HK1117571B - Genetic variants of vkorci predicting warfarin sensitivity - Google Patents

Description

Gene variants for predicting warfarin sensitivity

[ CROSS-APPLICATION ]

This application claims benefit of U.S. provisional application 60/638,837 filed on 21/11/2004 and U.S. provisional application 60/679,694 filed on 10/5/2005. The contents of these applications are incorporated herein by reference in their entirety.

[ technical field ] A method for producing a semiconductor device

The present invention relates to a method of determining the dosage of a drug, in particular warfarin.

Comparison document

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The disclosures of the publications, patents and patent applications cited above and elsewhere in the present application are hereby incorporated by reference in their entirety as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.

[ background of the invention ]

Warfarin is a widely used anticoagulant to prevent deep vein thrombosis (deep vein thrombosis), atrial fibrillation (atrial fibrillation) or replacement of artificial heart valve [1-4 ]]Of the subject (a). However, the Hua FangTreatment is problematic because warfarin therapeutic doses vary considerably, whether individually or racially [5-7 ]]. Asian populations, including Chinese, generally require much lower maintenance doses than Caucasian or Spanish [6-9 ]]. Bleeding is the most serious complication currently treated using warfarin [10-12]Efforts are therefore expended to monitor the safety of this oral anticoagulant. The dosage of warfarin is currently monitored closely by continuous measurement of prothrombin time (prothrombin time) using standard International Normalized Ratio (INR).

Cytochrome P450(Cytochrome P450) subfamily IIC, polypeptide 9(CYP2C9) is the major drug metabolizing enzyme catalyzing warfarin hydrolysis [13-15 ]. CYP2C9 x 2 and CYP2C9 x 3 are the most common polymorphisms in caucasian populations. Using CYP2C9 × 1 as the wild type, the haplotypes (Haplotype) probabilities for CYP2C9 × 1/CYP2C9 × 2 and CYP2C9 × 1/CYP2C9 × 3 were about 20% and about 12%, respectively [16, 17 ]. The reduced CYP2C9 enzyme activity in CYP2C9 x 2 and CYP2C9 x 3 variants leads to warfarin sensitivity and in more severe cases bleeding complications [18, 20 ]. However, CYP2C9 x 2 and CYP2C9 x 3 were not present at all or were rarely present in asian populations [9, 21 ]. Genetic variation in CYP2C9 only partially accounts for warfarin dose variability between individuals, but does not account for ethnic variability [6, 7, 16 ].

Coagulation factors (factors II, VII, IX and X) requiring gamma-carboxylation (gamma-carboxylation) of vitamin K are of considerable importance for coagulation. Gamma-carboxylase utilizes reduced vitamin K and oxygen to add carbon dioxide molecules to the glutamic acid side chain in coagulation factors. During the carboxylation, the reduced form of vitamin K is oxidized to vitamin K2, 3-epoxide, from which it is regenerated by vitamin K epoxide reductase for another catalytic cycle. Warfarin prevents the synthesis of clotting factors by inhibiting vitamin K epoxide reductase [22, 23 ]. Recently, the gene encoding vitamin K epoxide reductase complex subunit 1 (VKORC1) was cloned [24, 25], and VKORC1 gene was found in warfarin-resistant patients [25, 26 ]. In italian patients, intronic polymorphisms (intronic polymorphisms) have also been found to be associated with interindividual warfarin-sensitive variability.

However, these studies cannot account for the diversity of all warfarin doses, particularly the inter-ethnic differences, nor the Asian population that accounts for a significant portion of the world's population. It is therefore desirable to find a way to predict the appropriate warfarin dose range for widespread use.

[ summary of the invention ]

Applicants found that polymorphisms in the promoter of the VKORC1 gene are associated with warfarin sensitivity. This polymorphism may explain the inter-individual and inter-ethnic differences in warfarin dosage requirements. Furthermore, the polymorphism is also associated with promoter activity. The promoter of the VKORC1 gene has a G nucleotide at position-1639 (numbered relative to the first nucleotide of the start codon) which is 44% more active than the promoter having A at the same position. Patients with the-1639A polymorphism were more susceptible to warfarin. The dose requirement was lowest in patients with homozygous AA at position-1639, centered in patients with heterozygous AG at this position, and highest in patients with homozygous GG at this position. The promoter sequence or activity of the VKORC1 gene of a subject can thus be used to predict how much warfarin should be administered to the subject. The method can obviously improve the accuracy of warfarin dosage determination. Currently, patients are given an initial dose, whose INR is monitored periodically and warfarin doses are adjusted accordingly until a maintenance dose that is safe for the patient is reached. Using the method of the present invention, the predicted dose will be closer to the maintenance dose, and hence warfarin dosing will be faster, safer and more economical.

It is therefore an aspect of the present invention to provide a method for determining warfarin dose ranges in a subject, comprising studying the promoter sequence of the VKORC1 gene of the subject. Another aspect of the invention provides a method for assessing a patient's risk of developing complications after warfarin administration, comprising studying the promoter sequence of the VKORC1 gene of the subject. The more susceptible a subject is to warfarin, the higher its risk of developing complications such as bleeding.

Specifically, the sequence at position-1639 of the VKORC1 gene was investigated. If the nucleotide at this position is A, the subject is more sensitive to warfarin. Thus, subjects with homozygous AA at position-1639 of the VKORC1 gene were more sensitive than those with heterozygotes of A with other nucleotides (G, C or T) at the same position. But the heterozygotes were more sensitive to warfarin than subjects without a at this position.

The sequence can be studied by analyzing the-1639A or-1639G/C/T allele for an equivalent genetic marker (equivalent genetic marker), wherein the presence of the equivalent genetic marker indicates the presence of the corresponding allele. For example, the equivalent genetic marker may be a SNP selected from the group consisting of rs9934438, rs805894, rs2359612, and rs7294 of the VKORC1 gene, each of which indicates warfarin sensitivity.

In some embodiments of the invention, the sequence can be studied by using oligonucleotides that specifically hybridize to the VKORC1 gene promoter. Preferably, the oligonucleotide specifically hybridizes to at least 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides across position-1639 of the VKORC1 gene. The sequence can be studied using DNA prepared from the peripheral blood of a subject.

The subject of the invention is preferably a human, more preferably an asian, caucasian, african-american or hispanic.

Other methods of determining warfarin dosing can be combined with studies of the VKORC1 gene. For example, the sequence of the CYP2C9 gene can also be examined based on knowledge available in the art. For example, CYP2C9 x 2 and CYP2C9 x 3 are both associated with warfarin sensitivity.

Another aspect of the invention provides a method for determining warfarin dose ranges in subjects, comprising studying the activity of the VKORC1 gene promoter in the subjects. The higher the promoter activity, the higher the dose of warfarin required.

Another aspect of the present invention is to provide a kit for determining warfarin dosage range, comprising at least one component selected from the group consisting of: (a) means (means) for detecting sequence A at position-1639 of the VKORC1 gene; and (b) means for detecting sequence G at position-1639 of the VKORC1 gene.

The kit may further comprise means for detecting sequences C and T at positions-1639 of the VKORC1 gene, respectively. In certain embodiments, the kit may comprise means for detecting each of the four possible bases at that location. In other embodiments, the kit may contain means for detecting A at the location, and means for detecting G, C or T at the location, since the presence of A at the location indicates a higher sensitivity to warfarin. The means are preferably oligonucleotides. The kit may optionally comprise oligonucleotide hybridization reagents, PCR amplification reagents, and/or instructions for use.

Another aspect of the invention is to provide an oligonucleotide or a complementary strand thereof, which specifically hybridizes with a region of the promoter of the VKORC1 gene, wherein said region spans the-1639 position of the promoter and consists of at least 6 nucleotides. Preferably, the oligonucleotide specifically hybridizes to at least 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides across position-1639 of the VKORC1 gene. The oligonucleotide hybridizes to the region, wherein the nucleotide at position-1639 is A, G, C or T, preferably A or G. In some embodiments, the aforementioned oligonucleotide comprises about 15 nucleotides or less, 16-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-40 nucleotides. The invention also provides microarrays comprising the oligonucleotides of the invention.

The details of one or more implementations of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description, and from the drawings.

[ description of the drawings ]

FIG. 1 is a scatter plot of warfarin dose versus VKORC1-1639 genotype. The warfarin dose (see Table 1) of selected patients with warfarin sensitivity or resistance was plotted against the different genotypes at the VKORC1 promoter-1639 position. Individuals with CYP2C9 variations include CYP2C9 x 3, T299A (i.e. amino acid residue 299 changes from T to a) and P382L (i.e. amino acid residue 382 changes from P to L).

FIG. 2 is a relative measurement of luciferase activity levels in HepG2 cells. pGL3 luciferase reporter contains either an A (pGL3-A) or a G allele (pGL3-G) at promoter-1639 position. The mean of the data obtained from 9 experiments is shown, and the error bars (error bars) represent the standard deviation. pGL3-basic was used as a control without any promoter sequence inserted.

FIG. 3 shows the genomic sequence of the VKORC1 gene (Genb ank accession AY 587020). The transcription start position is nucleotide 5086 in the figure (bold and boxed), which is referred to in the traditional nomenclature as +1 of the gene. In the figure, A of the ATG translation initiation codon (bold) is located at nucleotide 5312 in the figure, which is defined as +1 according to the Human Genome Variation Society's recommendation in the new nomenclature system. The promoter polymorphisms described herein are underlined and in bold (nucleotide 3679 in this sequence), which is located at-1413 in the conventional system and at-1639 in the newly proposed system.

[ embodiment ] A method for producing a semiconductor device

We found that polymorphisms in the VKORC1 gene promoter are associated with warfarin sensitivity. This polymorphism may explain inter-individual and inter-ethnic variability in warfarin dose requirements. Further, this polymorphism is also associated with promoter activity. The activity of the promoter of the VKORC1 gene with G at nucleotide-1639 (first nucleotide relative to the start codon) is 44% higher than the activity of the promoter with A at the same position. Patients with the-1639A polymorphism are more susceptible to warfarin. Patients with AA homozygous at position-1639 had the lowest dose requirement, patients with AG heterozygous at position-1639 had the median dose requirement, and patients with GG homozygous at position-1639 had the highest dose requirement, so the promoter sequence or activity of the VKORC1 gene can be used to predict how much warfarin a subject should be given.

Before further describing the present invention, unless otherwise indicated, the terms used in this application are defined as follows.

Definition of

The term "warfarin" encompasses coumarin (coumarins) derivatives having anticoagulant activity. In a preferred embodiment, warfarin is 4-hydroxy-3- (3-oxo-1-

Phenylbutyl) -2H-1-benzopyranedione (4-Hydroxy-3- (3-oxo-1-phenylbutyl) -2H-1-benzopyran-2-one) (i.e., 3- α -phenyl (phenyl) - β -acetoacetyl-4-hydroxycoumarin). The current commercial product is a racemic mixture of the R isomer or the S isomer. The term "warfarin" encompasses the R isomer, the S isomer, any racemic mixture, and any salt thereof. In particular, it comprises coumadin (Couradin), Marvin (Marevan), Muvavin (Panwarfin), Prothromadin, Tintorane, Warflone, Waran, Athrombin-K, warfarin-dinol (deanol), Adoisine, warfarin acid (warfarin acid), Coumafen, Zoocoumarin, Phenprocolon, dicumarol (dicumarol), brodifacoum (brodifacoum), diphenylindandione (diphenadienone), chlorratine (chlorophacinone), bromodiphacin (brodomolone) and acetocoumarin (acetocoumarin).

An "oligonucleotide" according to the present invention is a polynucleotide comprising 2 to about 100 contiguous nucleotides connected via a nucleotide linkage. The oligonucleotide is at least about 10, 20, 30, 40, 50 or 60 nucleotides in length. Furthermore, the length of the oligonucleotide is preferably up to 200 nucleotides, preferably up to about 150, 100, 75, 50, 40, 30 or 20 nucleotides.

A hybridization "probe" is an oligonucleotide that binds in a base-specific manner to a complementary strand of nucleic acid. The probe comprises a peptide nucleic acid. The probe may be of any length suitable for specific hybridization to a target nucleotide sequence. The optimum probe length depends on the hybridization method in which it is used, e.g., a particular length may be more suitable for use in microfabricated arrays (microfabricated arrays), while probes of other lengths may be more suitable for use in conventional hybridization methods, such optimization being well known in the art. Suitable probes and primers are generally from about 8 nucleotides to about 100 nucleotides in length. For example, the length of the probe and the primer may be 8 to 20, 10 to 30, 15 to 40, 50 to 80 nucleotides, and preferably 12 to 20 nucleotides. The nucleotide sequence may correspond to the coding sequence of a gene or the complement of the coding sequence of a gene.

Methods and compositions

By comparing the CYP2C9 and VKORC1DNA sequences of Chinese patients with warfarin sensitivity or resistance (example 1), we found that CYP2C9DNA sequence variation occurs only in 5.4-7.3% of the Han nationality Chinese population. The variation is mainly CYP2C9 x 3. Furthermore, two missense mutations identified in this study, including a new mutation that has not been previously reported (P382L), are very rare and present only in warfarin-sensitive patients. CYP2C9 variation has varying incidence across different ethnic groups. CYP2C9 x 1/CYP2C9 x 2 was present in 20% of caucasian population [17 ]; however, it was completely absent in the population of people in this study. Thus, mutations in CYP2C9 may explain warfarin sensitivity in only a small proportion of people in the population of warfarin, but still fail to explain the differences in warfarin dosage requirements across ethnicities.

On the other hand, the inventors found that alterations in the VKORC1 gene can alter inter-individual warfarin dose requirement differences in the population of warfarin as well as inter-ethnic warfarin dose requirement differences between warfarin and caucasian. Patients with the AA genotype of the-1639 promoter polymorphism had lower dose requirements, while the AG/GG genotype had higher dose requirements (examples 1 and 2). Further, AA homozygotes are less abundant in the Caucasian population, however the genotype is found in a large part of the Hua population (example 3). This genotype frequency difference between caucasians and the warns, as well as the correlation of AA to warfarin sensitivity, is consistent with the clinically observed lower dose requirements for warfarin in the population of warfarin than the caucasians.

Accordingly, one aspect of the present invention provides a method for determining warfarin dose ranges in a subject, comprising studying the sequence of the promoter of the VKORC1 gene of the subject. And SEQ ID NO: 1, the promoter sequence preferably contains less than 10%, 8%, 6%, 4%, 3%, 2% or 1% variation within 3kb from the translation initiation site. More preferably, the promoter contains less than 20, 15, 12, 10, 8, 6, 4, 3 or 2 single base variations in the 3kb region. The above sequence variations in combination with altered promoter activity can be used to indicate the range of warfarin doses that should be administered.

Specifically, the sequence at position-1639 of the VKORC1 gene was investigated. Individual SNPs can be used to predict warfarin sensitivity without reference to any other genotype. Thus, in this position, AA homozygous indicates warfarin sensitivity. AG heterozygotes indicate moderate sensitivity, and GG homozygotes indicate relative resistance. The actual dosage of warfarin that should be administered to an individual who is sensitive, moderately sensitive, or relatively resistant to warfarin will vary depending on the warfarin formulation and the individual condition, and may be determined by the prescribing physician. For example, the study of the invention (example 1) shows that in a selected population of patients with warfarin sensitivity or resistance, patients with the lowest dose requirement (mean 1.19 mg/day, range 0.71-1.50 mg/day) had the AA genotype, whereas those with moderate dose requirements (mean 8.04 mg/day, range 6.07-10 mg/day) were the AG genotype, and those with the highest dose requirement (mean 9.11 mg/day, range 8.57-10 mg/day) were the GG genotype. In randomly selected patients (example 2), the AA genotype had a lower maintenance dose (2.61+/-1.10 mg/day) compared to AG and GG (3.81+/-1.24 mg/day). These ranges can serve as starting points for warfarin dosage determinations.

The gene sequence of the VKORC1 gene is available as Genbank accession number AY587020(SEQ ID NO: 1) (see FIG. 3). The sequence of VKORC1 sequence isoform 1mRNA is available as Genbank accession NM-024006.4. In the above two sequences, the transcription initiation point is marked as +1, and then the polymorphic promoter indicating warfarin sensitivity is positioned at position-1413. However, according to the recently proposed description of sequence variability (http:// www.genomic.unimelb.edu.au/mdi/mutunomen /) by the Human Genome Variation Society (HGVS), it was recommended to describe the promoter SNP with respect to A of the ATG translation start codon. According to this nomenclature, with A in the translation initiation codon ATG (Met) of NM-024006.4 or AY587020 as +1, the promoter SNP used according to the invention is located at-1639, where the G > A polymorphism is "NM-024006.4: c. -1639G > A ". It is clear here that the-1639G > A polymorphism (or more precisely NM-024006.4: c. -1639G > A) of the present invention is identical to the-1413G > A polymorphism previously named according to conventional promoter sequence numbering.

In addition to specific polymorphisms (e.g.: AA, AG or GG at position-1639), genetic markers linked to each specific SNP can also be used to predict the corresponding warfarin susceptibility. Because genetic markers in the vicinity of the SNP of interest tend to be co-segregated or show linkage disequilibrium. The presence of these markers (equivalent genetic markers) is therefore indicative of the presence of the SNP of interest, and thus of the degree of warfarin sensitivity.

Recently it was found that (D' Andrea et al.) [27] VKORC1-1639G > A promoter polymorphism is in linkage disequilibrium with the VKORC11173C > T intron polymorphism and can be used to explain the results that Italian patients with the 1173TT genotype have a lower mean daily dose compared to the CT or CC genotypes. The present invention also shows that in the population of Chinese people, the 3730G > A polymorphism located in the 3' untranslated region is in linkage disequilibrium with-1639A > G and 11173C > T. In particular, the 3730G allele is associated with the-1639A allele and with the 1173T allele. Other equivalent genetic markers for the-1639A SNP include rs9934438, rs805894, rs2359612, and rs 7294. Equivalent genetic markers can be any marker, including microsatellites (microsatellites) and single nucleotide polymorphism markers (SNPs). Preferably, useful genetic markers are about 200kb or less than 200kb from VKORC1-1639 position. More preferably, the marker is approximately equal to or less than 100kb, 80kb, 60kb, 40kb, 20kb, 15kb, 10kb or 5kb from the VKORC1-1639 position.

The present invention also provides oligonucleotides capable of hybridizing to the SNPs of the invention. The oligonucleotides can be used, for example, as hybridization probes or primers for detecting the promoter sequence of the VKORC1 gene. The oligonucleotide preferably comprises the sequence TGGCCGGGTGC (SEQ ID NO: 1 positions 3668 to 3678), or the complementary strand thereof. For hybridization, the sequence corresponding to position-1639 is preferably located near the middle of the oligonucleotide. Preferably at least 4 nucleotides are located on each side of the sequence corresponding to position-1639. More preferably, the sequence corresponding to position-1639 is flanked on each side by at least 5, 6, 7, 8 or 9 nucleotides.

Hybridization is usually carried out under stringent conditions, for example, at a salt concentration of not more than 1M and at a temperature of at least 25 ℃. For example, single nucleotide specific probe hybridization is suitably carried out in 5XS SPE (750mM NaCl, mM sodium phosphate, mM EDTA, pH7.4), at a temperature of 25 ℃ to 30 ℃ or the like. Preferably, hybridization may be followed by low stringency washes, including, for example, 42 ℃, 5xSSC and 0.1% SDS, or 50 ℃, 2xSSC and 0.1% SDS. High stringency wash conditions are most preferred and include, for example, 65 ℃, 0.1xSSC, and 0.1% SDS. It is well known to those skilled in the art that equivalent conditions can be determined by varying one or more parameters while maintaining a similar degree of identity or similarity between the target nucleotide and the primer or probe used.

The-1639 promoter SNP is located in the E-box (box) (consensus sequence for the E-box is CANNTG) and has three additional E-boxes within a short distance (200 bp). The E-box is an important factor mediating cell/tissue specific transcription, e.g.in muscle, neurons, liver, pancreas [28, 29 ]. As observed, changing the second base A to G at position-1639 disrupts the E-box consensus sequence and alters the promoter activity. It was clearly confirmed by promoter analysis that promoter activity increased by about 44% in HepG2 cells when the consensus sequence was disrupted (see FIG. 2). Without being bound by theory, this suggests that the function of the E-box may be a repressor binding site in HepG 2. Since HepG2 cells are derived from liver cancer cells, the-1639E-box may inhibit transcription in the liver.

The need for higher warfarin doses for a person with a genotype of-1639 GG may be explained as follows. The VKORC1 gene codes for vitamin K epoxide reductase subunit 1, which is responsible for the regeneration of reduced vitamin K. Gamma-carboxylase requires vitamin K in its reduced state, and gamma-carboxylation of vitamin K-dependent coagulation factors (factors II, VII, IX and X) is required for coagulation. When VKORC1 promoter activity is increased, elevated levels of VKORC1mRNA increase VKOR activity [25], thus increasing the efficiency of reduced vitamin K regeneration. Thus, high levels of reduced vitamin K lead to enhanced gamma-carboxylation of vitamin K-dependent coagulation factors. Warfarin acts by blocking the synthesis of coagulation factors, so having more coagulation factors would require more warfarin to achieve its anticoagulant effect. Since liver is the major organ for the synthesis of vitamin K-dependent coagulation factors and the expression of VKORC1 is highest in liver, a change in VKORC1 levels of about 44% in liver is likely to have a significant effect on the coagulation process. The warfarin dose required is therefore related to the VKORC1 gene polymorphism and promoter activity.

The inventors of the present invention believe that any sequence, other than A, at position-1639 of the VKORC1 gene disrupts the E box and increases promoter activity. While an increase in promoter activity increases the dosage required for warfarin, the promoter sequence, particularly at position-1639, can be used to indicate warfarin dosage. Further, other embodiments of the present invention provide methods for determining warfarin dosage or determining warfarin sensitivity by detecting promoter activity of VKORC1 gene. Likewise, the level of the VKORC1 gene product (mRNA or protein) or the activity of VKOR can also reflect warfarin dosage requirements. The individual VKORC1 promoter activity, mRNA amount, protein mass or VKOR activity is at least 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the subjects with AA genotype, indicating that a higher dose of warfarin is required compared to the subjects with AA genotype.

Methods for determining promoter activity or mRNA or protein quality are well known in the art. In some embodiments, PCR may be used as a method for detecting the amount of mRNA. In other embodiments, specific antibodies can be used to determine VKORC1 protein. The promoter activity can be detected, for example, by isolating a promoter sequence from a peripheral blood sample of a test subject, linking the promoter sequence to a reporter gene, expressing the reporter gene and quantifying the produced reporter. Methods for measuring VKOR activity are well known in the art.

It is a further object of the present invention to provide a method for determining whether a given mutation in the VKORC1 gene will affect warfarin dosage. In this method, the promoter activity, the amount of mRNA, the protein quality or the VKOR activity resulting from this mutation is measured and compared with the corresponding wild-type VKORC1 gene. If the promoter activity, mRNA amount, protein mass or VKOR activity is increased or decreased by 10%, 15%, 20%, 25%, 30%, 35% or 40% compared to the wild type, the physician should consider increasing or decreasing the warfarin dosage.

It is still another object of the present invention to provide a kit for determining warfarin dosage range, comprising at least one component selected from the group consisting of: (a) detecting a sequence A at a position-1639 of the VKORC1 gene; and (b) means for detecting sequence G at position-1639 of the VKORC1 gene.

The kit may further comprise means for detecting sequences C and T at positions-1639 of the VKORC1 gene, respectively. Thus in some embodiments the kit comprises means for detecting each of the four possible bases at this position. In other embodiments, because the presence of a at this position a indicates that warfarin sensitivity is most likely, this kit can comprise means for detecting a as well as means for detecting G, C or T at the same position. This means is preferably an oligonucleotide. The kit may optionally include oligonucleotide hybridization reagents, PCR amplification reagents, and/or instructions for use.

Another aspect of the invention is to provide an oligonucleotide or its complementary strand, which hybridizes to a region of the promoter of the VKORC1 gene, wherein said region spans the-1639 position of the promoter and consists of at least 6 nucleotides. Preferably, the oligonucleotide specifically hybridizes to at least 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides across position-1639 of the VKORC1 gene. The oligonucleotide can hybridize to the region, wherein the nucleotide at position-1639 is A, G, C or T, preferably A or G. In some embodiments, the oligonucleotide consists of about 15 nucleotides or less, 16-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-40 nucleotides. Microarrays comprising the oligonucleotides of the invention are also provided herein.

The following examples are provided to illustrate the present invention and should not be construed as limiting the scope of the invention. While the invention has been particularly shown and described with reference to a preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit of the invention and the scope of the appended claims.

Examples

The meanings of the abbreviations used in the following examples are as follows. Undefined abbreviations have a generally accepted meaning.

Degree centigrade

hr as hour

min is minutes

sec or s ═ sec

Micromolar concentration of

mM to millimolar concentration

M is molar concentration

ml to ml

μ l to μ l

mg ═ mg

Microgram of mug

mol to mol

pmol to picomole

ASO (allele specific oligonucleotide)

CYP2C9 ═ cytochrome P450 subfamily IIC, polypeptide 9(Cytochrome P450, subfamily IIC, polypeptide9)

IND is the International normalized ratio (International normalized ratio)

LD as linkage disequilibrium (linkage disequilibrium)

NTP ═ nucleoside triphosphate (nucleoside triphosphate)

Phosphate Buffered Saline (PBS)

PCR (polymerase chain reaction)

SNP (single nucleotide polymorphism)

VKORC 1-vitamin K epoxide reductase Complex subunit 1

Materials and methods

The patients: we found 16 patients receiving low-dose or high-dose warfarin from the cardiovascular outpatient clinic of four larger medical centers in taiwan (national taiwan university hospital, hero medical university hospital, taipei honor general hospital, new light hospital). The average maintenance dose of warfarin in China was about 3.3 mg/day [8, 9 ]. Thus, patients receiving a maintenance dose of ≥ 1.5 mg/day are considered to be sensitive to warfarin (see Table 1, 11 patients), while patients receiving a maintenance dose of ≥ 6 mg/day are considered to be resistant to warfarin (see Table 1, 5 patients). The definition of warfarin-resistant patients is supported by data that none of the randomly selected patients had warfarin doses exceeding 6 mg/day (see table 2). This is unlike in caucasian patients, whose average warfarin maintenance dose is between 5.1-5.5 mg/day [20, 27 ].

The average daily dosage by warfarin was calculated in units of one week period and the most recent International Normalized Ratio (INR) was recorded for each patient. Of the 104 warfarin-receiving patients randomized, all four hospitals tested the same target INR value, 1.4 to 3, regardless of dose. The warfarin indications are: prosthetic heart valve replacement (90 patients), deep vein thrombosis (5 patients), Atrial Fibrillation (5 patients), stroke (4 patients). Clinical data (including age, sex, weight, and daily average maintenance dose) for the patients were provided for each subject. At the time of blood draw, each patient had received a constant maintenance dose for at least three weeks. Patients with liver cancer, kidney cancer, gastro-intestinal cancer or abnormal bleeding prior to warfarin treatment will be excluded.

In addition, 92 unrelated caucasian DNA samples (C at.no, HD100CAU, research and medical science center (NIGMS) human gene cell preservation center, Camden, NJ, USA) served as a control group for caucasians. DNA samples of 95 healthy subjects were randomly selected from a biological database for national population studies, in which 3312 han were selected as a chinese control group based on geographical distribution in taiwan. The control group of warfarin and all subjects receiving warfarin treatment were unrelated taiwan-resident hans. Han constitutes the largest ethnic group in Taiwan, accounting for over 98% of the population, and the subject does not include the original people accounting for about 2% of the population in Taiwan.

The study was approved by the research institute and informed of all subjects and agreed.

DNA sequencing and genotyping with single nucleotide polymorphism markers (SNPs)

Utilization of genomic DNA by PUREGENE^TMDNA purification System (Gentrasystem, Minnesota, USA). CYP2C9 and VKORC1DNA sequence variants were first determined by direct sequencing (Applied Biosystem3730DNA Analyzer, Applied Biosystems, Foster City, CA, USA) in 16 Han patients with sensitivity (. ltoreq.1.5 mg/day, 11 patients) and resistance (. gtoreq.6.0 mg/day, 5 patients) to warfarin. Primers were designed using Primer3 PCR Primer program (http:// fokker. wi. mit. edu/cgi-bin/Primer3/Primer 3-www.cgi), specifically for the intron-exon (intron-exon) junction, exon, and 2kbp upstream of the transcription start site of CYP2C9 and VKORC 1. The sequences of the primers used to detect the variation in the VKORC1 promoter were 5 '-CAGAAGGGTAGGTGCAACAGTAA (SEQ ID NO: 2; sense strand 1.5kbps upstream of the transcription start site) and 5' -CACTGCAACTTGTTTCTCTTTCC (SEQ ID NO: 3; antisense strand 0.9kb upstream of the transcription start site). Polymerase Chain Reaction (PCR) was performed in a final volume of 25. mu.l containing 0.4. mu.M of each primer, 10mM Tris-HC1(pH8.3), 50mM KCl, 1.5mM MgCl₂0.2mM dNTPs and 1 unit HotStartTaq^TM(Qiagen Inc, Valencia, Calif., USA). Amplification conditions included an initial denaturation at 96 ℃ for 12min, 34 of the following PCR cycles: 30sec, 96 sec, 30sec, 60 ℃ 40sec, 7 sec2℃。

Following screening with MALDI-TOF mass spectrometer identification system (MassSpectrometry) (SEQUENOM MassARRAY System, Sequenom, san Diego, Calif., USA), variants were identified in 104 randomly selected warfarin-treated warfarin, 95 normal warfarin controls, and 92 normal caucasian controls. Primers and probes were designed using the Spectro DESIGNER software (S equinom). Subsequently, multiplex PCR (multiplex-PCR) was performed, and unincorporated dNTPs were dephosphorylated using shrimp alkaline phosphatase (Shriphalkaline phosphatase, SAP, Hoffman-LaRoche, Basel, Switzerland) followed by primer extension. The purified primer extension reaction product was spotted on a 384 pack silicon chip (SpectroCHIP, Sequenom) and analyzed on a Bruker Biflex III MALDI-TOF SpectroREADER mass spectrometer (Spequenom), and the resulting spectra were treated with SpectroTYPER (Spequenom).

Fluorogenic enzyme reporter assay

Plasmid construction

To test the VKORC1 promoter activity, the VKORC1 promoter from patients with the-1639 AA and-1639 GG genotypes, containing the-1639 polymorphism (-1798 to-35), was first cloned into a pGEM-T Easy vector (Promega, Madison, Wis., USA) using forward primers (forward primers): 5' -ccgctcgagtagatgtgagaaacagcatatgg (SEQ ID NO: 4; containing XhoI restriction sites) and reverse primer: 5' -cccaagcttaaaccagccacggagcag (SEQ ID NO: 5; containing a HindIII restriction site). The fragment containing the VKORC1 promoter was released from the pGEM-T Easy vector by digestion of the vector with XhoI and HindIII restriction enzymes, and subcloned into the multiple cloning site (XhoI and HindIII) of pGL3-basic vector (Promega). The pGL3-basic vector contains cDNA encoding firefly luciferase, and when fused to a promoter, can be used to analyze the activity of the inserted promoter when transfected into mammalian cells. This vector containing the-1639G/G genotype is referred to as pGL3-G, while the vector containing the-1639A/A genotype is referred to as pGL 3-A. The VKORC1 promoter sequence in both vectors was determined by direct sequencing analysis.

Cell culture and Dual luciferase reporter assays

HepG2 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal bovine serum supplemented with 100 units/mL penicillin, 100. mu.g/mL streptomycin and 2 mML-glutamine. 24 hours before transfection, 1.5X10 was implanted into each well of a 12-well plate⁵A cell. On the day of transfection, 1.5. mu.g of pGL3 vector and 50ng of pRL-TK vector (Promega) were co-transfected into each well using lipofectamine 2000(Invitrogen Corporation, Carlsbad, Calif., USA). The pRL-TK vector encodes an aequoria luciferase (Renilla luciferase) transcribed from the HSV-TK promoter, and was used as an internal control to normalize firefly luciferase expression. 48 hours after transfection, cells were lysed in passive lysis buffer (promega). The lysates were added to a luciferase matrix (dual luciferase reporter system, Promega) before detecting firefly and jellyfish luciferase activities with a luciferin meter (Luminometer, SIRIUS, Pforzheim, Germany).

Statistical analysis

The genotype frequency of each SNP was calculated. The Chi-square test was used to compare the genotype frequencies of each SNP in the three sample sets. A T-test and a Wilcoxon-Mann-Whitney test were performed to make multiple comparisons of mean dose levels between different genotype groups. Linkage disequilibrium of internal markers determined by two methods D' and r²For evaluation, a linkage disequilibrium Overview picture (Graphical Overview of LinkageDisequibrium) (GOLD, http:// www.sph.umich.edu/csg/abecasis/GOLD /) calculation was used.

Examples

Example 1 in selected warfarin-sensitive or resistant human patients CYP2C9 and VKORC1DNA sequence variants

The average maintenance dose of warfarin in China was 3.3 mg/day [8, 9 ]. Thus, patients receiving a maintenance dose of 1.5 mg/day or less were considered warfarin-sensitive (see patients 1, 11), while patients receiving a maintenance dose of 6 mg/day or more were warfarin-resistant (see patients 5, Table 1, see materials and methods previously described). Sequencing of the coding region, exon-intron junction, and promoter region of the CYP2C9 gene revealed three sequence variations in 4 of 11 warfarin-sensitive patients. The variation is as follows: 1075A > C (I359L is CYP2C9 x 3), 895A > G (T299A) and 1145C > T (P382L). CYP2C9 x 3 was detected in 3 patients (see table 1, subjects 1, 3, 5). Subject 5 also had 895A > G (T299A) alterations in addition to CYP2C9 x 3 as described previously [7 ]. A new exon mutation 1145C > T (P382L) was detected in the fourth patient (individual 6). As shown in table 1, CYP2C9 gene variation was not found in all warfarin-sensitive patients, and was completely absent in all warfarin-resistant patients.

TABLE I patient demographics and DNA sequence variation confirmation

Table 1: patient demographics and identified DNA sequence variants

We also sequenced the promoter, coding region and exon-intron junction of the VKORC1 gene. A variation (3730G > A) in the 3' untranslated region (UTR) of the VKORC1 gene was detected. Furthermore, the promoter polymorphism-1639G > A (or-1413 with the transcription initiation position being + 1) was detected in the upstream region of VKORC 1. This polymorphism was shown to be associated with warfarin sensitivity, as all warfarin-sensitive patients were AA homozygous at position-1639. Warfarin-resistant patients, on the other hand, were either AG heterozygous or GG homozygous (table 1). When warfarin doses were plotted against the-1639 genotype, patients with genotype AA had the lowest dose requirement (mean 1.19 mg/day, range 0.71-1.50 mg/day), patients with genotype AG-heterozygous had moderate dose requirements (mean 8.04 mg/day, range 6.07-10 mg/day), and patients with homozygous GG genotype had the highest dose requirement (mean 9.11 mg/day, range 8.57-10 mg/day) (see FIG. 1).

In addition to-1639G > A, the [27] intron 1 polymorphism 1173C > T described by D' Andrea et al was also identified in patients of the invention. These two polymorphisms were shown to be in stronger Linkage Disequilibrium (LD) (Table 1). In addition to one patient (subject 6), warfarin-sensitive patients with the-1639 AA genotype were found to be associated with 1173 homozygous TT. In warfarin resistant patients, patients with-1639 heterozygous AG genotype were found to be associated with 1173 heterozygous CT and homozygous-1639 GG was associated with 1173 homozygous TT (see Table 1).

Example 2 VKORC1-1639G in randomized warfarin patients>A Polymorphism

To further investigate whether the-1639G > A polymorphism is associated with warfarin dose differences between individuals, we genotyped 104-recipient warfarin patients with MALDI-TOF mass spectrometry regardless of their dose size. The AA and AG/GG populations did not differ in age, gender and INR (see Table 2). Only two patients were found to be homozygous for GG and were incorporated into the AG population at the time of statistical analysis. We analyzed the data without regard to the presence of other confounding factors, such as diet or other medication. As shown in Table 2, the AA group had a significantly lower dosage requirement (2.61 mg/day) than the AG/GG group (3.81 mg/day). This difference was significant between the AA and AG/GG populations, whether using the T test (p <0.0001) or the Wilcoxon-Mann-Whitney test (p ═ 0.0002).

TABLE 2 mean dose by genotype classification and other clinical characteristics for patients randomly selected for warfarin treatment

Comparison between AA and AG + GG groups^aAnd (4) P value. Checking P value by T<0.0001. P value was checked to 0.0002 using Wilcoxon Mann Whjtney. Data are presented as mean ± SD.

Example 3 VKORC1-1639G from Chinese and Caucasian>A polymorphism and genotype frequency of CYP2C9 variation

The warfarin maintenance doses are known to be much lower in the population of warfarin compared to the caucasian race. To examine whether frequency differences in VKORC1-1639 genotypes could account for differences between ethnic groups in warfarin doses, 95 normal Han and 92 normal Caucasian subjects were genotyped. In the caucasian population, the frequency of AA homozygotes was lowest, while the AG and GG genotypes accounted for the majority of the population (14.2%, 46.7% and 39.1%, respectively) (see table 3). In contrast, in the population of Chinese, homozygous AA accounted for the majority of the population (82.1%), with the remainder being AG heterozygotes (17.9%). No GG homozygotes were found in the randomly selected population. This ratio was similar to that in 104 patients receiving warfarin treatment, with 79.8% being AA homozygote, 18.3% being AG heterozygote, and only 1.8% being GG homozygote. The genotype difference was significant between the caucasian population and the two chinese populations with p-values below 0.0001. The differences between the two ethnicities (huaman versus caucasian) and the association of warfarin sensitivity with AA are consistent with the clinically observed lower dose requirement for huaman versus caucasian.

TABLE 3 polymorphism of VKORC1 promoter (-1639G > A) and CYP2C9 variants in Chinese and Caucasian

P value <0.0001, compare caucasian to chinese population.

P value is less than 0.0001, and the caucasian population and the Chinese population which receive warfarin treatment are randomly selected.

P-value of 0.817, compare warfarin with randomly selected patients receiving warfarin treatment.

^aCYP2C9 x1 is a wild type of CYP2C 9. CYP2C9 x 2 and CYP2C9 x 3 are each

A variant in which arginine is substituted with cysteine at residue 144 and isoleucine is substituted with leucine and leucine, leucine and leucine at residue 359. The frequency in caucasians was derived from public data [17 ].

^bCYP2C9 × 3 frequency was from genotyping 74 patients receiving warfarin.

In addition to-1639G > A, we also screened the Chinese population for three intron polymorphisms (rs9934438, intron 11173C > T; rs8050894, intron 2g.509+ 124C; rs2359612, intron 2g.509+837C) and for one 3' UTR polymorphism (rs7294, 3730G > A). Therefore, all four polymorphisms were in linkage disequilibrium with the-1639 promoter polymorphism (inter-marker D' and r2 values of 1.0).

CYP2C9 variation was also genotyped in the population of people in china. The proportion of CYP2C9 x 1/' 3 was 7.3% in the normal population of warfarin and 5.4% in randomly selected patients receiving warfarin. CYP2C9 x 2 variants were not found in either the human patients or the control group. Compared to published data on the frequency of CYP2C9 variation in caucasians, the caucasian population had much higher frequency of CYP2C9 variation compared to warfarin (about 30% versus 7%), and caucasians were more resistant to warfarin. Other missense mutations 895A > G (T299A) and 1145C > T (P382L) (table 1) detected in warfarin-sensitive patients were not found in any of the randomly selected patients and controls, indicating that they are rare mutations.

Example 4 VKORC1 promoter Activity

The differences in susceptibility to warfarin in patients with AA and GG genotypes can be explained by the altered activity of the VKORC1 promoter. The-1639 promoter SNP is located at the second nucleotide of the E-box (CANNTG), and therefore this polymorphism will alter the consensus sequence of the E-box and can cause an alteration in the activity of the VKORC1 promoter. To test this hypothesis, the VKORC1 promoter containing the-1639 position was amplified by PCR from patients with the-1639 AA or GG genotype and cloned into pGL-3 vector. HepG2 cells (human hepatoma cells) were selected for promoter experiments because VKORC1 has high expression in the liver. A total of nine experiments were performed with consistent results (shown in figure 2). the-1639G VKORC1 promoter has higher luciferase activity (about more than 44%) than the-1639A promoter. The pGL-3-basic vector control does not contain any promoter inserted into the multiple cloning site and has a negligible amount of luciferase activity. Thus, the sequence at the-1639 position is important for promoter activity, and the higher the promoter activity (e.g., -1639G), the higher the warfarin dosage requirement.

Example 5 determination of the warfarin combining VKORC1-1639SNP with CYP2C9 variant Dosage of

Although the VKORC1-1639SNP alone can be used to predict warfarin sensitivity, this SNP can alternatively be combined with other genetic markers. For example, the sequences of the VKORC1 gene and the CYP2C9 gene can be tested in a warfarin dose determination. When both genes were considered, the following table shows the recommended dose of coumadin for each haplotype.

Other embodiments

Various embodiments of the invention have been described. It will be understood that various modifications may be made without departing from the spirit and scope of the invention.

Claims (8)

1. Use of an oligonucleotide, or a complementary sequence thereof, that specifically hybridizes to the VKORC1 gene promoter for the manufacture of a kit for determining warfarin dose ranges for a subject by studying the sequence of the VKORC1 gene promoter of the subject, wherein the oligonucleotide is used to study the nucleotides at positions-1639 of the VKORC1 gene, wherein a homozygous for AA at positions-1639 of the VKORC1 gene indicates warfarin sensitivity, a heterozygous AG for a nucleotide at positions-1639 of the VKORC1 gene indicates warfarin moderate sensitivity, and a homozygous for GG at positions-1639 of the VKORC1 gene indicates warfarin resistance.

2. The use of claim 1, wherein the oligonucleotide specifically hybridizes to at least 6 nucleotides across position-1639 of the VKORC1 gene.

3. The use of claim 1, wherein the oligonucleotide is used for studying the nucleotide at position-1639 of the VKORC1 gene by using DNA prepared from the peripheral blood of a subject.

4. The use of claim 1, wherein the kit further comprises means for detecting the sequence of the CYP2C9 gene.

5. Use of a tool for studying the promoter activity of the VKORC1 gene of a subject for the preparation of a kit for determining the warfarin dose range of the subject, wherein the tool is used for studying the nucleotides at positions VKORC 1-1639.

6. The use of claim 5, wherein the promoter activity ratio is determined if the promoter activity ratio comprises the amino acid sequence of SEQ ID NO: 1 by at least about 20% less, then the subject requires a lower dose.

7. A kit for determining warfarin dosage ranges comprising at least one component selected from the group consisting of:

(a) detecting a sequence A at a position-1639 of the VKORC1 gene; and

(b) and (3) detecting the sequence G at the position-1639 of the VKORC1 gene.

8. The kit of claim 7, wherein the means in (a) and the means in (b) are oligonucleotides.