VKORC1 Asp36Tyr geographic distribution and its ...

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Americans. Egyptian carriers of the VKORC1 Tyr36 showed higher war- farin dose requirement (57.1 ± 29.4 mg/week) than those with the. Asp36Asp genotype ...
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Blood Coagulation, Fibrinolysis and Cellular Haemostasis

VKORC1 Asp36Tyr geographic distribution and its impact on warfarin dose requirements in Egyptians Mohamed Hossam A. Shahin1; Larisa H. Cavallari2; Minoli A. Perera3; Sherief I. Khalifa4; Anne Misher5; Taimour Langaee1; Shitalben Patel2; Kimberly Perry3; David O. Meltzer3; Howard L. McLeod5; Julie A. Johnson1 1Department

of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, Florida, USA; 2Department of Pharmacy Practice, University of Illinois at Chicago, Chicago, Illinois, USA; 3Department of Medicine, University of Chicago, Chicago, Illinois, USA; 4Pharmaceutical Sciences Section, College of Pharmacy, Qatar University, Doha, Qatar; 5Univeristy of North Carolina Institute for Pharmacogenomics and Individualized Therapy, Chapel Hill, North Carolina, USA

Summary The VKORC1 Asp36Tyr single nucleotide polymorphism (SNP) is one of the most promising predictors of high warfarin dose, but data on its population prevalence is incomplete. We determined the frequency of this SNP in participants from seven countries on four continents and investigated its effect on warfarin dose requirement. One thousand samples were analysed to define the population prevalence of this SNP. Those samples included individuals from Egypt, Ghana, Sudan, Kenya, Saudi Arabia, Peru and African Americans from the United States. A total of 206 Egyptian samples were then used to investigate the effect of this SNP on warfarin dose requirements. This SNP was most frequent among Kenyans and Sudanese, with a minor allele frequency (MAF) of 6% followed by Saudi Arabians and Egyptians with a MAF of 3% and 2.5%, respectively. It was not detected in West Africans, based on our data from Ghana, and a large cohort of African Correspondence to: Julie A. Johnson, PharmD Center for Pharmacogenomics, University of Florida Health Science Center, PO Box 100486 Gainesville, FL 32610, USA Tel.: +1 352 273 6007, Fax: +1 352 273 6121 E-mail: [email protected]

Americans. Egyptian carriers of the VKORC1 Tyr36 showed higher warfarin dose requirement (57.1 ± 29.4 mg/week) than those with the Asp36Asp genotype (35.8 ± 16.6 mg/week; p=0.03). In linear regression analysis, this SNP had the greatest effect size among the genetic factors (16.6 mg/week increase in dose per allele), and improved the warfarin dose variability explained in Egyptians (model R2 from 31% to 36.5%). The warfarin resistant VKORC1 Asp36Tyr appears to be confined to north-eastern Africa and nearby Middle-Eastern populations, but in those populations where it is present, it has a significant influence on warfarin dose requirement and the percent of warfarin dose variability that can be explained.

Keywords Warfarin, pharmacogenetics, VKORC1 Asp36Tyr, polymorphism, Egyptians Financial support: This study was supported by U01 GM074492 NIH grant (J.A.J.), University of Illinois at Chicago College of Pharmacy Hans Vahlteich Research Award (L.H.C.); K23 HL089808–01A2 NIH grant (M.P.). Received: October 30, 2012 Accepted after major revision: March 3, 2013 Prepublished online: March 21, 2013 doi:10.1160/TH12-10-0789 Thromb Haemost 2013; 109: 1045–1050

Introduction Warfarin was first approved for clinical use in 1954, and since then, it has been the mainstay oral anticoagulant therapy for treatment and prevention of thromboembolic events. However, its narrow therapeutic index and wide inter-patient variability in dose requirements for a therapeutic warfarin effect make it an extremely challenging pharmacological agent to utilise clinically (1-3). Numerous pharmacogenetic studies have been conducted with warfarin, and have shown that warfarin dose requirements, risk of bleeding and time to reach a stable warfarin dose are influenced by demographic factors (age, gender and ethnicity), clinical factors (smoking, concurrent medications, illness and diet) and genetic variables (4-7). In 2007, and again in 2010, the Food and Drug administration (FDA) updated the warfarin label with information on warfarin pharmacogenetics. The 2010 update included a dosing © Schattauer 2013

guidance based on genetic factors, specifically CYP2C9 and VKORC1 polymorphisms, which are strongly associated with warfarin dose requirements, with the variant alleles leading to lower warfarin dose (1, 8-11). The inclusion of the CYP2C9 and VKORC1 warfarin sensitivity polymorphisms with clinical factors explain more than 50% of the warfarin dose variability in those of European ancestry; however, less variability was explained in other ethnic populations (1, 9, 12, 13). Thus, it is important to identify other genetic or clinical factors that may help improve the prediction of warfarin dose requirements in non-Europeans. It is also clear that even in whites, there is a substantial portion of the variability yet to be explained, and it is important to note that most of the genetic factors identified to date help to explain requirements for a low dose of warfarin; the genetic underpinnings for high warfarin dose requirements, or warfarin resistance, are poorly understood. Thrombosis and Haemostasis 109.6/2013

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Shahin et al. VKORC1 Asp36Tyr distribution and promise for a better warfarin dose prediction

The one variant that has been most strongly associated with high warfarin dose requirements is the VKORC1 coding Asp36Tyr (D36Y; rs61742245) variant. This variant appears to exhibit large differences in population prevalence. For example, it is relatively common in Ethiopians with minor allele frequency (MAF) of 15%, and Ashkenazi Jews (MAF 4%), less common in Israeli Jews (MAF 1.5%) and Arab Muslims in Israel (MAF 1%), and has a MAF of 0.5% in Sephardic, Yemenite, and North African Jews (10, 14-18). On the other hand, it was absent in over 700 non-Jewish Caucasian controls, 180 Israelis of Druze descent, 220 Han Chinese, 240 Southeast Indians and 213 South African individuals (17, 19-22). The primary objective of this study was to better define the population frequencies of this variant, through testing of populations in seven countries on four continents, including five African and Middle Eastern countries, the United States (African Americans), and Peru. We also investigated the effect of VKORC1 Asp36Tyr polymorphism on warfarin dose requirements in Egyptians.

Methods Study population A total of 1,000 samples were included in the analysis to define population prevalence. Those samples included individuals from Egypt, Ghana, Sudan, Kenya, Saudi Arabia, Peru and African Americans from the United States, as shown in ▶ Table 1. All participants provided informed consent, and the study protocol was approved by relevant local Institutional Review Boards. A total of 207 Egyptian patients were enrolled while taking chronic warfarin therapy (Marevan®; GlaxoSmithKline, Cairo, Egypt) for various indications as previously described (23). Eligible patients were those who were taking stable weekly doses of warfarin for three consecutive clinic visits, occurring over a minimum time period of two months. A stable weekly maintenance Table 1: VKORC1 Asp36Tyr genotype prevalence in the seven studied populations.

Population

Na

Asp36Tyr genotypes Tyr/Tyr Asp/Tyr Asp/Asp

Tyr36allele frequency, %

Kenya

41

1

3

37

6

Sudan

43

0

5

38

6

Saudi Arabia

85

0

5

80

3

Egypt

206

0

10

196

2.5

Ghana

85

0

0

85

A) warfarin sensitivity polymorphisms along with clinical factors explain more than 50% of the warfarin dose variability in those of European ancestry; however, less variability is explained in other ethnic populations. Most of the genetic factors identified to date help to explain requirements for a low dose of warfarin, while the identification of predictors significantly associated with higher warfarin dose are limited. VKORC1 also contains a SNP associated with high warfarin dose, Asp36Tyr, but data on its population prevalence is incomplete. Little is known about the percent of warfarin dose variability explained by the presence of this variant in African or Mediterranean populations.

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The warfarin resistant VKORC1 Asp36Tyr appears to be confined to populations from north-eastern Africa and nearby Middle-Eastern populations. It appears to be absent or very rare in West Africans, based on our data from Ghana, and a large cohort of African Americans. Egyptian carriers of the VKORC1 Tyr36 showed higher warfarin dose requirement (57.1 ± 29.4 mg/week) than those with the Asp36Asp genotype (35.8 ± 16.6 mg/week; p=0.03). The addition of this variant to our linear regression model improved the warfarin dose variability explained in Egyptians (model R2 increased from 31% to 36.5% with addition of Asp36Tyr). Also, it had the largest effect size among the significantly associated genetic variants in our model, resulting in a 16.6 mg/week higher warfarin dose per allele. The VKORC1 Asp36Tyr is of great importance in those populations where it is present, since it has a significant influence on warfarin dose requirement, but other factors that lead to high warfarin dose requirement remain to be defined.

© Schattauer 2013

Table 2: VKORC1 -1639G>A, and CYP2C9*2*3*4*5 and *8 genotypes distribution among the 10 VKORC1 Asp36Tyr variant carriers in the Egyptian cohort.

Sample VKORC1 VKORC1 CYP2C9# Weekly warfarin number Asp36Tyr -1639G>A# dose (mg/week) 1

Asp/Tyr

WT§

WT

98

2

Asp/Tyr

WT

WT

87.5

3

Asp/Tyr

G/A

WT

84

4

Asp/Tyr

WT

WT

77

5

Asp/Tyr

G/A

WT

70

6

Asp/Tyr

WT

WT

45.5

7

Asp/Tyr

G/A

WT

38.5

8

Asp/Tyr

G/A

WT

24.5

9

Asp/Tyr

G/A

WT

24.5

10

Asp/Tyr

G/A

*3/*3

21

#VKORC1

(-1639G>A, rs9923231), CYP2C9 tested for *2, *3, *4, *5, and *8 which were previously published (23). §WT refers to a wild-type genotype (G/G for VKORC1 and *1/*1 for CYP2C9).

Table 3: Linear regression stepwise modelling association between mean weekly warfarin dose (square root transformed) as a dependent variable and genetic and non-genetic factors as independent variables.

Predictor

Coefficient Standard Partial R2 Error (%)

Intercept

8.19

0.319

VKORC1# variant

-0.62

0.131

11.5