Study on a Novel Polymorphism in the VKORC1 Promoter Region Using Bioinformatic Tools and Warfarin Dosing Data

Highlights
• Optimal dose of warfarin should be personalized for each person.
• Polymorphism in the promoter region of VKORC1 is effective in warfarin medication.
• Changes in the binding sites of the transcription factors can affect the expression of the VKORC1 gene.

Introduction
Warfarin, an antagonist of vitamin K, interferes with coagulation by disrupting the vitamin K cycling pathway [1]. The VKOR enzyme which is the target of warfarin is encoded by the VKORC1 gene [2]. Several studies have demonstrated that genetic variation in VKORC1 led to differences in the amount of required warfarin doses between geographically-distinct or ethnic groups [3]. Oldenburg, et al. found that polymorphism in this gene is responsible for 30% difference in warfarin therapeutic dose among various races [4]. However, reports on the VKORC1 role in thrombogenicity are apparently suffering from the notable gaps of information about enriched transcription factor-binding sites (TFBSs) in the promoter and the potential transcription factors, which would influence the gene expression. Finding effects of polymorphisms in warfarin sensitive or resistant patients on TFBSs of the VKORC1 gene promoter can offer valuable clues in order to determine transcription factors involved in VKORC1 gene expression. In this study, for the first time, variants of VKORC1 gene promoter and the relation between these variants and warfarin maintenance dose in Iranian patients taking warfarin were investigated.

Section snippets

Materials and methods
This study was approved by the Scientific and Ethical Committee of Urmia University of Medical Sciences (code: 1394.282). 2 ml blood samples obtained from 29 warfarin receiving patients of Seyyed alshohada Hospital in Urmia, Iran. Genomic DNA extracted with DNA Extraction mini kit (YTA Company, Iran). To investigate VKORC1-1639G > A polymorphism in the studied cohort PCR-RFLP analysis was carried out according to Sconce et al. [11]. Additionally, primers were specifically designed for 849 bp of

Results
Patients who received warfarin dose less than 1.5 mg/day considered as warfarin sensitive (12 patients), between 1.6 and 7.5 mg/day as control group (8 patients) and more than 7.5 mg/day as high dose taker (10 patients). Among 29 patients, there were 15 males and 14 females.
All patients were genotyped for rs9923231 in the promoter region of VKORC1. A novel mutation with a single base substitution (located at -330 C → A in the upstream region of VKORC1), along with the VKORC1-1639G > A in 3 out of the

Discussion
A specific dose of warfarin cannot be applied for all patients due to differences between individuals, one of which is genetic differences. Appropriate dose was determined according to the individual's resistance or sensitivity to warfarin. This study included patients with different warfarin dose requirements, thus allowing the determination of the genetic variants related to pharmacological response in sensitive, resistant and control groups. This study introduced the genetic polymorphism

Acknowledgements
The present study was supported by the Research Vice Chancellors of Urmia University of Medical Sciences, Urmia, Iran (grant number 1799). The authors are grateful to Dr. Jeannette Koschmann for her valuable remarks concerning the bioinformatic analysis. Special thanks go to Professor Ann K. Daly for her helpful comments on the manuscript.

Conflict of interest statement
None declared.