A study of correlation between CYP 2 C 9 gene polymorphism and Warfarin maintenance dose in anticoagulant therapy among Han people in Yunnan of China

1 Affiliated Yan An Hospital of Kunming Medical University, Kunming, Yunnan Province, P.R. China. 2 Neuroscience Institution of Kunming Medical University, Kunming, Yunnan Province, P.R. China. 3 Institute of Criminal Science and Technology of Lin Cang Police, Bureau, Lin Cang, Yunnan Province, P.R. Cina. 4 Forensic Medicine College of Kunming Medical University, Kunming, Yunnan Province, P.R. China.


INTRODUCTION
As a kind of common oral anticoagulant, Warfarin is extensively applied for the anticoagulant therapy in various diseases, including valvular heart disease or pathological conditions, valve replacement, fibrillation atrial, electrical conversion, coronary heart disease, pulmonary embolism, deep vein thrombosis and stoke e.t.c.Along with the elevation of incidence rate in chronic fibrillation atrial, cardiovascular and cerebrovascular diseases related to thrombo embolism occur more than ever before.Additionally, the popularity of artificial cardiac valve replacement results in more and more patients receiving long-term oral administration of Warfarin for anticoagulant *Corresponding author.E-mail: bingying_xu@126.com.# ZhiYu Chen and Jintao Li contributed equally to this work.therapy.However, in the clinical practice of anticoagulant therapy, it is difficult to control the effective maintenance dose of Warfarin, due to the large variation with regard to intra-individual difference in maintenance dose of anticoagulant agent, its therapeutic effect and side effect (Aithal et al., 1999).
Inadaptable dose of Warfarin administration, especially over dose may lead to some serious complications including hemorrhage or thrombosis, even a threat to life.In the early stage of Warfarin administration, the incidence rate of hemorrhage is about 12%, and death rate resulted from hemorrhage in patients is 2% (Levine et al., 2001).It is reported that in America, there are about 2 million patients who were suffered from the side effect of drugs, half of which were lethal (Lazarou et al., 1998).Therefore, to make a change for traditional medication mode into personalized medicine will shed a new light on the settlement of this difficulty by adjusting the Warfarin dose as optimal one suitable for patients and, at the same time, reducing the side effect of it.
To date, among the studies of gene polymorphism of CYP2C9, the most studied and extensive ones were involving in the correlation between mutation of either CYP2C9*2 or CYP2C9*3 and clinical Warfarin maintenance dosage (Higashi et al., 2002).Furthermore, there exists large variation in Allele Frequency of CYP2C9*2 and CYP2C9*3 among different races.However, until now, there are few reports about the roles of gene loci of CYP2C9 in Warfarin administration, and the guidance of Warfarin personalized medication needs intensive study.
In this study, we investigated the correlation between the gene polymorphism of three important gene loci (CYP2C9*2, CYP2C9*3 and CYP2C9*c_65) in CYP2C9 gene and Warfaring maintenance dosage administered in anticogulatant thrapy, so as to pave a new way for the personalized medicine of Warfarin according to different genotypes of different patients.

Sample harvesting and Admission standard of patients
A total of 300 patients of Han population who were subjected to cardiac valve replacement from 2008 to 2009 in Affiliated Yan An Hospital of Kunming Medical University in Yunnan Province of China were registered and recruited according to the strict standard (see below) in this study.After informed consent was signed, a total of 3ml peripheral veinous blood was extracted from each patient.The blood samples were anticoagulated by addition of ethylenediaminetetraacetic acid (EDTA) and preserved at -48°C.
All 300 patients, more than 18 year-old, were orally administered with Warfarin for anticoagulant therapy under strict monitor persisted for one month post of operation, whose intertional normalized ratio (INR) range from 1.5 to 3.0.In the retrospective whole therapies of the patients were performed at this Hospital.The Warfarin tablets administered to all these patients were produced from the same pharmaceuticals company (Orion Corporation).The strict monitor index consisted of normal hepatic function and obeying dietetic contraindication according to the medical order.Clinical therapeutic and laboratory data were recorded in detail.

Exclusion standard of samples
Under following circumstances, the patients were excluded outside of this study:The patients who had liver diseases at present and before, or his (her) serum transaminase was 1.5 fold more than that of normal level.Patients with impaired renal function, whose serum creatinine >120 μmol/L.In the prospective cases, Warfarin was orally administered three months prior to this test.Patients who were administered or administering the drugs influenced the metabolism of Warfarin Herman et al. (2006) Basal INR scale>1.4patientswho were not appropriate for Warfarin administration due to other reasons.

International normalized ratio (INR)
Since Professor Armand Quick (1935aQuick ( , 1935b) ) set up routine prothrombin time (PT) blood cogulation assay in 1935, untill now, it is still an important screen test to measure factors and associated inhibitors in exogenous blood coagulation system.However, the outcome of PT assay was influenced by various factors.Therefore, it must undergo standardization and quality control so as to elevate its precision, accuracy and reliability.In recenty years, international normalized ratio (INR) detection was adopted extensively to measure the clotting time, which avoids the differential outcomes in different detections due to different reagents used in this assay.

Target International normalized ratio (INR) in this study
In this study, the scales of INR measured from blood samples of patients were screened by the standard ranging from 1.5 to 3.0, downward for 0.2 was considered as the normal.The patients whose INR were outside of this range were excluded from this study, because under this situation, the Warfarin dose could not attain stablility, which is unfavorable to our study and may produce a misleading and even false outcome.

Stable dosages of Warfarin
Stable dosage of Warfarin was referred to: under the same dosage of Warfarin, sequential INR detection of patients ranged from 1.5 to 3.0.The interval of two INR tests was at least above 7 days.

DNA extraction
Saturated phenol/ chloroform method was employed according to traditional procedure described previously by Joanna et al. (2012) to extract DNA from the blood samples of 300 patients who were recruited for this study.DNA purification and concentration assaying were performed as described in Alessandra et al. (2011) report.

Primer dilution
The pairs of primers used in PCR amplification were diluted and adjusted at the concentration of 20 pmol/μl referred to following fomula: Following dilution, the primer solution was subpackaged and stored at -48°C, awaits further usage.

Digestion by nucleate endonuclease
Restrictive endonuclease AvaⅡ, Mva1 and HpaⅠwere used for digestion in target fragment of CYP2C9*2, CYP2C9*3 and CYP2C9*c_65 gene loci respectively.The contents and quantities of the digestion system of CYP2C9*2, CYP2C9*3 and CYP2C9*c_65 gene loci were shown in Table S2.The digestion reaction was perfomed in a thermostatic waterbath at 37°C for 14 h.

Electrophoresis test for products from PCR amplication and digestion
In this test, 8% vertical native polyacrylamide gel electrophoresis was employed.Silver Nitrate staining was used to observe the outcomes of the electrophoresis.By using 50 and 100 bp DNA ladder as standard molecular weight markers, the lengths of DNA fragments of target genes were ascertained and their genotypes were determined.Silver Nitrate staining method was used for coloration.Camera (Canon, IXY DIGITAL) was used to take pictures of the staining outcome.Dry gelatin was made so as to preserve the outcome.

DNA sequencing of three loci of CYP2C9 gene following PCR amplification
The products of PCR amplification of CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910) and CYP2C9*c_65 (rs9332127) were subjected to DNA sequencing.A total of 5 DNA samples from each locus were used for DNA sequencing in order to ascertain whether the PCR amplification products was the expectant target fragments.3130-Genetic Analysis Apparatus (ABI Company, America) was employed to perform automatic DNA sequencing.

DNA sequencing of three loci of CYP2C9 gene following PCR amplification and digestion
The digestive products underwent DNA sequencing at CYP2C9*3 (rs1057910) locus consisted of 5 cases of A/A wild type (127 bp, 75 bp), 1 case of C/C homozygote mutation type (105 bp, 75 bp , 22 bp) and 2 cases of A/C heterozygote mutant (127 bp , 105 bp , 75 bp ,22 bp).
The DNA sequencing for the digestive products of CYP2C9*c_65 (rs9332127) locus following PCR amplification included 5 cases of G/G wild type (317 and 54 bp) and 2 cases of G/C heterozygote mutant (371, 317 and 54 bp).

Statistical analysis
Experimental data were expressed as mean±SD and analyzed by SPSS15.0 and PLINK software.Hardy-Weinberg law of genetic equilibrium was employed to detect the goodness of fit-test of genetic balance.The genotypes and allelotype frequency of three gene loci of CYP2C9 were calculated.χ 2 -test was used to analyze the data of genotypes and allelotype-frequency.A level of p<0.05 was considered as statistical significance.

Products of PCR amplification and genotypes of three loci of CYP2C9 gene
The segment size of CYP2C9*2 locus derived from the PCR and digestion in 300 DNA samples was all 309 bp (Figure 1) With the digestion by restrictive endonuclease AvaⅡ, when restriction enzyme cutting site was at base C, fragment of 309 bp-product following PCR was digested into three frangments, whose segment size was 195, 91 and 23 bp respectively.When restriction enzyme cutting site was at base T, the fragment of product following PCR could be digested into two fragments, whose segment size was 286 and 23 bp respectively.Only one allele-C (195, 91 and 23 bp) and one kind of genotype-C/C wild type (Figure 1) was checked out in the 300 samples.Among the 300 DNA samples, there was no mutational site was detected at CYP2C9*2 gene locus.As the digestive product of 23 bp fragment at CYP2C9*2 locus was the smallest one, when polyacrylamide gel electrophoresis (PAGE) finished, this fragment have run out of the gelatin, image of this fragment could not be observed within the gelatin.Only the 195 and 91 bp DNA fragments could be observed.
The segment size of CYP2C9*3 locus obtained from the PCR and digestion in 300 DNA samples was all 202  bp (Figure S1).With restrictive nuclease va1 digestion, when the restriction enzyme cutting site was at base A, the 202 bp DNA fragments were digested into two fragments, whose segment size was 127 and 75 bp respectively.When the restriction enzyme cutting site was at base C, the DNA fragments (202 bp) were digested into three fragments, with segment size of 105, 75 and 22 bp respectively.Among 300 samples, two kinds of alleles were detectable, which were A and C.There were three kinds of genotypes found in these 300 samples, exhibiting A/A wild type (127 bp, 75 bp), C/C homozygosis mutant (105, 75 and 22 bp) and A/C heterozygote mutant (127, 105, 75 and 22 bp) in genotypes (Figure S1).Among these 300 DNA samples, there were 276 cases of A/A wild type for CYP2C9*3 locus, which accounted for 92%.There were 22 cases of A/C heterozygote mutant, accounting for 7.3%.There were 2 cases of C/C homozygote mutant, accounting for 0.7%.The frequency of allele A was 95.67%, and that of allele C was 4.03%.
The DNA fragment of PCR and digesitive product at CYP2C9*3 locus was 22 bp.As it was too small, when the gel electrophoresis finished, this fragment has run out of the gelatin, this fragment could not be seen within the galatin.Only DNA fragments sized 127, 105 and 75 bp could be observed.
The segment size of CYP2C9*c_65 locus derived from the PCR and digestion in 300 DNA samples was all 371 bp (Figure S3).By using restrictive nuclease HpaⅠ, when enzyme cutting site located at base G, 371 bp sized DNA fragment obtained from PCR was digested into two fragments, whose segment size was 317 and 54 bp respectively.When the enzyme cutting site located at base C, the DNA fragment of 371 bp could not digested by HpaⅠ, leaving a single fragment of 371 bp.Among the 300 DNA samples, there were two kinds of alleles detectable, which were G and C.There were two kinds of genetypes found in these 3000 DNA samples, exhibiting G/G wild type and G/C heterozygote mutant (Figure S2).Among these 300 DNA samples, there were 281 cases showing G/G wild type, accounted for 93.7%.There were 19 cases showing heterozygote mutant, accounting for 6.3%.The frequency of allele G was 96.83%, and that of allele C was 3.17% (Figure S3).

Statistical analysis
Hardy-Weinbergbalance test (HWSIM) statistical analysis revealed that the observed number coincided well with the expected value in CYP2C9*3 and CYP2C9*c_65 locus (P>0.05), which was in accordance with the Hardy-Weinbergbalance law, suggesting that the DNA samples possessed group representativeness (Tables 1  and 2).Sex distribution of CYP2C9*3 and CYP2C9*c_65

DISCUSSION
In the present study, only the population of patients subjected to the operation of cardiac valve replacement was investigated.Because, in this population, patients routinely administered Warfarin, which provides an appropriate to examine the correlationship of genotypes of special loci in CYP2C9 gene and Warfarin maintenance dosage.As for other diseases apart from cardiovascular disorders, such as diabetes, hypertension, were not involved in this study due to their independent with our study.

Influence of CYP2C9 gene mutation on Warfarin dosage in anticoagulant therapy and possible mechanism
The molecular mechanism of CYP2C9 gene polymorphism leading to metabolic defect of Warfarin lies on the base mutation, which results in alteration of sequences of DNA base, and then, amino acid replacement occurs, ultimately changes the catalytic activity of proteins, expressing as weak metabolic pattern and enzyme deficiency pattern.So far, researches involving in the correlation between CYP2C9 mutation and maintenance dosage of Warfrarin in anticoagulant therapy demonstrated that CYPC29 gene mutation could decrease the Warfarin metabolism.Therefore, patients with CYPC29 gene mutation needed relatively lower dosage of Warfarin.Higashi et al. (2002) firstly reported that the correlation between genotype of CYP2C9 gene and anticoagulation or hemorrhage.Subsequently, Sconce et al. (2005) found mutation of CYP2C9*2 or CYP2C9*3 reduced the Warfarin dosage needed in patients subjected to anticogulantant therapy, and CYP2C9*3 mutation led to a more lage extent of Warfarin dosage reduction (30% reduced).There exists much variation of allele frequency in CYP2C9*2 and CYP2C9*3 loci among different races (Margaglione, 2000;Taube, 2000;Loebstein, 2001;Sanderson, 2005;Yu, 2004;Mizutani, 2003;Hong, 2005;Bae, 2005) as well.In White People, obvious ununiformity is commonly seen in the distribution of allele frequency of CYP2C9*2 gene in that the allele frequency ranges from 8 to 19% (Nakai, 2005;Scordo, 2001;Garcia-Martin, 2001).Higashi et al. (2002) reported CYP2C9 gene has high genetic polymorphism, especially in the mutation of CYP2C9*2 and CYP2C9*3, because the activity of enzymes encoded by them decreased 30 and 80% respectively when compared with that of wild type CYP2C9*1, which is the main cause of CYP2C9 mutation, leading to the lower dosage of Warfarin administered in patients with CYP2C9 mutation.Although the correlationship between CYP2C9*2 and/or CYP2C9*3 and Warfarin maintenance dose was involved in several studies by other authors, as for the correlation between CYP2C9*2 and/or CYP2C9*3 mutation and Warfarin maintenance dose in the Han people in China was few reported .Additionally, the crucial role of gene polymorphism of CYP2C9*2 and/or CYP2C9*3 in the intra-individual variation of Warfarin dose attracts more and more attention.Therefore, in this study, our finding that the influence of CYP2C9 gene polymorphism on the substrate drug metabolism has gene dosage effect sheds a new light on the personalized medicantion of Warfarin by applying Warfarin according to different genotypes of patients.Importantly, results from CYP2C9*3 locus study showed that the maintenance dose of Warfarin administered in anticoagulant therapy among the CYP2C9*3 genetypes exhibited: A/A wild type >A/C heterozygote>C/C homozygote, suggesting that patients carried with C/C mutation needed the lowest maintenance dose of Warfarin among the three genotypes found in this study.Our investigation is the first time to elucidate the conclusive correlationship between the mutatant in CYP2C9*3 gene locus and Warfarin maintenance dosage in Han people for clinical practice, providing a novel, useful and effective guaidance for Warfairin personalized medication according to different genotypes in different patients.

Correlationship between CYP2C9*c_65 locus mutation and Warfarin maintenance dosage
In the present study, CYP2C9*c_65 locus-genotypes and their corresponding distributed rates suggesting the mutation at CYP2C9*c_65 locus may not correlated with maintenance dosage of Warfarin.However, Chern et al.

Figure 1 .
Figure 1.Samples 1 to 5 showed the digestive products following PCR, their genotypes were all C/C wild type Sample 6 showed PCR product of CYP2C9*2 locus, whose segment size was 309 bp.Number 1 was heterozygotemutant A/C (127, 105, 75 and 22 bp), Number 2 was homozygote mutant C/C (105, 75 and 22 bp), Number 3 to 5 was wild type A/A (127 and 75 bp), Number 6 was the product of PCR amplification of CYP2C9*2 locus，with segment size of 202 bp.

Figure S1 .
Figure S1.Samples 1 to 5 showed the digestive products following PCR, their genotypes were all C/C wild type.Sample 6 showed PCR product of CYP2C9*2 locus, whose segment size was 309 bp.

Table S1 .
Contents and quantities of PCR reaction system.

Table 1 .
Hardy-Weinberg goodness of fit test of genotypes of CYP2C9*3 locus.

Table 2 .
Hardy-Weinberg goodness of fit test of genotypes of CYP2C9*c_65 locus.