Weight and the vitamin K expoxide reductase 1 genotype primarily contribute to the warfarin dosing in pediatric patients with Kawasaki disease

Accepted Manuscript Weight and the vitamin K expoxide reductase 1 genotype primarily contribute to the warfarin dosing in pediatric patients with Kawasaki disease

Zhouping Wang, Li Zhang, Ping Huang, Xiaoqiong Gu, Xiaofei Xie, Yanfei Wang, Wei Li, Qiyi Zeng PII: DOI: Reference:

S0049-3848(18)30337-2 doi:10.1016/j.thromres.2018.05.002 TR 7023

To appear in:

Thrombosis Research

Received date: Revised date: Accepted date:

16 February 2018 30 April 2018 4 May 2018

Please cite this article as: Zhouping Wang, Li Zhang, Ping Huang, Xiaoqiong Gu, Xiaofei Xie, Yanfei Wang, Wei Li, Qiyi Zeng , Weight and the vitamin K expoxide reductase 1 genotype primarily contribute to the warfarin dosing in pediatric patients with Kawasaki disease. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tr(2017), doi:10.1016/j.thromres.2018.05.002

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ACCEPTED MANUSCRIPT Weight and the vitamin K expoxide reductase 1 genotype primarily contribute to the warfarin

dosing in pediatric patients with Kawasaki disease

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Zhouping Wang , Li Zhang , Ping Huang , Xiaoqiong Gu , Xiaofei Xie , Yanfei Wang , Wei Li , Qiyi b

Department of Pediatric Cardiology, the Second Medical College of Southern Medical University,

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Zeng *

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Affiliated Guangzhou Women and Children’s Medical Center of Guangzhou Medical University,

Guangzhou, Guangdong Province, China

Pediatric Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province,

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China

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*Corresponding author

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Qiyi Zeng

Pediatric Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province,

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China

Email address: [email protected]

Word count

3800 words

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Abstract

Introduction: Warfarin therapy is recommended in children with giant coronary artery aneurysms (GCAAs)

after Kawasaki disease (KD). Large individual variability makes it difficult to predict the warfarin dose.

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Polymorphisms in the vitamin K expoxide reductase 1 (VKORC1) and cytochrome P4502C9 (CYP2C9)

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genes have been reported to influence the warfarin dose. We investigated the effects of the VKORC1

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and CYP2C9 genotypes on the warfarin dose in pediatric patients with giant CAAs after KD. We

attempted to create a dosing algorithm.

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Materials and Methods: The clinical and genetic data of patients were documented. VKORC1 (rs

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9923231) and CYP2C9 *3 (rs 1057910) were genotyped using TaqMan real-time polymerase chain

reaction. A linear regression analysis was performed to evaluate the contribution of clinical and genetic

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factors to the warfarin maintenance dose.

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Results: Forty-seven patients were enrolled. Patients with the CT or CC genotype of VKORC1 had a

relatively higher warfarin dose than did those with the TT genotype (p<0.05). Three patients with

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CYP2C9*1/*3 had a lower warfarin dose than did those with the wild CYP2C9*1/*1 genotype, but the

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difference did not reach significance (p>0.05). Weight and the VKORC1 genotype predominantly

contributed to the warfarin dose, with 33.0% and 11.2% of variability, respectively. The observed warfarin

dose was correlated with the predicted dose based on the algorithm used in our study (r=0.45, p<0.01).

Conclusions: Weight and the VKORC1 genotype primarily determined the warfarin dose in Chinese

pediatric patients with KD. Further studies are warranted to verify the findings of our study.

Keywords: warfarin, vitamin K expoxide reductase 1, cytochrome P4502C9, Kawasaki disease

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Abbreviations

KD: Kawasaki disease; CAA: coronary artery aneurysm; GCAA: giant coronary artery aneurysm; INR:

international normalized ratio; SNP: single nucleotide polymorphism; VKORC1: vitamin K expoxide

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reductase; CYP2C9: cytochrome P4502C9; BSA: body surface area; OR: odds ratio; SD: standard

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deviation

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Introduction

Kawasaki disease (KD) is a systemic form of vasculitis and the predominant cause of acquired heart

disease in children [1]. Although intravenously administered immunoglobulin has reduced the incidence

of coronary artery aneurysms (CAAs), giant coronary artery aneurysms (GCAAs) develop in

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approximately 1% of children with KD [1]. GCAAs are associated with high morbidity and mortality when

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accompanied by coronary artery events including thrombosis, stenosis, occlusion, and myocardial

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infarction [2]. In addition to antiplatelet therapy, anticoagulant therapy such as warfarin is recommended

in patients with GCAAs [1, 3].

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Warfarin is a frequently used oral vitamin K antagonist. It is applied to prevent thromboembolic events in

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patients with cardiac valve replacement, CAAs after KD, deep thrombosis, and other diseases. Warfarin

has a narrow therapeutic index that is adjusted according to the same narrow target international

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normalized ratio (INR), and inter- and intra-individual variability make it difficult to precisely predict the

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necessary warfarin dose [4]. Determining the appropriate warfarin dose is important, because over- or

under-anticoagulation will lead to severe complications such as bleeding or thromboembolism.

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Genetic variations and demographic characteristics have been reported to contribute to the variability of

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the warfarin dose. Single nucleotide polymorphisms (SNPs), which are located in vitamin K expoxide

reductase (VKORC1) [5-7] and cytochrome P4502C9 (CYP2C9) [8-9], have been identified as

predominant genetic variations that influence the dose of warfarin. VKORC1 and CYP2C9 have been

proven to contribute to the metabolism of warfarin and interfere with the activation of clotting factors [10].

In adults, two SNPs, VKORC1 (1173C>T, rs 9923231) and CYP2C9 (*2/*3), accounted for 30%-40%

[11-13] of variations in the dose, resulting in the establishment of genetically guided dosing algorithms [12,

14]. However, the ideal warfarin dose in pediatric patients is still empiric. Only a few studies have

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investigated the influence of VKORC1, CYP2C9, and demographic factors such as age, weight, height,

and body surface area (BSA) on the dose of warfarin in children [4-5, 15-16]. In contrast to adults, in

pediatric patients, demographic factors have a greater effect on the dose of warfarin than genetic

variations do.

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The purpose of this study was to evaluate the effects of the VKORC1 and CYP2C9 genotypes on the

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dose of warfarin in pediatric patients with GCAAs after KD and, if possible, to create a dosing algorithm.

Materials and Methods

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Ethical considerations

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The present study was conducted in accordance with the ethical principles of the Declaration of Helsinki

and approved by our local ethics committee. Written informed consent was obtained from the

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participants’ legal guardians.

Study design and participants

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In this retrospective study, we investigated patients with GCAAs after KD who were seen at the

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cardiology department of Guangzhou Women and Children’s Medical Center, China, from April 2014 to May 2017. A GCAA were defined as an aneurysm with an absolute dimension ≥ 8 mm or a z score > 10.

We included participants who were 16 years or younger who received warfarin for at least 3 months and

had a stable dose that was within the INR (1.5-2.5). The criterion for a stable maintenance dose of

warfarin was no change in the dose’s target INR for at least three consecutive over a period of 1 week.

The demographic data, including sex, age, weight, height, BSA, target INR, warfarin dose (mg/kg),

concomitant treatment, and bleeding or thrombosis complications, were collected when a stable warfarin

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dose was achieved.

Genotyping

DNA was extracted from blood samples using the QI Aamp DNA Blood Mini Kit (QIAGE,USA) according

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to the manufacturer’s protocols and then stored at -20℃ for analysis. VKORC1 (rs 9923231) and

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CYP2C9 (rs 1057910) were genotyped using TaqMan real-time polymerase chain reaction (Applied

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Biosystems, ABI,USA).

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Statistical analyses

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Quantitative parameters are presented as a mean ± standard deviation, and qualitative values are shown as numbers and percentiles. The Mann-Whitney’s U test was used to compare the differences between

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the genotypes. Fisher’s exact test was used to compare ratios between the groups. An initial univariate

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linear regression analysis was performed to evaluate the relationship between a stable warfarin

maintenance dose (mg/kg.d) and covariates including genetic polymorphisms and demographic data.

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Covariates with a p-value <0.2 in the univariate regression analysis were entered into a final multiple

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regression analysis to develop a novel algorithm. A stepwise method was chosen to avoid

multicollinearity. Pearson’s correlation coefficient (r) was used to test the relationship between the

observed warfarin dose and predicted dose based on the algorithm that was created. All of the tests were

two-sided, and p-values <0.05 were considered statistically significant. All statistical analyses were

performed using SPSS software version 17.0.

Results

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We enrolled 47 patients in south China; most were male. They were all diagnosed with GCAAs after KD

and received warfarin to prevent thrombosis and ischemia, in accordance with a 2014 guideline [3]. All of

these patients took aspirin concomitantly with warfarin. Two received beta blockers and captopril. The

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detailed demographics and genetic polymorphisms of the patients are demonstrated in Table 1.

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Table 1. Demographic and genetic characteristics of the study patients

48.46±42.01

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Age, mean±SD (months)

37/10

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Sex, M/F

Weight, mean±SD (kg)

17.23±10.69

94.02±29.23

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Height, mean±SD (cm)

Target INR

0.67±0.35

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BSA, mean±SD (m )

1.89±0.34

0.108±0.032

Time to a stable maintenance dose

13.80±12.50

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Warfarin dose, mean±SD ( mg/kg.d)

VKORC1 genotype

N (%)

TT

37 (78.72)

CT

9 (19.15)

CC

1 (2.13)

CYP2C9

N (%)

*1/*1

44 (93.62) 7

Concomitant treatment

N (%)

Aspirin

47 (100)

Beta blockers

2 (4.25)

Captopril

2 (4.25)

Adverse events* N (%)

7 (14.89)

Dermatorrhagia

5 (10.63)

Epistaxis

3 (4.26)

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3 (6.38)

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*1/*3

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*One patient suffered from both dermatorrhagia and epistaxis

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SD: standard deviation; INR: international normalized ratio; VKORC1: vitamin K expoxide reductase 1,

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Comparison between genotypes

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CYP2C9: cytochrome P4502C9

Patients with the CT or CC genotype of VKORC1 (rs 9923231) had a relatively higher warfarin dose than

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did those with the TT genotype (0.130±0.033 vs. 0.103±0.030 mg/kg.d, p<0.05) (Fig. 1a). Only three

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patients carried the allele of CYP2C9*3 (rs 1057910), and they had a lower warfarin dose than did those

with the wild CYP2C9*1/*1 genotype (0.127±0.039 vs. 0.107±0.032 mg/kg.d, p>0.05) (Fig. 1b). Patients

combined with VKORC1 CC/CT and CYP2C9*1/*1 genotype had higher warfarin dose than did those

with the VKORC1 TT and CYP2C9*1/*1 genotype (0.127±0.336 vs. 0.104±0.292 mg/kg.d, p<0.05). No

significant difference was found among other groups (Fig. 1c).

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Fig. 1. Difference in the warfarin maintenance dose between genotypes. a. The VKORC1 genotype, TT

type vs. the CT/CC type; b. The CYP2C9 genotype, *1/*1 vs. *1/*3; c. The distribution of warfarin dose by

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combined VKORC1 and CYP2C9 genotype. The numbers above the whiskers indicate mean values.

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VKORC1: vitamin K expoxide reductase 1; CYP2C9: cytochrome P4502C9

Patients with the VKOCR1 TT genotype reached the target INR earlier than those with the CT or CC

genotype did (10.38±8.88 vs. 27.89±15.70 day, p<0.01) (Fig. 2a). In patients with CYP2C9*1/*1 and *1/*1,

the target INR was reached at a similar time (13.23±12.47 vs. 25.67±2.08 day, p>0.01) (Fig. 2b).

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the CT/CC type; b. The CYP2C9 genotype, *1/*1 vs. *1/*3

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Fig. 2. Difference in time to the target INR between genotypes. a. The VKORC1 genotype, TT type vs.

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VKORC1: vitamin K expoxide reductase 1; CYP2C9: cytochrome P4502C9

Contribution of clinical and genetic variables to the warfarin dose

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In the univariate regression analysis, the warfarin dose was significantly associated with age, weight,

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height, BSA, and the VKORC1 genotype. The INR for those with the CYP2C9 genotype did not reach

statistical significance. Candidate variables were entered into the final multiple regression analysis.

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Weight and the VKORC1 genotype accounted for 44.2% of the total variation in the final model. Weight

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had a greater influence on the warfarin dose than did the VKORC1 genotype (33.0% and 11.2%,

respectively). Weight was negatively correlated with the warfarin dose (r=-0.58, p<0.01) (Fig. 3)

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Fig. 3. The relationship between weight and the daily warfarin maintenance dose

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The prediction algorithm of warfarin dose for south Chinese children was as follows:

Daily warfarin dose (mg/kg) = 0.133 - 0.002*weight (kg) + 0.026*VKORC1

CC/CT type for 1;

TT type for 0

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VKORC1:

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Fig. 4 shows that the observed warfarin dose was correlated with the predicted dose, based on the

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algorithm that was used in the current study (r=0.45, p=0.002).

Fig. 4. The relationship between the observed and predicted warfarin doses 11

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Complications

Four patients experienced spontaneous petechiae and one had traumatic purpura. Three patients had

spontaneous epistaxis. All the bleeding events were self-limited. None of them suffered from

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life-threatening bleeding events. There was no correlation between genotypes and bleeding events

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(VKORC1: odds ratio [OR]=1.32, p>0.05; CYP2C9: OR=1.18, p>0.05). Twenty-five patients developed a

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coronary artery thrombus before the warfarin therapy. Twelve of them had partial regression of the

coronary artery thrombus, and the thrombus did not change during the follow-up period in 10 patients.

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Coronary artery stenosis or obstruction occurred in three patients.

Discussion

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In the current research study, the effects of clinical and genetic factors on the dose of warfarin were

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assessed in a group of Chinese pediatric patients for the first time. The results indicated that weight, age,

height, BSA, and the VKORC1 genotype were significantly associated with the dose of warfarin, and

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weight and the VKORC1 genotype principally determined the warfarin dose. Finally, we identified a novel

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algorithm for the warfarin dose in patients with KD.

In our study, weight was the predominant contributor to variations in the warfarin dose (33.0% of the total

variability). Some previous studies demonstrated that clinical variables contribute to the warfarin dose in

children, ranging from 26% [17] to 48% of these patients [15]. Most of these studies reported that age primarily determined variations in the warfarin dose [4, 16-17], whereas Shaw et al.’s findings [9] and

ours indicated that weight was the most important contributor. All evidence showed that the warfarin dose

was negatively correlated with clinical variables, as confirmed in our study. In contrast, clinical variables

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were attributable to less than 20% of the dose variability in adults [11-12, 18]. Additionally, other reports

indicated that the maintenance dose of warfarin was negatively correlated with weight or age. Takahashi

et al. found that the body weight-normalized unbound oral clearance of warfarin for children was

significantly greater than that for the adults[19]. The vitamin K-dependent coagulation factors were

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decreased were decreased during infancy and childhood [20]. These findings may partly explain the

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reason why children need more warfarin than adults do.

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We confirmed that the VKORC1 genotype was the major genetic predictor of the warfarin dose,

accounting for 11.1% of the total variability. In previous pediatric studies, VKORC1 polymorphisms were

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associated with variations in the dose of 3.7% [17] to 47% [15], less than the findings that were reported

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in adults [12, 18]. Individuals with the CT or CC genotype require a higher maintenance dose than those

with the TT genotype do, as described before. Our findings showed that patients with the VKOCR1 TT

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genotype reached the target INR earlier than did those with the CT or CC genotype. Adult patients with

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the VKOCR1 TT genotype had a decreased time to the target INR within the therapeutic range [6, 21]. Shaw et al.’s results in pediatric patients were similar to ours [9]. However, other studies reported that the

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VKOCR1 did not influence the time to the target INR [17, 22]. This suggests that a further investigation of

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pediatric patients is needed.

Three patients with the CYP2C9*1/*3 genotype required a lower warfarin dose than did those with the

wild CYP2C9*1/*1 genotype. Due to the rare number of cases, the CYP2C9 polymorphism was not

included in the final regression model in this study. The CYP2C9 genotype has been reported to account

for 2% [15] to 12.8% [16] of the dose variability in children. Although the contribution of the CYP2C9

genotype was less than that of the VKORC1 genotype, the difference of the dose was more obvious

among patients with different CYP2C9 alleles. Patients with the CYP2C9*1/*1 genotype required a

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six--fold greater dose than did those with the CYP2C9*3/*3 genotype [4]. Additionally, patients with the

VKORC1 CC genotype required a two-fold greater dose than did those with the VKORC1 CC genotype.

The CYP2C9*2 and CYP2C9*3 alleles are absent or rare in Asian people; therefore, the effects of

CYP2C9 polymorphisms on the warfarin dose are hard to identify in Asian people. There was no

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difference in the time to the target INR between CYP2C9 polymorphisms. This agrees with the findings of

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previous studies [17-18].

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The International Warfarin Pharmacogenetics Consortium recommended a warfarin dose algorithm in

adults that overestimated the warfarin dose in children. Overestimation of the warfarin dose could be

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explained by a greater contribution of age to variability in children than in adults. Several warfarin dose

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algorithms were proposed for children, all of which showed that there was a beneficial correlation

between the predicted and observed doses [4, 9, 15, 23]. However, the algorithms were diverse and their

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use in children is still controversial. In addition to developmental variables and genetic polymorphisms,

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the indications of medicine often influence the warfarin dose. Streif et al. [24] indicated that patients who

were administered fontan required a 25% lower dose than did other patients with the same target INR.

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Shaw et al. [9] suggested that the effect of VKOCR1 was distinct among those with different indications.

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In our patients, a coronary aneurysm that occurred after KD was the only indication for treatment.

However, coronary aneurysms only occurred in a small proportion of patients in previous studies. We

created an algorithm to determine the warfarin dose in patients with coronary aneurysms after KD. This

may allow us to diversify algorithms, to some extent.

Our study has some limitations. First, the small sample size prevented us from evaluating the

contribution of CYP2C9 to the warfarin dose. Second, we only investigated patients with coronary

aneurysms, which limited the application of the warfarin dose algorithm in patients with other diseases.

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Furthermore, CYP2F4, GGCX, and EPHX1 [21, 23], which were reported to influence the warfarin dose,

were not incorporated into the analysis.

To our knowledge, this is the first report that assessed the contribution of clinical and genetic factors to

the warfarin dose in children with KD. Our study provides evidence that weight and the VKOCR1

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genotype significantly influence the warfarin dose in children. An algorithm that is based on weight and

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the VKORC1 genotype may be useful to accurately predict the necessary dose. The effectiveness of the

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algorithm must be verified in people of other ethnicities with varied indications. In the future, we will

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employ the algorithm in a prospective study.

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Declarations of interest: none

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Authors' contributions: Zhouping Wang，Li Zhang and Qiyi Zeng contributed to the concept and design

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of the study. Ping Huang, Xiaoqiong Gu and Xiaofei Xie collected the clinical data. Yanfei Wang and Wei

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Li performed statistical analyses. Zhouping Wang wrote the first draft of the manuscript.

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Funding: The work was supported by the Traditional Chinese Medical Bureau of Guangdong province

[grant number 20171204].

Acknowledgements

we thank Xiaoyi Cai for her help in field of statistic analysis and literary

improvement.

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[21] E. Pautas, C. Moreau, I. Gouin-Thibault, J.L. Golmard, I. Mahe, C. Legendre, E. Taillandier-Heriche, B. Durand-Gasselin, A.M. Houllier, P. Verrier, P. Beaune, M.A. Loriot, V. Siguret, Genetic factors (VKORC1, CYP2C9, EPHX1, and CYP4F2) are predictor variables for warfarin response in very elderly, frail inpatients, Clin Pharmacol Ther 87(1) (2010) 57-64. [22] T.T. Biss, P.J. Avery, M.D. Williams, L.R. Brandao, J.D. Grainger, F. Kamali, The VKORC1 and CYP2C9 genotypes are associated with over-anticoagulation during initiation of warfarin therapy in children, J Thromb Haemost 11(2) (2013) 373-5. 18

ACCEPTED MANUSCRIPT [23] T. Wakamiya, T. Hokosaki, S. Tsujimoto, K. Kadota, Y. Nakano, S. Watanabe, M. Iwamoto, M. Yanagimachi, S. Ito, Effect of VKORC1, CYP2C9, CFP4F2, and GGCX Gene Polymorphisms on Warfarin Dose in Japanese Pediatric Patients, Mol Diagn Ther 20(4) (2016) 393-400. [24] W. Streif, M. Andrew, V. Marzinotto, P. Massicotte, A.K. Chan, J.A. Julian, L. Mitchell, Analysis of

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warfarin therapy in pediatric patients: A prospective cohort study of 319 patients, Blood 94(9) (1999)

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Conflict of interest There are no conflicts of interest to declare. thank you!

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ACCEPTED MANUSCRIPT Highlights

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We investigated the effects of VKOaRC1 and CYP2C9 on warfarin dosing in children We identified a novel warfarin dosing algorithm for patients with KD

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Weight and the VKORC1 genotype primarily determine the warfarin dose in KD

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patients

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