This (Shukla et al., 2013). 2.1.1 Pharmacokinetic

This study is conducted to develop the genotyping method which is Polymerase Chain Reaction (PCR) in the detection of rs9923231 VKORC1 gene polymorphism. Before explain further about PCR, it is important to know the overview of the warfarin, how it causes bleeding and the background of VKORC1 gene. 2.1 INTRODUCTION TO WARFARIN Warfarin is the most widely used anticoagulant drug which used by approximately 1.5 million individuals (Bertram G. Katzung, 2015).  Warfarin can be used to treat many diseases such as atrial fibrillation, heart valve prosthesis, recurrent stroke, deep vein thrombosis (DVT), myocardial infarction, and pulmonary embolism (Shukla et al., 2013). 2.1.1 Pharmacokinetic of warfarinUpon administration, warfarin is totally absorbed and reach the maximum plasma concentration between 2 and 6 hours. This drug binds extensively to plasma protein thus having a small volume of distribution (Vd) which is 10L/70kg. R-warfarin and S-warfarin are the racemic mixtures of warfarin (Gan et al., 2011). This anticoagulant drug will present into two enantiomers that will be metabolized differently by human cytochromes P450 (CYP) in the liver (Kaminsky & Zhang, 1997). For S-warfarin, it is about 3-5 times more potent in inhibiting the Vitamin K epoxide reductase (VKOR) compared to R-warfarin and it is metabolized via CYP2C9 to 7-hydroxywarfarin. While for R-warfarin, it is mainly metabolized by CYP1A2 to 6-hydroxywarfarin and 8-hydroxywarfarin and by CYP3A4 to 10-hydroxywarfarain as well as carbonyl reductase which metabolizes R-warfarin to diastereoisomeric alcohols (Kaminsky & Zhang, 1997). While for the elimination, warfarin has small clearance which is 0.2 L/h/70kg by the hepatic metabolism (Holford, 1986).2.1.2 Mechanism of actionBefore going into details on how warfarin act, it is crucial to know the function of vitamin K (a fat-soluble vitamin) in the human body. Without regular intake of vitamin K, this vitamin tends to rapidly deplete as human body stores very small amount even though vitamin K is fat-soluble vitamin (Cheung, Sahni, Cheung, Sing, & Wong, 2015). In the coagulation cascade, vitamin K plays the essential role in the synthesis coagulation factors. In the intrinsic and common pathway, factors II, VII, IX, and X are all important and they are all vitamin K dependent factors (“Hemostasis | Boundless Anatomy and Physiology,” n.d.). In order to avoid from excessive thrombosis following the initial coagulation cascade, vitamin K also synthesize the anticoagulant proteins such as Protein S, Protein C and Protein Z which can inactivate the coagulation factors.Upon administration of warfarin, this drug can inhibit the role of vitamin K which is also known as an antagonist for vitamin K. In the human body, the vitamin K is recycled by the body through a process called the vitamin K-epoxide cycle (Cheung et al., 2015). To be specific, warfarin will disrupt the vitamin K cycle by blocking vitamin K epoxide reductase (VKOR) complex in the liver which results in depletion of the vitamin K hydroquinone (reduced form). Briefly, in the normal reaction the reduced dorm of vitamin K (Vitamin K hydroquinone) is needed in the cycle in order to oxidize to vitamin K epoxide (oxidized form), this reaction eventually will trigger the ?-glutamylcarboxylase to carboxylate selective glutamic acid residues on vitamin K-dependent proteins (Cheung et al., 2015). The reduced form of vitamin K is also very important as it acts as a cofactor in the gamma carboxylation process for vitamin K dependent factors (Ansell et al., 2008). While for the vitamin K epoxide reductase (VKOR), it is required for recycle vitamin K between reduced and epoxide forms.If the gamma-carboxylation process is absence, the vitamin K dependent factors which are factors II (prothrombin), VII, IX, and X, are immunologically detectable, but these factors fail to be activated, as the result, they cannot bind to calcium and phospholipid membranes which are needed for hemostatic function (Ansell et al., 2008). 2.2 PROBLEMS RELATED TO WARFARINWarfarin has a narrow therapeutic window and has a wide variability of inter-individuals which contribute to many problems (Gan et al., 2011). Under-coagulation will cause thrombosis while over-coagulation will cause dangerous bleeding episodes which can be life-threatening (Gan et al., 2011). In short, the best dose of warfarin is difficult to achieve due to several factors which influence the individual’s response to warfarin, including non-genetic and genetic factors.2.2.1 Non-genetic factors that contribute to the problems in warfarin.2.2.1.1 AgeGenerally, geriatric (>65 years) are more prone to have a sensitivity to warfarin effect, thus they need a lower mean daily dose to achieve the target anticoagulant effect (Gan et al., 2011). The systematic review that was done by Jinhua Zhang and his colleagues reported that, pediatric that age 0 to 6 years require a lower dose of warfarin compare to >7 years due to the abnormal liver function that can affect the metabolism of warfarin (Zhang, Tian, Zhang, & Shen, 2015). Besides, this is also due to the increasing in body weight and body surface area (BSA) that can lead to increase in warfarin hepatic clearance (Zhang et al., 2015). However, this study has some limitations as the study just involves a small number of articles which is only eight that is not enough. Furthermore, most of the studies were on Caucasian patients with just only one study involved Japanese patients and this systematic review does not conduct subgroup analysis according to different ethnicity.2.2.1.2 Disease State2.2.1.2.1 Liver diseaseThe liver has important roles which involve the production of the clotting factors and metabolism of warfarin through cytochrome CYP450 enzyme system. In the liver disease patient, the production of clotting factors is disrupted which result in the reduction in synthesis of coagulation factors V, VII, X, and prothrombin (White, 2010). Thus, the patients with liver disease warrant a lower initiation dose of warfarin which is ? 5mcg due to depletion of clotting factors synthesis, along with decrease albumin production (White, 2010). 2.2.1.2.2 Renal diseaseA study has suggested, at the beginning of the outpatient warfarin therapy, the patients with kidney failure tend to have a higher risk of the major bleeding event. The patients with severe kidney failure with the creatinine clearance <30 mL/min are more likely to have unstable and supratherapeutic INR. Furthermore, the incidence of bleeding is higher compare to the mild renal insufficiency patients with the creatinine clearance > 60 mL/min. Severe and mild renal failure patients also require a different average dose of warfarin which are 3.9 mg/d and 4.8 mg/d respectively (White, 2010). 2.2.1.2.3 Thyroid disease The hyperthyroid patients are more likely to face with warfarin sensitivity due to either reduced in the synthesis of vitamin K-dependent clotting factors or increase catabolism of prothrombin and factor VII. While in hypothyroid patients, at the starting of thyroid replacement therapy, it is crucial to always monitor the INR. This is because, a report has shown that, the need for significant dose reduction when the normal thyroid function is obtained (Demirkan, Stephens, Newman, & Self, 2000).2.2.1.3 Nutritional statusThe patients with low vitamin K stores are more likely to sensitive to the pharmacodynamics effect of warfarin compared to the patients with higher vitamin K stores. During maintenance of warfarin therapy in the patients, the fluctuation in dietary vitamin K can have an impact on INR stability. In order to reduce intra-patient variability in the INR response, the patients should maintain the right and consistent intake of vitamin K in their diet (White, 2010)2.2.1.4 Drug interactionsAnother factor which can affect the warfarin response is the drug interactions which may inhibit or potentiate the warfarin response through pharmacokinetics and pharmacodynamics mechanism when the drug therapy is initiated or discontinued. Pharmacodynamics interaction may affect hemostasis as well as alteration of clotting factors synthesis (White, 2010). The administration of NSAIDs or aspirin with warfarin may have the pharmacodynamics interaction which can increase the risk of getting bleeding by inhibiting the platelet function without altering the anticoagulant effect on INR response (Howard, Ellerbeck, Engelman, & Patterson, 2002). The retrospective study has been conducted and describes that 54% to 79% of warfarin patients are prescribed a potentially interacting medication (Snaith, Pugh, Simpson, & McLay, 2008). There was a case-control study that was done among 38,762 patients with aged 65 years concluded that, there is the interaction between antibiotics particularly antifungals drugs with warfarin which can lead to 2-fold increased risk of bleeding in patients (Baillargeon et al., 2012).  Furthermore, this study also mentioned that cotrimoxazole and fluconazole was proved can cause increasing in gastrointestinal bleeding (Baillargeon et al., 2012). But, this study has some limitations in which, there is not enough information on the intensity of the anticoagulation therapy.2.2.2 Genetic factors responding to warfarin.A single base pair mutation in the DNA sequence is called as single-nucleotide polymorphisms (SNPs). In the human genome, there are approximately 3 billion of the base pairs located in the 23 pairs of the chromosomes which specifically in the nucleus of the human cell (Venter et al., 2001). SNPs are the most common variations that can occur in coding or non-coding region of the genome. If SNPs occur in the coding region (nonsynonymous SNPs), this will result in the alteration of the amino acid sequence which eventually lead to the dysfunctional of the protein (Venter et al., 2001).   There are two important genes had been studied in the patients with warfarin which are CYP2C9 and VKORC1 (Vitamin K epoxide oxidase reductase complex subunit 1). The polymorphisms of these genes may affect pharmacokinetics and pharmacodynamics of the warfarin (Gan et al., 2011) and when combining with the clinical factors, this polymorphism had shown to affect over 50% of the variability of warfarin dose (Sconce et al., 2005). 2.2.3 The pharmacogenetics and pharmacogenomics of Warfarin 2.2.3.1 Cytochrome P450 enzymesMany genes encode this enzyme and they mainly found in the liver. Their roles are very important as they involve in the drug and other xenobiotic metabolism and also involve in detoxifications process as they eliminate the foreign substances.  They are 70 cytochrome P450s (CYPs) in every species. However, in the human genome, there is only 57 CYP genes and 33 pseudogenes are found. The most important enzyme in the drug metabolism is CYP2C9 that metabolize approximately 60 drugs including warfarin. When SNPs occur in CYP2C9 gene, this will cause the slow in the metabolism of warfarin, need longer time to reach INR stabilization, higher INR in the beginning of warfarin therapy as well as the lower maintenance dose (Aithal, Day, Kesteven, & Daly, 1999). Furthermore, during initiation of warfarin, 2-3 fold increasing of bleeding risk had been observed in patients (Aithal et al., 1999). There are two common SNPs that had been identified in CYP2C9 which are CYP2C9*2 and CYP2C9*3. At the position 144 in exon 3 in the CYP2C9*2 variant has an arginine replaced with a cysteine which causes enzymatic activity to fall by 30% equivalent to 17% reduction in dose (Limdi & Veenstra, 2008). In CYP2C9*3 variant at exon 7, leucine has replaced the isoleucine that causes the reduction in enzymatic activity by 80% corresponding to 37% reduction in dose (Limdi & Veenstra, 2008). These variants cause the reduction of the dose had been proven in the Caucasian patients account for 6-12% of the variation in warfarin dose. A review from Lee reported that there is 10–15% and 5–10% frequency of CYP2C9*2 and CYP2C9*3 in Caucasian patients respectively. While in the ethnic group of Asian populations (Malays and Indian), both alleles are detected with higher frequency in the Indians compare to Malays, the other studies reported that the CYP2C9*2 allele is not detected in the Chinese (Gan et al., 2011). 2.2.3.2 Vitamin K Epoxide Reductase (VKORC1)VKORC1 gene encodes a small transmembrane protein of the endoplasmic reticulum which is vitamin K epoxide reductase complex subunit-1 and it is the target site of warfarin (Ross et al., 2010). VKORC1 is well established in many research papers in which polymorphisms of VKORC1 gene has been showed to have a major effect on the warfarin metabolism. The single nucleotide polymorphism of VKORC1 has been found to be located at strong linkage disequilibrium (LD) region (Bodin et al., 2005). A number of studies have established that warfarin dose requirement is associated with several intronic and promoter polymorphism (Dang, Hambleton, & Kayser, 2005). Various VKORC1 polymorphisms are been discovered but there are two most widely studied which are C1173T and G-1639A (Limdi & Veenstra, 2008). In a study that had been conducted has proved that VKORC1 -1639 SNPs has significantly effect on warfarin maintenance dose while VKORC1 1173 SNPs has no significant effect (Zhang et al., 2015). Therefore, focusing on only one of this polymorphism which is G-1639A SNPs is enough to characterize the genetic variability in the population since G-1639A has been found to account high variability in the warfarin dose (Gan et al., 2011). The mutation occurs when G allele is replaced with A allele at gene position 3673 and the minus sign of 1639 G>A indicating that this is in an upstream promoter (Christine Kasper PhD, n.d.).A study by Gin Gin Gan and colleagues in Asian population reported that patients with GG genotype require a higher dose, patients with AA genotype need a lower dose and patients AG genotype require intermediate dose (Gan et al., 2011). Another report also demonstrated that lower dose is associated in the patients with A allele (Miao, Yang, Huang, & Shen, 2007). The study in the Chinese and Japanese population reported that the frequency of AA genotype is 79% which is higher compare to the Western population with 14% and demonstrated that AA genotype requires lower dose (OBAYASHI et al., 2006).The location of VKORC1 SNP with reference sequence number rs9923231 is at chromosome 16 (16p11.2) as shown in the figure 2.3 in the promoter region of the gene. Since SNP is located in the promoter region, it does not code for amino acid (Christine Kasper PhD, n.d.).The finding from the systematic review suggest that the adverse drug reactions including bleeding can be reduced through the use of the pharmacogenomics knowledge (Phillips, Veenstra, Oren, Lee, & Sadee, 2001). So, it is crucial to use the patients’ genetic make-up in order to determine the patients’ response toward the specific drug especially drug with the narrow therapeutics index, so that the therapeutic goals of the drugs may successfully achieve.  2.3 THE EXTRACTION OF DNA FROM HUMAN BLOODAs mentioned previously the association of VKORC1 SNPs with the bleeding event in the patients who are taking warfarin, hence it is crucial to understand a technique which can be used in the detection of the VKORC1 gene polymorphism in the Malaysian population. The method that will be used are Deoxyribonucleic acid (DNA) extraction from blood, primer design, Nested Polymerase Chain Reaction (PCR) which is two-step PCR and agarose gel electrophoresis. In order to study DNA, the very first thing that needs to do is to get out DNA from the human cell. The human cell is eukaryotic cell where DNA is located in the nucleus as chromosomes. The purpose of the DNA extraction process is to acquire DNA in a purified form which can be used in PCR. DNA extraction involves the separating of DNA from the protein, membranes, and other cellular material contained in the cell from which it is recovered (Howard et al., 2002). Low quality of the DNA may not perform well in the PCR. So to prevent the contamination of the sample and crossover, DNA extraction process requires extra careful handling of the biological material. There are three basic steps in the DNA extraction process which are lysed (break open) the cells, precipitation and purifications. The first step that involves is the break open of the cell and the nucleus to allow the DNA release from the nucleus. The way to do this is by using detergents and enzymes that can dissolve cellular proteins and allow DNA to free from the nucleus. After DNA is free from the nucleus, DNA has mixed with mashed up cell parts. The precipitation step is crucial which can separate the DNA from the cell debris. Sodium ions (Na+) is used to neutralize the anion charges of the DNA which can make DNA stable. While alcohol (isopropanol) can make the DNA to precipitate out of the aqueous solution since it is insoluble in the alcohol. Now DNA is said already separated from the aqueous solution and to remove the unwanted material and cellular debris to obtain pure DNA, alcohol is used.2.4 OVERVIEW OF POLYMERASE CHAIN REACTIONThe specific genes can be amplified or make millions of copies by using a tool which is Polymerase Chain Reaction (PCR). Kary B. Mullis, the Japan Biochemist was the one who invented PCR process in 1983 and he won a Nobel Prize in Chemistry in 1993 for his pioneering work (Hannah Wilgar, n.d.). PCR is a powerful technique and has many advantages including it is quick, inexpensive and simple. Nowadays, PCR has become the most widely use technique in molecular biology.2.4.1 The components in PCRThe main ingredients used in PCR are primers, DNA templates that contain specific region to be amplified, deoxynucleotide triphosphates (dNTPs), Taq polymerase and buffer. The primers are the short stretches of the DNA that contain 18 – 28 bases nucleotides which complementary to the target sequence. Primers also have a hydroxyl group at 3’end which allow the elongation process to occur. There are two primers used in PCR which are forward and reverse primers. Other main components are dNTPs which are four nucleotides that contain triphosphate group and are also the building block for the elongation process to form new DNA strand (Introduction et al., 2011). Buffers used in PCR reaction to give the suitable chemical environment for the optimum activity and also for the stability of Taq polymerase (Introduction et al., 2011). 2.4.2 STEPS IN PCRThere are three basic steps of PCR which are denaturation, annealing and extension as described in Figure 2.4. These steps can be done by PCR thermal cycler which can rapidly heat and cool the reaction mixture. The first step is denaturation where the double-stranded of the DNA are separated into two single strands at high temperature (from 90 – 97 degrees Celsius) by breaking the hydrogen bond between the complementary bases (Introduction et al., 2011). The second step is annealing which is the binding of the primers to the DNA template to primer extension (Joshi & Deshpande, 2010). PCR process does not amplify all the DNA in the sample, it just only copies the specific DNA sequence that is targeted by DNA primers. In the annealing process, the temperature is lowered to the 50-60°C. the temperature reaction depends on the Tm (melting temperature) of the primers used in PCR. The temperature is typically 3-5°C lower than the Tm of the primers (Introduction et al., 2011). This step allows the primers to hybridize to their respective complementary template strands (Joshi & Deshpande, 2010).In step three, Taq polymerase will bind to the 3’end of the primers and initiate the elongation to create a complimentary copy strand of DNA by adding dNTPs in 5′ to 3′ direction (Joshi & Deshpande, 2010). The temperature reaction depends on the Taq polymerase that is used in PCR which has an optimum temperature at 75–80 °C, however mostly 72°C is used with this enzyme (Introduction et al., 2011). 2.5 NESTED POLYMERASE CHAIN REACTIONPCR also has some limitations which include, the DNA polymerase are susceptible to the errors and the fragment generated lead to mutation (Garibyan & Avashia, 2013). Another limitation is alteration of the specificity of the PCR product due to the nonspecific binding of the primers to other similar sequences on the template DNA (Garibyan & Avashia, 2013).Thus, nested PCR is designed to improve sensitivity and specificity of the reactions and it is the modifications of PCR which use two pairs of primers (rather than just a single) and two successive PCR reactions (Carr, Williams, & Hayden, 2010). The first pair of the primers will be used in the first PCR to amplify the target fragment and the amplicon will be used as the template in the second PCR reaction with the second different set of the primers to generate a product of the expected size  (James, Reid, & Rybicki, 2001).If one of the primers in the first PCR is used again for the second PCR, this called as semi-nested PCR when only one of the primers is replaced and the other primer is similar with first PCR. Nested polymerase reaction which uses two round of PCR reaction can reduce contamination in products due to the amplification of unexpected primer binding sites. 2.6 OPTIMIZATION OF POLYMERASE CHAIN REACTIONWhen handling with PCR, lack of optimization may lead to many drawbacks which may affect the result. For example, PCR product cannot be detected or low-efficiency amplification, the nonspecific band is presented and the formation of “primer-dimers”. In order to reduce nonspecific amplification and to increase the yield of the desired PCR product, optimization of PCR condition is very important. The parameters that involve in the PCR optimization include quality and the concentration of DNA template, concentration of primers, magnesium chloride ions and deoxynucleotides, selection and concentration of DNA polymerase and cycling temperature (Introduction et al., 2011). 2.7 AGAROSE GEL ELECTROPHORESISElectrophoresis is the most common technique used in the laboratory to separate the charged molecules which depend on the size and charge of separated particles, as well as the pore size present in the gel (Drabik, Bodzo?-Ku?akowska, & Silberring, 2016). Due to the negative charged, DNA fragment will migrate to the positively charged anode when place in an electric field (Lee, Costumbrado, Hsu, & Kim, 2012). The migration of the DNA fragment on the gel depends on the size. The larger the size of the DNA, the slower it will migrate toward positively charged than the shorter molecules. Agarose gel can be used for separation of the fragment of the DNA and its concentration that used to make the gel depend on the size of the DNA fragments. The matrix is denser when a higher concentration of agarose is used. Higher concentration of agarose can be used to separate the smaller fragments of DNA while low concentration of the agarose can be used to separate the larger DNA fragment because it results in greater separation between bands that are close in size (Hannah Wilgar, n.d.). The most commonly buffer used in the gel electrophoresis are Tris-acetate EDTA, Tris/Borate/EDTA and sodium borate. The buffer with specified ionic strength will bathe the gel to allow an electric current to run from one end to another. For visualization of the DNA bands, the most common dye used is ethidium bromide. Extreme caution needs to be taken during handling with ethidium bromide as it is a potent mutagen.Thus, the factors that can affect the migration of the DNA molecule through the gels is determined by DNA molecular weight, voltage, the concentration of agarose, electrophoresis buffer as well as the presence of ethidium bromide (Lee et al., 2012).