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The Cytochrome 4F2 rs2108622
Genetic Variant among Healthy
Jordanian Volunteers
3/24/2016
Danya Iqtait and Rana Sadieh

2. The Cytochrome 4F2 rs2108622 Genetic Variant
among Healthy Jordanian Volunteers
Abstract
Background: The rs2108622 genetic variant in CYP4F2 gene has been recently
shown to influence stable warfarin dose requirements. Objective: To determine the
frequency of CYP4F2alleles and genotypes among the Jordanian
population.Methods:The present study extracted the DNA from 90 healthy Jordanian
volunteers and determined the frequency of CYP4F2 rs2108622 by polymerase chain
reaction (PCR) followed by restriction fragment length polymorphism assays
(RFLP).Results: The frequency (95% confidence interval) of rs2108622 C and T
allele was 0.55 (0.48-0.62) and 0.45 (0.38- 0.52), respectively. The rs2108622C>T
frequency was high among the Jordanian population and almost similar to Caucasians
but different than other ethnic populations. Conclusion:The high frequency of
CYP4F2rs2108622 polymorphism among Jordanian may associate with
cardiovascular diseases and warfarin response.

3. Introduction
Cytochrome 4F2 (CYP4F2) is monooxygenase enzyme localized in the endoplasmic
reticulum of cells. It metabolizes mainly endogenous compounds(1). The CYP4F2
influences the blood vessel and platelet function through oxidizing of arahidonic acid
and leukotriene-B4 to 20-hydroxyeicosatetraeonic acid and 20-hydroxyleukotriene-
B4, respectively(2, 3). In addition, CYP4F2 oxidizes saturated and unsaturated fatty
acids such as lauric and palmitic acids(4). Furthermore, CYP4F2 is involved in
vitamin K metabolism and hence affects on the blood coagulation and the
anticoagulant activity of warfarin. Therefore, CYP4F2 plays a role in the
cardiovascular homeostasis (5, 6).
The expression of CYP4F2 is different among the body tissues. It is found that
CYP4F2 is predominantly expressed in the liver, kidney and human platelets(7),
while it couldn't be detected in human megakaryoytes (8).
The CYP4F2 is encoded by CYP4F2 gene located on chromosome 19 at position
13.12. The CYP4F2 gene is 20096 (from 15,878,024 to 15,898,120) base pair size
with 13 exons and 12 introns (1, 9, 10). Genetic variants on CYP4F2 gene had been
reported to affect on CYP4F2 function and on the cardiovascular homeostasis. It is
reported that 1347 C>T polymorphism (rs2108622) in the 11th exon region of
CYP4F2 gene were associated with cardiovascular diseases, such as hypertension
(11). It is found in multiple studies that differences in CYP4F2 genotype account for
approximately 1-7% of the inter-individual variability in warfarin response(12). The
exonic non-synonymous CYP4F2rs2108622 genetic variant showed high influence on
warfarin response (13). It is characterized by the substitution of 1393 cytosine to
thymine on exon 11 which replaces valine to methionine at position 433 on CYP4F2

4. amino acid sequence. This amino acid substitution reduces CYP4F2 metabolic
activity (2).
There is an inter-ethnic variation in the frequency of CYP4F2rs2108622 among
different ethnic groups. It is found that the frequency of CYP4F2rs2108622 T allele is
higher among Caucasians (0.44) and Asians (0.35) than Africans (0.07) (1). This
difference may affect on the susceptibility to cardiovascular diseases and warfarin
response among different ethnic groups. Among Middle Eastern countries, it is found
that the frequency of CYP4F2rs2108622 among Egyptian population was high (0.42)
in comparison with other Caucasian and African ethnic groups (14).
Although CYP4F2rs2108622 genetic variant plays a role in cardiovascular
homeostasis and warfarin response (5, 6), no study on CYP4F2rs2108622 was done
on Jordanian population. Therefore, the aim of the present study was to find the
frequency of warfarin sensitive CYP4F2rs2108622 genetic variant among healthy
unrelated Jordanian population.

5. Method
Sample collection
A total of 90 healthy Jordanian volunteers ( 30 males and60 females, with an average
age ± standard deviation of 23± 3 years) agreed to participate in the study and signed
the admittance paper. The study was approved by the College of Pharmacy, Al-
Zytoonah University. From each volunteer 2 – 3 ml of venous blood were obtained in
EDETA tubes from each volunteer. Individuals were healthy, unrelated Jordanian.
Most of them were from Al-Zytoonah university students and workers.
DNA extraction
DNA was extracted using Wizard DNA extraction kit (Promega, Madison, WI, USA)
according to the manufacturer’s method. Briefly, leukocytes were isolated from whole
blood through 3000 rpm centrifugation and then incubated with cell lysis solution.
After that, the cell nucleus pellet was lysed through lysis solution. Then, the proteins
in the samples were precipitated. Then, the DNA was precipitated by isopropanol and
washed with %70 ethanol. Lastly, the DNA was dissolved in nuclease free water and
was stored at -20˚C until used.
Amplification of CYP4F2 gene fragment
The CYP4F2 gene fragment in the DNA samples was amplified using polymerase
chain reaction (PCR) with the following reaction system : a sample of 100 ng of
genomic DNA was reconstituted in a total 50 μl reaction mixture containing 1 μl of 10
mM dNTPs; 2 μl of 25mM MgCl2; 10μl of 10X Taq polymerase buffer, 1 unit of
TaqDNA polymerase and 10 pmole from each of The following forward

6. (ATCAACCCGTTCCCACCT) and reverse (CGTCTGGGAGCACAATGT) PCR
primer sequences (15).
After that, the PCR mixture was heated to 94 °C for 5 min (denaturation), and then it
incubated in 35 thermal cycles of: denaturation step at 94°C for 1 min, annealing step
at 55 °C for 1min and elongation step at 72 °C for 1 min. Then, the PCR product was
completed by incubating it at 72 °C for 7 min as a final step of elongation. The PCR
produt size was 505bp, as shown on Figure 1.
Restrictionfragment length polymorphism
A sample of 1 μg of DNA from PCR product was subjected to restriction enzyme
analysis (PvuII-HF; new England bio labs). Breifly, 1 μl of PvuII-HF, 5 μl (x1) of 10x
NEBuffer was added to 10 μl PCR product then incubate it at 37 °C for overnight to
detect the genetic variation (rs2108622). The PvuII restriction enzyme cut the PCR
product to 2 fragments with size of 185 and 320 base pair.
After cell digestion products were separated on 3% agarose gel for detection of
rs2108622 variation after gel staining with ethidium bromide as shown on Figure 2.

7. Figure 2. The CYP4F2 rs2108622 genotype among sample of healthy Jordanian
volunteers
Statisticalanalysis
Chi-square (χ2 test) was used to determine whether the CYP4F2 allele distribution
was in Hardy-Weinberg equilibrium. A p-value < 0.05 was used to reject the null
hypothesis and z- core test was used for comparison of CYP4F2 genotypes frequency
among Jordanians with other populations.
DNA
ladder Heterozygote
Heterozygote
Heterozygote
Heterozygote
Homoozygote
Wild 505 bp
320bp
185bp

8. Results
The frequency of CYP4F2rs2108622 C and T alleles among healthy unrelated
Jordanians were 0.55 and 0.45, respectively ( Table 1 ).
Table 1. The allele frequency of CYP4F2rs2108622C > T genetic variant among
healthy Jordanian population
95% confidenceFrequency observedAllele
0.48-0.620.55C
0.38- 0.520.45T
1Total
The CYP4F2rs2108622 genotype frequencies among healthy Jordanian volunteers
were wild CC (0.27), heterozygote CT (0.55), homozygote TT (0.18) as presented in
Table 2. The data showed that heterozygote CT genotype is the most common
genotype with frequency of 55% of the total CYP4F2rs2108622 genotypes among
these volunteers of Jordanian. As it shown on Table 2, all of CYP4F2rs2108622
genotype frequencies were within Hardy-Weinberg equation.
Table 2. The CYP4F2rs2108622 genotype frequency among healthy Jordanian
population
†test2XPredicted number
(frequency)
Observed number
(frequency)
Genotype
>0.05
27 (0.30)24 (0.27)Wild (C / C)
45 (0.50)50 (0.55)Heterozygote (C / T)
19 (0.20)16 (0.18)Homozygote (T / T)
90 (1)Total
Weinberg-All of CYP4F2rs2108622 genotype frequencies were within Hardy†
equation
In comparison with other ethnic populations, the current study found that the
frequency of CYP4F2rs2108622C > T was similar to white Caucasians and slightly

9. higher than Middle Eastern Kuwaiti and Turkish populations, while it was higher than
African ethnic group using z-test, p value <0.05 (Table 3).
Table 3. The comparison between CYP4F2rs2108622 allele frequencies among
healthy Jordanians with other ethnic populations
ReferenceIs there statistical
difference
in comparison with
Jordanian
population
Different than
2
Jordanian (X
test)
CYP4F2rs2108622
T
Frequency
CYP4F2rs2108622
C
frequency
Ethnic group
This study->0.050.450.55Jordanian
(1)No> 0.05*0.4470.553Caucasian
(1)Yes<0.05*0.0700.93African
(1)Yes<0.05*0.3570.643Asian
(16)Yes<0.050.4**0.6Kuwait
(15)Yes<0.050.397***0.603Turkish
((* (1), **(17),***(16) ))
Discussion
CYP4F2 plays a role in warfarin response (18). However the frequency was reported
in many ethnic populations, the frequency of CYP4F2rs2108622 genetic variant had
not been investigated before in Jordanian population. In this study, we showed that
Jordanian population has high frequency of this genetic variant (45%), Which may
affect significantly on warfarin response and susceptibility to cardiovascular diseases
among Jordanian.
Because CYP4F2 is the major cytochrome responsible for synthesis of the
vasoconstrictor and platelet activator 20-hydroxyeicosatetraenoic acid (20-HETE),
genetic variants on CYP4F2 gene may affect on 20-HETE production. The rs2108622
polymorphism in CYP4F2 gene affected enzyme activity and decreased 20-HETE

10. production (19). Therefore, rs2108622 variant was associated with cardiovascular
diseases (19).
The cardiovascular diseases are major chronic dieses in Jordan and are considered
one of the most common causes of death in this country by approximately 35% of
total deaths (20). For instance, ischemic heart disease and strokes occupy the first
place among diseases causing death in Jordan by 18% (21), 11.95 (22) of total death,
respectively. As the frequeny of rs2108622 variant is high among Jordanian
population, it is suggested to investigate the association of rs2108622 variant with
Jordanian cardiovascular patients.
Warfarin , a vitamin k antagonist (23), is the most frequently prescribed anticoagulant
with large inter-individual variability in dosage (18). The warfarin dosing is required
to achieve therapeutic efficacy and minim
ize drug toxicity to avoid serious complications associated with bleeding or
thromboembolism (23). The CYP4F2 is the vitamin K oxidase involved in the
metabolism of vitamin K (19). Patients with CYP4F2rs2108622 T/T genotype
required approximately 1 mg/day more warfarin than patients with 2 C/C genotype
(19)
Conclusion
This study reported a high frequency of CYP4F2rs2108622 polymorphism among
Jordanians which may contribute to the inter-individual variation in warfarin response
and susceptibility to cardiovascular diseases.

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decreases 20-HETE production. Physiological Genomics. 2007;30(1):74-81.
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1.Alvarellos ML, Sangkuhl K, Daneshjou R, Whirl-Carrillo M, Altman RB, Klein TE.
PharmGKB summary: very important pharmacogene information for CYP4F2.
Pharmacogenetics and genomics. 2015;25(1):41-7.
2.StecDE, RomanRJ, FlaschA,RiederMJ. Functional polymorphism in human CYP4F2
decreases 20-HETE production. Physiological Genomics. 2007;30(1):74-81.
3.Lasker JM, Chen WB, Wolf I, Bloswick BP, Wilson PD, Powell PK. Formation of 20-
Hydroxyeicosatetraenoic Acid, a Vasoactive and Natriuretic Eicosanoid, in Human Kidney:
ROLE OF CYP4F2 AND CYP4A11. Journal of Biological Chemistry. 2000;275(6):4118-26.
4.Hardwick JP. Cytochrome P450 omega hydroxylase (CYP4) function in fatty acid
metabolism and metabolic diseases. Biochem Pharmacol. 2008;75(12):2263-75.
5.Edson KZ, Prasad B, Unadkat JD, Suhara Y, Okano T, Guengerich FP, et al.
Cytochrome P450-DependentCatabolismof VitaminK:ω-HydroxylationCatalyzedbyHuman
CYP4F2 and CYP4F11. Biochemistry. 2013;52(46):8276-85.
6.Munshi A, SharmaV, Kaul S,Al-Hazzani A,Alshatwi AA,Shafi G, et al. Association of
1347 G/A cytochrome P450 4F2 (CYP4F2) gene variant with hypertension and stroke. Mol
Biol Rep. 2012;39(2):1677-82.
7.Hirani V,YarovoyA,KozeskaA,MagnussonRP,LaskerJM. EXPRESSION OFCYP4F2 IN
HUMAN LIVER AND KIDNEY: ASSESSMENT USING TARGETED PEPTIDE ANTIBODIES. Archives
of biochemistry and biophysics. 2008;478(1):59-68.
8.Jarrar YB, Shin JG, Lee SJ. Expression of arachidonic acid-metabolizing cytochrome
P450s inhuman megakaryocyticDami cells. In Vitro Cell Dev Biol Anim. 2013;49(7):492-500.
9.gen n. CYP4F2 cytochrome P450 family 4 subfamily F member 2 [ Homo sapiens
(human) ]
Available from: https://ghr.nlm.nih.gov/gene/CYP4F2
http://www.ncbi.nlm.nih.gov/gene/8529.

13. 10.KikutaY, Miyauchi Y, Kusunose E,Kusunose M.Expressionand molecular cloning of
human liver leukotriene B4 omega-hydroxylase (CYP4F2) gene. DNA Cell Biol.
1999;18(9):723-30.
11.Meng C, Wang J, Ge W-N, Tang S-C, Xu G-M. Correlation between CYP4F2 gene
rs2108622 polymorphism and susceptibility to ischemic stroke. International Journal of
Clinical and Experimental Medicine. 2015;8(9):16122-6.
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Warfarin. cpmc; Available from: https://cpmc.coriell.org/about-the-cpmc-study/health-
conditions-and-drug-response/warfarin-coumadin/risk-factors.
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Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant.
Molecular Pharmacology. 2009;75(6):1337-46.
14.ShahinMHA, KhalifaSI,GongY, Hammad LN,Sallam MTH, Shafey ME, et al. Genetic
and nongenetic factors associated with warfarin dose requirements in Egyptian patients.
Pharmacogenetics and genomics. 2011;21(3):130-5.
15.Primer3. Available from: Primer3
16.Ozer M, Demirci Y, Hizel C, Sarikaya S, Karalti I, Kaspar C, et al. Impact of genetic
factors (CYP2C9, VKORC1 and CYP4F2) on warfarin dose requirement in the Turkish
population. Basic Clin Pharmacol Toxicol. 2013;112(3):209-14.
17.Alsmadi O, John SE, Thareja G, Hebbar P, Antony D, Behbehani K, et al. Genome at
Juncture of Early Human Migration: A Systematic Analysis of Two Whole Genomes and
Thirteen Exomes from Kuwaiti Population Subgroup of Inferred Saudi Arabian Tribe
Ancestry. PLoS ONE. 2014;9(6):e99069.
18.Li S, Zou Y, Wang X, Huang X, Sun Y, Wang Y, et al. Warfarin Dosage Response
Related Pharmacogenetics in Chinese Population. PLoS ONE. 2015;10(1):e0116463.
19.Caldwell MD, Awad T, Johnson JA, Gage BF, Falkowski M, Gardina P, et al. CYP4F2
genetic variant alters required warfarin dose. Blood. 2008;111(8):4106-12.
20.Organization WH.Noncommunicable Diseases(NCD) CountryProfiles World Health
Organization
http://www.who.int/nmh/countries/jor_en.pdf2014; Available from:.
21.CDC. CDC in Jordan. 2013; Available from:
http://www.cdc.gov/globalhealth/countries/jordan/pdf/jordan.pdf.
22.WHO. WORLDHEALTHRANKINGS. 2014; Available from:
disease-heart-coronary-http://www.worldlifeexpectancy.com/jordan.
23.BakerWL, JohnsonSG.Pharmacogeneticsand oral antithrombotic drugs. Curr Opin
Pharmacol. 2016;27:38-42.