TITLE: Characterization of the VKORC1 and CYP2C9 genotypes
AUTHORS: Mirsada Causevic, Edin Begic

[DESCRIPTION]
Vitamin K antagonists (e.g. warfarin) are anticoagulants which represent widely prescribed drugs for prevention and treatment of thromboembolic disorders. 

Warfarin's molecular target is vitamin K epoxide reductase enzyme and its metabolism is performed by the cytochrome P-450 2C9 enzyme, both of which are encoded by polymorphic genes - vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P-450 2C9 (CYP2C9), respectively. 

Identification of the VKORC1 and CYP2C9 genotypes was recommended by the U.S. Food and Drug Administration in 2007, in order to guide the initial dosing of warfarin, the first oral vitamin K antagonist drug, to achieve the optimum anticoagulation and prevent hemorrhagic events. 

In an effort to provide a lab protocol that will provide training in pharmacogenomics/pharmacogenetics (PGx) for undergraduate students of pharmacology or medicine, that can be performed in a laboratory that contains basic molecular biology equipment and by using reagents that are available worldwide at low cost, we revisited protocols published previously by other researchers. 

We hope that it will provide the elementary understanding of the simplest molecular biology methods used to identify different VKORC1 (-1639G>A) and CYP2C9 genotypes, as well as understanding of a link between a specific genotype (or combination of genotypes) and phenotype relevant for a patient’s drug response.

The protocol represents a step-by-step guide to using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method in determining VKORC1 -1639G>A, CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, and CYP2C9*2/*3 genotypes. 

Based on the combination of the VKORC1 -1639G>A and CYP2C9 genotypes, that is, the pattern of DNA fragments observed at the end of an experiment, which are visualized using agarose gel electrophoresis and DNA staining, a conclusion on an individual’s sensitivity to warfarin and appropriate drug dose is reached. 

A dataset containing the VKORC1 -1639G>A and CYP2C9 genotypes characterized in 32 patients using this protocol was deposited at the Harvard Dataverse Repository that can be accessed at the following digital object identifier: https://doi.org/10.7910/DVN/SFPYCD. In the dataset, the patients were grouped into the following anticoagulant sensitivity/dosing categories, as recommended by the U.S. Food and Drug Administration’s (FDA) label for warfarin: 1) standard anticoagulant sensitivity/dosing genotypes (N=20), 2) intermediate anticoagulant sensitivity/dosing genotypes (N=11), and 3) low anticoagulant sensitivity/dosing genotypes (N=1).

[BEFORE_START]
In order to protect the individuals from the potential HepB virus infection while working with human blood, the U.S.A. Centers for Disease Control and Prevention (CDC) issued a recommendation for HepB vaccination to be carried out in individuals before they start working with human blood. Vaccine-induced antibody titer to the HepB virus of ≥10 mIU/mL should be present in individuals before commencing work with human blood.

[GUIDELINES]
This protocol uses human whole blood as a starting material for extracting human, genomic DNA. Human whole blood could be a source of hepatitis B (HepB) virus infection (if taken from persons with HepB virus infection) to the individuals who work with human blood and, as such, represents health and safety or biological hazard.

[STEPS]
SECTION: Genomic DNA extraction
1. Patients' whole blood was collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes and stored at -20°C until use. Genomic DNA extraction from the human whole blood, that is, leukocytes, was carried out according to the protocol described by Subbarayan PR and colleagues (doi: 10.2144/02336bm10), with modifications. 

Upon thawing of the blood, for every patient's blood sample, five (5) sterile tubes were labeled and 300 μl of the whole blood was added. This was the amount of the starting material that yielded ample amount of human, genomic DNA at the end of the DNA extraction protocol.
SECTION: Genomic DNA extraction
2. Concentrations of human, genomic DNA was determined by using Qubit 4 Fluorometer (Q33226, Invitrogen and Thermo Fisher Scientific). 

Subsequently, for polymerase chain reactions (PCRs), 100 ng of genomic DNA was used in every PCR reaction.
SECTION: Identification of the VKORC1 -1639G&gt;A genotypes
3. Identification of the VKORC1 -1639G>A genotypes was performed according to the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method-based protocol published by Sconce EA and colleagues (doi: 10.1182/blood-2005-03-1108).
SECTION: Identification of the VKORC1 -1639G&gt;A genotypes
4. PCR reactions were assembled as follows: reactions of 25 μl contained 1) sterile, milliQ-H2O; 2) 1xPCR buffer (for GoTaq G2 DNA polymerase, Promega), from a stock of 5xPCR buffer; 3) 0.2 mM deoxyribonucleotide triphosphates (dNTPs) (U1330, Promega), from a stock of 2.5 mM; 4) 1 μM forward primer, from a stock of 25 μM; 5) 1 μM reverse primer, from a stock of 25 μM; 6) 100 ng of genomic DNA and 7) 2.5 U GoTaq DNA polymerase (M7845, Promega). 

Sequences of the PCR primers were as follows: 1) Forward primer: 5'-GCCAGCAGGAGAGGGAAATA-3'; 2) Reverse primer: 5'-AGTTTGGACTACAGGTGCCT-3'.

PCR cycles used for the amplification of the VKORC1 gene promoter, which were based on the touchdown PCR protocol published by Korbie and Mattick in 2008, were as follows: 
1) 95°C for 3 min (1 cycle); 
2) 95°C for 2 s, 60°C for 2 s, 72°C for 30 s (2 cycles); 
3) 95°C for 2 s, 59°C for 2 s, 72°C for 30 s (2 cycles); 
4) 95°C for 2 s, 58°C for 2 s, 72°C for 30 s (2 cycles); 
5) 95°C for 2 s, 57°C for 2 s, 72°C for 30 s (2 cycles); 
6) 95°C for 2 s, 56°C for 2 s, 72°C for 30 s (2 cycles); 
7) 95°C for 2 s, 55°C for 2 s, 72°C for 30 s (30 cycles); 
8) 72°C for 7 min (1 cycle); 
9) 4°C for 2 h (1 cycle). 

 

SECTION: Identification of the VKORC1 -1639G&gt;A genotypes
5. In order to characterise the VKORC1 -1639G>A genotypes by using the PCR-RFLP method, the following reactions were set up with MspI restriction endonuclease enzyme: 
1) 25 μl of the PCR product; 
2) 3 μl of the 10xCutSmart Buffer (B7204S, New England Biolabs); 
3) 1 μl of sterile, milliQ-H2O; 
4) 1 μl of MspI enzyme (R0106S, New England Biolabs). Total reaction volume: 30 μl. 

Reactions were incubated in a 37°C water bath overnight. 

DNA fragments were examined by using 2% agarose gel electrophoresis with 1xTris, boric acid, EDTA (TBE) buffer and agarose gels containing 0.5 mg/ml ethidium bromide (E1510-10ML, Sigma). The results were viewed with a UV lamp (UVstar, Biometra, Analytik Jena). Gel images were captured by using BioDocAnalyze (BDA) camera (Biometra, Analytik Jena).


 



SECTION: Identification of the CYP2C9 genotypes
6. Identification of the CYP2C9 genotypes (*1/*1, *1/*2, *1/*3, and *2/*3) was performed according to the PCR-RFLP method-based protocol published by Sullivan-Klose TH and colleagues (doi: 10.1097/00008571-199608000-00007) and Yasar U and colleagues (doi: 10.1006/bbrc.1998.9992).


SECTION: Identification of the CYP2C9 genotypes
7. PCR reactions were assembled as follows: reactions of 25 μl contained 1) sterile, milliQ-H2O; 2) 1xPCR buffer (for GoTaq G2 DNA polymerase, Promega), from a stock of 5xPCR buffer; 3) 0.2 mM deoxyribonucleotide triphosphates (dNTPs) (U1330, Promega), from a stock of 2.5 mM; 4) 1 μM forward primer, from a stock of 25 μM; 5) 1 μM reverse primer, from a stock of 25 μM; 6) 100 ng of genomic DNA and 7) 2.5 U GoTaq DNA polymerase (M7845, Promega). 



SECTION: Identification of the CYP2C9 genotypes
7.1. Sequences of the PCR primers for AvaII restriction endonuclease enzyme reactions were as follows: 1) AvaII-Forward primer: 5'-TACAAATACAATGAAAATATCATG-3'; 2) AvaII-Reverse primer: 5'-CTAACAACCAGACTCATAATG-3'. 

PCR cycles used with the above primers were as follows:
1) 94°C for 5 min (1 cycle);
2) 94°C for 60 s; 55°C for 90 s; 72°C for 30 s (35 cycles);
3) 72°C for 7 min;
4) 4°C for 2 h (1 cycle).

 


SECTION: Identification of the CYP2C9 genotypes
7.2. In order to characterise the CYP2C9 *1/*1 or *1/*2 genotypes by using the PCR-RFLP method, the following reactions were set up with AvaII restriction endonuclease enzyme: 
1) 10 μl of the PCR product; 
2) 2 μl of the 10xCutSmart Buffer (B7204S, New England Biolabs); 
3) 7 μl of sterile, milliQ-H2O; 
4) 1 μl of AvaII enzyme (R0153S, New England BioLabs). Total reaction volume: 20 μl.  

Reactions were incubated in a 37°C water bath overnight. 

DNA fragments were examined by using 2% agarose gel electrophoresis with 1xTBE buffer and agarose gels containing 0.5 mg/ml ethidium bromide (E1510-10ML, Sigma). The results were viewed with a UV lamp (UVstar, Biometra, Analytik Jena). Gel images were captured by using BioDocAnalyze (BDA) camera (Biometra, Analytik Jena).


 


SECTION: Identification of the CYP2C9 genotypes
7.3. Sequences of the PCR primers for NsiI restriction endonuclease enzyme reactions were as follows: 1) NsiI-Forward primer: 5'-AATAATAATATGCACGAGGTCCAGAGATGC-3'; 2) NsiI-Reverse primer: 5'-GATACTATGAATTTGGGACTTC-3'.

PCR cycles used with the above primers were as follows: 
1) 94°C for 5 min (1 cycle);
2) 94°C for 60 s; 60°C for 90 s; 72°C for 30 s (35 cycles);
3) 72°C for 7 min;
4) 4°C for 2 h (1 cycle).

 


SECTION: Identification of the CYP2C9 genotypes
7.4. In order to characterise the CYP2C9 *1/*3 or *2/*3 genotypes by using the PCR-RFLP method, the following reactions were set up with NsiI restriction endonuclease enzyme: 
1) 10 μl of the PCR product; 
2) 2 μl of the 10xNEBuffer 3.1 (B7203S, New England Biolabs); 
3) 7 μl of sterile, milliQ-H2O; 
4) 1 μl of NsiI enzyme (R0127S, New England BioLabs). Total reaction volume: 20 μl.  

Reactions were incubated in a 37°C water bath overnight. 

DNA fragments were examined by using 2% agarose gel electrophoresis with 1xTBE buffer and agarose gels containing 0.5 mg/ml ethidium bromide (E1510-10ML, Sigma). The results were viewed with a UV lamp (UVstar, Biometra, Analytik Jena). Gel images were captured by using BioDocAnalyze (BDA) camera (Biometra, Analytik Jena).

 

SECTION: Identification of the CYP2C9 genotypes
7.5. Sequences of the PCR primers for KpnI restriction endonuclease enzyme reactions were as follows: 1) KpnI-Forward primer: 5'-AATAATAATATGCACGAGGTCCAGAGGTAC-3'; 2) KpnI-Reverse primer: 5'-GATACTATGAATTTGGGACTTC-3'.

PCR cycles used with the above primers were as follows: 
1) 94°C for 5 min (1 cycle);
2) 94°C for 60 s; 60°C for 90 s; 72°C for 30 s (35 cycles);
3) 72°C for 7 min;
4) 4°C for 2 h (1 cycle).

 


SECTION: Identification of the CYP2C9 genotypes
7.6. In order to characterise the CYP2C9 *1/*3 or *2/*3 genotypes by using the PCR-RFLP method, the following reactions were set up with KpnI restriction endonuclease enzyme: 
1) 10 μl of the PCR product; 
2) 2 μl of the 10xBuffer J (R009A, Promega); 
3) 0.5 μl Bovine Serum Albumin Acetylated, 10mg/ml (R3960, Promega);
4) 6.5 μl of sterile, milliQ-H2O; 
5) 1 μl of KpnI enzyme (R6341, Promega). Total reaction volume: 20 μl.  

Reactions were incubated in a 37°C water bath overnight. 

DNA fragments were examined by using 2% agarose gel electrophoresis with 1xTBE buffer and agarose gels containing 0.5 mg/ml ethidium bromide (E1510-10ML, Sigma). The results were viewed with a UV lamp (UVstar, Biometra, Analytik Jena). Gel images were captured by using BioDocAnalyze (BDA) camera (Biometra, Analytik Jena).