.Until now, the aetiology and pathogenesis of KC were still staying ill-defined. Furthermore, surveies on the association between PON1 192Q/R polymorphism with KC are missing. Hence, this survey aims to measure the association between PON1 192Q/R polymorphism with KC by executing polymerase concatenation reaction ( PCR ) and limitation enzyme digestion on the extracted Deoxyribonucleic acid from blood specimens of KC patients.
OBJECTIVES OF THE STUDY
To analyze the allelomorph and genotype frequences of PON1 192Q/R polymorphism in KC patients and normal controls.To analyze the significance of association of the PON1 192Q/R polymorphism with KC.To supply informations on familial polymorphisms of KC patients for possible forecast and diagnosing of KC.
MATERIALS AND METHOD
A sum 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 marks of KC were recruited from the household members and relations of propositi, every bit good as other patients who were ascertained at Ophir Eye Clinic and Surgery and squad members of the survey.
The research protocol was approved by the Ethics Committee of the University of Malaya. Written informed consent signifiers were obtained from all participants. All participants were interviewed utilizing a questionnaire that includes information on human ecology, medical history and household history.Of these topics, 9 KC patients, 2 forme fruste KC patients and 56 normal controls were examined utilizing the autorefractormeter to find the spectacle power, followed by blood pickings, so utilizing the keratometer to obtain the corneal topography, the Snellen ‘s chart to execute the ocular sharp-sightedness trial and in conclusion the Haag-Streit Slit Lamp to transport out biomicroscopy test.. The diagnosing of KC was based on clinical scrutiny and was performed by Dr. Jenny Parameshvara Deva, a adviser eye doctor and refractile sawbones. KC patients were those who had one or more of the undermentioned clinical marks with no other pathology in one oculus: distinguishable stromal cutting, Vogt striae, or a Fleischer ring detected by slit-lamp scrutiny ; obvious scissoring of the ruddy physiological reaction or the Charleaux oil droplet mark as identified by retinoscopy.
The blood samples were obtained by venipuncture and were drawn into EDTA vacutainer tubings. The blood samples were stored in a -20 & A ; deg ; C freezer prior to DNA extraction.
Deoxyribonucleic acid Extraction
Deoxyribonucleic acid was isolated from the peripheral blood samples utilizing the conventional phenol-chloroform method. First, DNA was extracted from the blood samples utilizing 10 % w/v Na dodecyl sulfate ( 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 Deoxyribonucleic acid was so purified by utilizing phenol-chloroform. Last, the Deoxyribonucleic acid was precipitated with 4M Na chloride ( NaCl ) and ethanol.The concentration and the purification of the purified DNA were determined by utilizing the undermentioned expression:The Deoxyribonucleic acid samples were so diluted to a concentration of about 0.
4µg/µl which was tantamount to 400ng of Deoxyribonucleic acid.
Polymerase Chain Reaction ( PCR )
Deoxyribonucleic acid elaboration was carried out to magnify a 99-bp PCR merchandise incorporating a native AlwI limitation enzyme at codon 192. Deoxyribonucleic acid elaboration was performed utilizing GoTaq® Green Master Mix. The maestro mix contains GreenTaq Reaction Buffer pH8.5, 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP and 3mM Mg chloride ( MgCl2 ) .
The constituents of PCR and the primers that were used were shown as follows:ComponentsVolume ( µl )GoTaq® Green Master Mix23.42100µM frontward primer0.20100µM contrary primer0.38Deoxyribonucleic acid templet ( 400ng )1.00PrimersPCR merchandiseForward: 5’TAT TGT TGC TGT GGG ACC TGA3 ‘Reverse: 5’CAC GCT AAA CCC AAA TAC ATC TC3 ’99-bpThe amplified Deoxyribonucleic acid samples were electrophoresed in 1.
0 TBE buffer at a electromotive force of 5V/cm on a 1.5 % w/v agarose gel incorporating ethidium bromide against a 20-bp ladder. The ladder was used to find the DNA quality and to gauge the DNA measure every bit good. The agarose gel was viewed under UV ( UV ) visible radiation and an image of the agarose gel were captured utilizing Polaroid.
Restriction Fragments Length Polymorphism ( RFLP )
PON 1 genotyping was performed by limitation digestion utilizing the limitation endonuclease AlwI where the amplicons were digested into fragments. The 192Q/R polymorphism was determined by limitation enzyme digestion analysis of PCR merchandises amplified from genomic Deoxyribonucleic acid.
Agarose Gel Electrophoresis ( AGE )
The ensuing fragments of the digested PCR merchandises were separated by cataphoresis utilizing a 3 % w/v of agarose gel in 1.
0 TBE buffer at a electromotive force of 5V/cm until the ethidium bromide ( bluish dye ) reaches about 75 % down the gel. The Deoxyribonucleic acid marker that was used was the 20-bp DNA ladder. The agarose gel was viewed under UV ( UV ) visible radiation to find the genotypes.
An image of the agarose gel was captured utilizing Polaroid.
The informations obtained from the survey was analyzed utilizing SPSS package version 14. The distribution of allelomorph and genotype frequences between KC instances and normal controls were evaluated utilizing Genepop version 4.0. The association between KC and genotypes were so evaluated utilizing Chi-square ( ?2 ) trial, Fisher ‘s trial and Microsoft Excel.
Polymerase Chain Reaction ( PCR )
Using a forward and change by reversal primer 192 for elaboration of the PON1 192 cistron, a 99 bp PCR merchandise was amplified. All amplified DNA were electrophoresed by 1.5 % agarose gel before digestion to guarantee that the 99 bp set was amplified.
Restriction Fragments Length Polymorphism ( RFLP )
Amplified DNA were genotyped by RFLP utilizing limitation 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 sets.
Agarose gel cataphoresis was used to divide the digested fragments. In the presence of the mutant-type base CGA ( RR ) , two sets of 63-bp and 36-bp sets were seen on complete digestion occurred ; in the presence of the wild-type base CAA ( QQ ) , merely 99-bp set was seen as no digestion occurred ; in the presence of both mutant- and wild-type bases ( QR ) , three sets of 99, 63 and 36-bp sets were observed. The consequences were indicated in Figure 1:Digested fragments( bp )GenotypesQQ( wild type )QR( mutant + wild type )RR( mutant type )99
Figure 1: Genotypes of PON1 polymorphism as determined by RFLP.
3.1 ) Demographic Distribution
A sum of 67 topics were recruited in this survey. There were 9 KC patients ( 13.4 % ) , 2 forme fruste KC patients ( 3.0 % ) , 20 myopic patients ( 29.
8 % ) and 36 normal topics ( 53.7 % ) . The age scope of the recruited topics was 5 to 68 old ages old. The average ages for the several 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 topics ) . In the sample population, the male to female ratio was 36:31 with the per centum of 53.7 % of males and 46.3 % of females.In the pooled sample population, the Malay ( 41.
2 % ) and Indian ( 44.8 % ) were every bit 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 frequences were 0.
1791 ( QQ ) , 0.5522 ( QR ) and 0.2687 ( RR ) . The allele frequences for Q allelomorph and R allele were 0.4552 and 0.5448 severally while the frequences for Q bearer and R bearer were 0.7317 and 0.
3.3 ) Association between KC and genotypes
In this survey, the genotypic distribution of 192Q/R polymorphism indicates no important difference with KC patients in Malaysia and suggests that there may perchance be no relation between PON1 192Q/R polymorphism and the hazard of an person to KC.
The ?2 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 frequence in both of the KC patients and non-KC topics.
3.4 ) Association between KC and allelomorphs
The allelomorphic distribution of 192Q/R polymorphism besides shows no important difference with KC patients in Malaysia. This suggests that there may be no relation between PON1 192Q/R polymorphism and the hazard of an person to KC. The ?2 value for 192Q/R polymorphism was 0.282, and the p-value was more than 0.05.
The allele frequence between Q allelomorph and R allele show non much difference between KC patients and non-KC topics.
3.5 ) Association between KC and bearers
In this survey, the bearer distribution of 192Q/R polymorphism indicates no important difference with KC patients in Malaysia and suggests that there may perchance be no relation between PON1 192Q/R polymorphism and the hazard of an person to KC. The ?2 value for 192Q/R polymorphism was 0.270 and the p-value was more than 0.05. From the analysis, the Q and R bearers were every bit distributed in both of the KC patients and non-KC topics.
6 ) Association between Ethnics and Genotypes
Comparison of genotypes between Malays and Indians showed no important difference. The ?2 value obtained was 5.818 ( p=0.055 ) . Chinese topics were ignored in the analysis as there were merely 9 Chinese topics being recruitmed and no Chinese with the QQ genotype was recruited in this survey.
There are few documents published on PON1 polymorphisms in other optic diseases such as aged-related macular devolution ( . However, there are no published documents reported on PON1 polymorphism with KC. This survey on the association of PON1 and KC may be the first to transport out. In the present survey, there was no important grounds that 192Q/R was associated with KC hazard as our informations did non observe statistical significance in the distribution of allelomorph and genotype frequences of 192Q/R between KC patients and non-KC topics.The restriction of this survey was that the KC sample size was excessively little to obtain a important association between PON1192Q/R polymorphism and KC. KC patients recruited in this survey were from two selected countries: THONEH in Petaling Jaya and Ophir Eye Clinic and Surgery in Klang.
Future work has been discussed to roll up KC informations from all provinces in Malaysia.