CN111004811B - CYP2C9 gene segment containing 637A > - Google Patents

Abstract

The invention belongs to the field of biology, and relates to single base mutation. More particularly, the invention relates to a mutation site of CYP2C9 gene corresponding to the 637 th site of SEQ ID NO.2, wherein the site is mutated from wild type A to G, a nucleic acid fragment containing the mutation site, a protein fragment coded by the nucleic acid fragment and application of the nucleic acid fragment. The invention also provides an allele-specific oligonucleotide, a kit and a detection method for detecting the mutation site.

Description

CYP2C9 gene segment containing 637A >

Technical Field

The invention belongs to the field of biology, and relates to single base mutation. More specifically, the present invention relates to a single base mutation of the CYP2C9 gene.

Background

CYP2C9 is the most important member of the CYP2C subfamily of the large family of cytochrome P450 enzymes, and represents approximately 20% of the total human liver microsomal CYP enzymes. Approximately 10-16% of the clinically used drugs are metabolized by CYP2C9 oxidation, and mainly include tolbutamide, S-warfarin, phenytoin, glipizide, glyburide, tolazamide, losartan, irbesartan, and many non-steroidal anti-inflammatory drugs (e.g., ibuprofen, lornoxicam, diclofenac, and naproxen) (see references 1-5).

The CYP2C9 gene is highly polymorphic. According to the current clinical research, the polymorphism of the CYP2C9 gene is the main reason for causing the activity of the CYP2C9 enzyme to be greatly different among individuals, so that the drug curative effect can be greatly different among individuals carrying different CYP2C9 genotypes, and even serious drug toxic and side effects or insufficient treatment can be generated. Therefore, the research on the influence of the CYP2C9 gene polymorphism on the curative effect of the medicament provides important scientific basis for clinical rational medicament application.

Disclosure of Invention

The invention aims to provide a novel single base mutation site of CYP2C9 gene, a nucleic acid fragment containing the mutation site, a protein fragment coded by the nucleic acid fragment and application of identifying the mutation site in medication guidance.

The first aspect of the present invention provides a nucleic acid fragment comprising a mutation site corresponding to position 1001 of SEQ ID No.1 and being at least 10 consecutive nucleotides of the nucleotide sequence shown in SEQ ID No.1, wherein the nucleotide at position 1001 is G; or the nucleic acid fragment comprises a mutation site corresponding to the 637 th position of SEQ ID No.2 and is at least 10 consecutive nucleotides of the nucleotide sequence shown in SEQ ID No.2, wherein the 637 th nucleotide is G; or a complementary sequence fragment of the above-mentioned nucleic acid fragment.

In a second aspect of the present invention, there is provided a primer for detecting and/or analyzing a single base mutation corresponding to position 1001 of SEQ ID NO.1 or to position 637 of SEQ ID NO.2, said primer being capable of amplifying said single base mutation.

A third aspect of the present invention provides a kit for detecting and/or analyzing a single base mutation, the kit comprising the primer of the present invention.

A fourth aspect of the present invention provides the use of a nucleic acid fragment of the present invention in the preparation of a test marker or a formulation for detecting mutations in the CYP2CP gene.

A fifth aspect of the present invention is to provide a method for analyzing a nucleic acid, comprising analyzing the nucleotide corresponding to position 1001 in a nucleic acid comprising a sequence corresponding to SEQ ID NO.1 in a sample to be tested or analyzing the nucleotide corresponding to position 637 in a nucleic acid comprising a sequence corresponding to SEQ ID NO.2 in a sample to be tested.

In a sixth aspect, the invention provides a CYP2C9 protein or fragment or variant thereof, said protein having the sequence shown in SEQ ID No. 3; the fragment or variant comprises a valine corresponding to position 213 of SEQ ID No.3 and is at least 10 consecutive amino acids of the amino acid sequence shown in SEQ ID No. 3.

The present invention provides CYP2C9 gene and coding sequence containing new single base mutation. The gene is mutated from A to G (637A > -G) at a 637-th nucleotide corresponding to SEQ ID NO.2, so that the coded amino acid is mutated from isoleucine to valine, namely valine corresponding to 213-th nucleotide of SEQ ID NO. 3. The mutant CYP2C9 protein (designated I213V) has higher metabolic activity to drugs than the wild type. The single base mutation has guiding significance for the administration of individuals carrying the mutation site.

Drawings

FIG. 1 is a nucleotide sequencing map at position 1001 corresponding to a sequence of SEQ ID NO.1 of the present invention in example 1;

FIG. 2 is a diagram of the structure of the insect expression vector pFastBac-dual;

FIG. 3 is a Western result chart of each microsomal expressed protein in example 2;

FIG. 4 is a graph of data for each microsomal metabolized tolbutamide from example 3;

figure 5 is a graph of data for the metabolism of losartan by each microsome in example 4.

Detailed Description

The present invention will be described with reference to the following specific embodiments, but the present invention is not limited thereto.

Unless otherwise specified, the "nucleic acid fragment" of the present invention is composed of nucleotides or analogs thereof, and may be a fragment of DNA, RNA or analogs thereof; may be single-stranded or double-stranded; may be natural (e.g. genomic) or synthetic.

In the present invention, the "mutation" refers to the presence of a nucleotide site different in sequence from the wild-type CYP2C9 gene in the gene to be detected, i.e., the CYP2C9 gene. "mutation site" refers to the position at which a base is mutated. In the present invention, the mutation site is a site corresponding to position 1001 in the sequence represented by SEQ ID NO.1 or position 637 in the sequence represented by SEQ ID NO. 2.

The present disclosure relates to non-synonymous mutations of the CYP2C9 gene. Since the mutation site is located in the coding sequence of the gene, it will be appreciated by those skilled in the art that the mutation site may be expressed in either the genomic DNA or the coding sequence (i.e., CDS, corresponding to the mRNA sequence). The skilled person can, depending on the sample to be tested, detect this mutation site on the genomic DNA or mRNA level. In the present application, SEQ ID NO.1 is a genomic DNA sequence of 1kb before and after the mutation site of the present application as a center, that is, the 1001 st position of SEQ ID NO.1 is the mutation site related to the present invention. SEQ ID NO.2 is a cDNA sequence of the CYP2C9 gene having said mutation site, wherein the 637 th position is the mutation site related to the present invention. As is known to those skilled in the art, herein, the 637 th site corresponding to SEQ ID NO.2 and 1001 st site corresponding to SEQ ID NO.1 are used synonymously.

In the present invention, the abbreviations for nucleotides and amino acids are used in the manner known in the art, such as nucleotides wherein A represents adenine, G represents guanine, C represents cytosine and T represents thymine. In the amino acids, A represents alanine, R represents arginine, N represents asparagine, D represents aspartic acid, C represents cysteine, Q represents glutamine, E represents glutamic acid, G represents glycine, H represents histidine, I represents isoleucine, L represents leucine, K represents lysine, M represents methionine, F represents phenylalanine, P represents proline, S represents serine, T represents threonine, W represents tryptophan, Y represents tyrosine, and V represents valine.

The invention relates to a novel single base mutation site based on CYP2C9 gene. The mutation site is positioned in the coding region of the CYP2C9 gene, corresponds to the 637 th site of SEQ ID NO.2, and is mutated from wild type A to G (637A > -G); in addition, the 213 th position of the protein encoded by the mutated CYP2C9 gene is mutated from isoleucine to valine (I213V).

In a first aspect, the present invention provides a nucleic acid fragment comprising a mutation site corresponding to position 1001 of SEQ ID No.1 and being at least 10 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID No.1, wherein the nucleotide at position 1001 is G; or the nucleic acid fragment comprises a mutation site corresponding to the 637 th position of SEQ ID No.2 and is at least 10 consecutive nucleotides of the nucleotide sequence shown in SEQ ID No.2, wherein the 637 th nucleotide is G; or a complementary sequence fragment of the above-mentioned nucleic acid fragment.

In one embodiment, the nucleic acid fragment may be, for example, 10-100, 101-200, 201-500, or 501-1000 nucleotides in length. Preferably, the nucleic acid fragment is 10-20, 221-30, 31-40, 41-50, 51-60, or 61-100 nucleotides in length.

The mutation site may be located at any position of the nucleic acid fragment.

In another embodiment, the nucleic acid fragment is the sequence set forth in SEQ ID NO. 1.

In another embodiment, the nucleic acid fragment is the sequence set forth in SEQ ID NO. 2.

A second aspect of the present invention is to provide a primer for detecting and/or analyzing a single base mutation corresponding to position 1001 of SEQ ID NO.1 or to position 637 of SEQ ID NO.2, which is capable of amplifying the single base mutation.

In one embodiment, the primer has the sequence shown in SEQ ID NO.8 and SEQ ID NO. 9; in another embodiment, the primer has the sequence shown in SEQ ID NO. 18. Wherein SEQ ID NO 8 and SEQ ID NO 9 are amplification primers and SEQ ID NO 18 is a sequencing primer.

A third aspect of the present invention provides a kit for detecting and/or analyzing a single base mutation, the kit comprising the primer of the present invention. Those skilled in the art can configure other reagents in the kit according to actual needs.

A fourth aspect of the present invention provides the use of a nucleic acid fragment of the present invention in the preparation of a test marker or a formulation for detecting mutations in the CYP2C9 gene.

A fifth aspect of the present invention is to provide a method for analyzing a nucleic acid, which comprises analyzing the nucleotide corresponding to position 1001 in a nucleic acid comprising a sequence corresponding to SEQ ID NO.1 in a sample to be tested or analyzing the nucleotide corresponding to position 637 in a nucleic acid comprising a sequence corresponding to SEQ ID NO.2 in a sample to be tested.

In one embodiment, the method may be restriction fragment length polymorphism analysis (RFLP). One skilled in the art can design experiments to analyze whether the nucleotide at position 1001 in the nucleic acid of the sequence of SEQ ID NO.1 or the nucleotide at position 637 in the nucleic acid of the sequence of SEQ ID NO.2 is G, based on the disclosure of the present invention.

In another embodiment, the method may be a sequencing method comprising isolating and determining the nucleic acid sequence from genomic DNA or RNA, analyzing whether the nucleotide corresponding to position 1001 in the nucleic acid comprising the sequence corresponding to SEQ ID No.1 or the nucleotide corresponding to position 637 in the nucleic acid comprising the sequence corresponding to SEQ ID No.2 is G. The sequencing method can be any available sequencing method known in the art. The sequencing primer may be designed according to the common knowledge of those skilled in the art, for example, primers are designed at appropriate positions upstream and downstream of the site to be detected to expand the fragment containing the site to be detected, thereby determining the nucleotide of the site. The oligonucleotides of the invention may also be used as primer sequences.

In another embodiment, the method is a method of specifically identifying whether the nucleotide corresponding to position 1001 in a nucleic acid comprising a sequence corresponding to SEQ ID NO.1 or the nucleotide corresponding to position 637 in a nucleic acid comprising a sequence corresponding to SEQ ID NO.2 in a test sample is G using probe hybridization; the probe employed in the method is an oligonucleotide of the invention. For example, nucleic acids are isolated from a test sample, and the probe is contacted with the nucleic acids under conditions that allow hybridization of the probe to specific target sequences that may be present in the nucleic acids; detectable hybridization can be achieved by using probes labeled with a detectable agent; for example, the probe may be labeled with a radioisotope, a fluorescent dye, or an enzyme that catalyzes the formation of a detectable product. Methods for labeling probes, and for using labeled probes to detect the presence of a target sequence in a sample are well known to those skilled in the art.

In the present invention, the sample may be any sample containing nucleic acids, such as blood; preferably the sample is from a human. The nucleic acid may be DNA or coding RNA, preferably genomic DNA. The method for analyzing nucleic acid of the present invention may use DNA or RNA as a target. As known to those skilled in the art, when DNA is used as a detection target, a nucleotide corresponding to position 1001 in a nucleic acid containing a sequence corresponding to SEQ ID NO.1 in a sample to be detected is analyzed, and a probe or a primer used is designed according to the sequence of SEQ ID NO. 1; when RNA is taken as a detection target object, the nucleotide corresponding to the 637 th position in the nucleic acid containing the sequence corresponding to SEQ ID NO.2 in a sample to be detected is analyzed, and the probe or primer is designed according to the sequence of SEQ ID NO. 2.

In a sixth aspect, the invention provides a CYP2C9 protein or fragment or variant thereof, said protein having the sequence shown in SEQ ID No. 3; the fragment or variant comprises a valine corresponding to position 213 of SEQ ID No.3 and is at least 10 contiguous amino acids, such as 10-20, 21-50 or 51-100 amino acids, of the amino acid sequence shown in SEQ ID No. 3.

The CYP2C9 protein (named I213V) with mutation has higher metabolic activity to drugs than the wild type, thereby having guiding significance to the drug administration of individuals carrying the mutation sites. The drug for CYP2C9 metabolism provided by the invention comprises: anticancer drugs such as cyclophosphamide, ifosfamide or taxol; anticoagulants, such as warfarin, viniferin, anticonvulsants, or mephenytoin; hypoglycemic agents such as tolbutamide, nateglinide, pioglitazone or rosiglitazone; antiepileptic drugs such as phenytoin or zonisamide; antimalarial/antiparasitic agents such as amodiaquine, proguanil hydrochloride or quinine; antipsychotics, such as amitriptyline, citalopram, imipramine, perospirone, sertraline, thioridazine, or venlafaxine; hypotensive agents such as losartan, irbesartan or valsartan; non-steroidal anti-inflammatory drugs such as diclofenac, aminopyrine, antipyrine, celecoxib, flurbiprofen, ibuprofen, indomethacin, lornoxicam, mefenamic acid, naproxen, piroxicam or tenoxicam; analgesics such as loperamide, methadone or morphine; proton pump inhibitors such as lansoprazole or omeprazole; sedatives such as clobazam, meparbital or zopiclone.

The invention will be further illustrated by the following specific examples, which are intended to be exemplary only.

Examples

Example 1: identification of novel mutation site of human CYP2C9 gene

In the embodiment, a patient blood sample clinically using propofol is collected, genomic DNA in blood is extracted, a sequencing primer is designed to perform sequence amplification and sequencing on 9 exons of the CYP2C9 gene, and whether mutation sites exist in the CYP2C9 gene is analyzed.

1) And (3) extracting DNA:

taking 5ml of venous EDTA anticoagulant liquid sample from a tested person; the genomic DNA of the blood sample to be tested is then extracted according to the ordinary salting-out method and/or by using a special DNA extraction kit (DNA extraction kit from Omega, USA).

2) And (3) PCR amplification:

and designing an amplification primer, and amplifying 9 exon sequences of the CYP2C9 gene in the obtained genomic DNA sample. The sequences of the amplification primer pairs are shown in Table 1.

A50. Mu.l PCR reaction was used, including: 1 XPCR buffer, 1.5mM MgCl ₂ 100 to 150ng of genomic DNA, 0.2. Mu.M of upstream and downstream primers, 0.4mM of dNTP, and 1.5U of LATaq DNA polymerase from TaKaRa. The PCR amplification cycle parameters were as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 30 sec, extension at 72 ℃ for 2min and 30 sec, and re-extension after 30 cycles for 5 min. The annealing temperature is related to the length of the primer, and the specific temperature is shown in Table 1.

The amplification was carried out using a GeneAmp PCR System 9700 amplification apparatus from ABI, USA.

Table 1: sequencing primer pair and annealing temperature

3) And (3) purifying an amplification product:

50 ul of PCR amplification product was separated by agarose gel electrophoresis and the band of interest was excised with a razor blade. The DNA of the target band was recovered and purified according to the E.Z.N.A. gel recovery kit (Omega).

4) Sequencing:

using the recovered product as a template, and using a sequencing primer according to CEQ ^TM The DTCS-Quick Start Kit sequencing Kit (Beckman, USA) requires sequencing PCR reaction, and after the reaction is finished and purified, the sequence of the amplified product is identified by separating with a CEQ8000 type gene sequencer (Beckman, USA). The sequencing primers are shown in Table 2.

Table 2: sequencing primer

Region(s)	Sequencing primer (5 '-3')
		Exon 1	TACCTCTAGGGATACAC(SEQ ID NO.16)
Exon 2&3	CTAACAACCAGGACTCATAAT(SEQ ID NO.17)
		Exon 4	TTGCTGTTAAGGGAATTTGTAGGTAAGATA(SEQ ID NO.18)
Exon 5	TAGTGGTCTATTTTGTTATTCATTCAT(SEQ ID NO.19)
		Exon 6	TTCCAGTTTCTATGTTG(SEQ ID NO.20)
Exon 7	ACCCGGTGATGGTAGAGGTT(SEQ ID NO.21)
		Exon 8	ACGGGATTTCCTCATCTG(SEQ ID NO.22)
Exon 9	CGATACACTGAACAGTTATTGC(SEQ ID NO.23)

5) And (3) data analysis:

the determined sequence was aligned with the wild-type CYP2C9 x 1 sequence (GenBank accession No. NM — 000771.3).

Through alignment analysis, the 637 th nucleotide of the coding region of the CYP2C9 gene is changed from A to G (as shown in FIG. 1, wherein R represents A or G), and the mutation is located in the 4 th exon of the CYP2C9 gene. Accordingly, it was concluded that in the protein encoded by the CYP2C9 gene, the 213 th amino acid was mutated from isoleucine (I) to valine (V). The mutation is now not named by the P450 naming Committee and is not published externally.

Methods for identifying new mutation sites are presented in this example. The person skilled in the art will clearly understand from the above that the method for specifically detecting the nucleotide 1001 of the sample to be tested, which nucleotide corresponds to SEQ ID NO.1: isolating the nucleic acids in the sample and carrying out the amplification reaction under the experimental conditions corresponding to this example, using the primer pair SEQ ID NO.8 and 9; sequencing the amplified product by using a sequencing primer SEQ ID NO. 18; the sequencing result was compared with the wild type result, and the nucleotide corresponding to the 1001 st position of SEQ ID NO.1 was analyzed.

Example 2: expression of target genes

Plasmid vector connected with open reading frame of wild type CYP2C9 x 1 (presented by Zhou tref professor of the university of south Florida) is used as template, and site-directed mutagenesis technology is utilized to obtain open reading frames of CYP2C9 x 2 (430C > T), CYP2C9 x 3 (1075A >) and I213V mutant of the invention. Site-directed mutagenesis techniques are well known in the art and one skilled in the art would know without doubt how to accomplish this step based on the defined template and target.

Then, the ORFs of the CYP2C9 x 1 gene and the three mutant genes subjected to site-directed mutagenesis are cloned into a vector pFastBac-dual connected with cytochrome P450 Oxidoreductase (OR), so that the CYP2C9 gene and the OR are respectively placed in a PH promoter and a P10 promoter, and a double expression vector for simultaneously expressing the OR and the CYP2C9 (OR mutants thereof) is constructed. The structure of the pFastBac-dual vector and the insertion sites of the CYP2C9 gene and OR are shown in FIG. 2.

According to the use instruction of a Bac-to-Bac baculovirus expression system kit (purchased from Invitrogen company in America and used for expressing a large amount of exogenous target genes in insect cells), P1 generation insect viruses and P2 generation insect viruses are respectively packaged by using a constructed double expression vector and a control vector, and the obtained P2 generation viruses are infected into sf21 insect cells according to the infection amount of MOI (multiple-of-infection) of 4 after the titer is determined. After 72 hours of infection, the cells were collected by centrifugation, and the cells were disrupted by ultrasonication using an ultrasonication apparatus (SONIC, USA) at an energy of 40%, and the insect cell microsomes were extracted by differential centrifugation. Detecting the expression quantity of CYP2C9 and OR in each microsome by using a Western method; the total enzyme concentration of CYP2C9 in each microsome was determined by the reduced CO differential spectrometry.

The Western results are shown in FIG. 3. The first row shows the amount of CYP2C9 expression, and the second row shows the amount of OR expression. As can be seen from the figure, all 4 dual expression vectors expressed the OR protein and expressed 1, 2, CYP2C9 of type 3 and I213V protein of the present invention, respectively. The expression product is sequenced and is consistent with the wild type and mutant type sequences preset in the experiment.

Enzyme metabolic activity assay

According to the results of the present studies, the metabolic activity of the wild type (. X.1 type) was higher for various drugs, while the metabolic activity of the.x.2 and.x.3 types was significantly altered from that of the wild type. Thus, there is a consensus in the art that: the metabolic activity of an enzyme expressed by the same genotype on a specific substrate may represent the metabolic activity on other substrate drugs. Thus, the metabolic activity of an enzyme expressed by a certain genotype on a specific substrate can be analogized from the metabolic activity data of the enzyme expressed by that genotype on other substrate drugs (e.g., the metabolic activity of the enzyme expressed by that genotype can be compared with the metabolic activity of the enzyme expressed by the wild-type).

Example 3: the metabolic characteristics of the p-tolbutamide were analyzed in vitro using the obtained insect microsomes:

1) Chromatographic and mass spectrometric conditions: the analysis was performed by the LC-MS method. The column was a Waters Acquity UPLC BEH C18 reverse phase column (2.1 x 50mm,1.7 μm, waters Corp, usa); mobile phase a is 0.1% formic acid; the mobile phase B is acetonitrile; the column temperature was 40 ℃ and the flow rate was 0.4ml/min, and gradient elution was carried out: 0-1.4min, A (60% -10%), 1.4-2.6min and A (10% -60%). Electrospray ion source (ESI) selecting a positive ion mode scan; the ion source temperature is 500 ℃, the signal acquisition mode is multi-stage reaction monitoring, and the parameters of metabolites and internal standards are shown in the following table:

2) Incubation conditions were as follows:

the total volume of the reaction was 200. Mu.L, including: 100mM Tris-HCl (pH 7.4), 1 XNADPH coenzyme generation system, 2pmol cytochrome b5 and tolbutamide (purchased from Sigma, USA, final concentration 50-2000. Mu.M). After preincubation at 37 ℃ for 5min, 1pmol of the recombinant microsome constructed in example 2 was added to initiate the reaction. After incubation at 37 ℃ for 40min, 200. Mu.L of 0.1M ACN and 30. Mu.L of 200 ng/. Mu.L internal standard midazolam (purchased from Sigma, USA) were added and centrifuged at 10,000 Xg for 10min at 4 ℃ with vortexing. Taking 2 mu L to be detected by a Waters XEVO TQD liquid chromatography-mass spectrometer.

The results of the Michaelis-Menten data analysis of this example are shown in FIG. 4. Further pharmacokinetic analysis was performed and the results are shown in table 3:

table 3: pharmacokinetic analysis results of tolbutamide metabolized by each microsome

As can be seen from fig. 4 and table 3, the overall enzymatic activity of I213V of the present invention was higher than that of the wild type x 1 type and the mutant type x 2 and the mutant type x 3.

Example 4: in vitro analysis of the metabolic characteristics of losartan by using the obtained insect microsomes

1. Chromatographic and mass spectrometric conditions: the analysis was performed by the LC-MS method. The column was a Waters Acquity UPLC BEH C18 column (2.1 x 50mm,1.7- μm, waters Corp, USA); mobile phase a was 0.1% formic acid; the mobile phase B is acetonitrile; the column temperature was 40 ℃, the flow rate was 0.4ml/min, and the mobile phase was a: B = 25. Electrospray ion source (ESI) selecting a positive ion mode scan; the ion source temperature is 500 ℃, the signal acquisition mode is multi-stage reaction monitoring, and the parameters of metabolites and internal standards are shown in the following table:

name (R)	Parent ion (m/z)	Sub-ion (m/z)	Collision energy (eV)
				Losartan carboxylic acid (E-3174)	437.2	235.2	15
Midazolam	326.1	291.1	30

2. Incubation conditions were as follows:

the total reaction volume was 200. Mu.L, which included: 100mM Tris-HCl (pH 7.4), 1 XNADPH coenzyme production system, 2pmol cytochrome b5 and losartan (purchased from Sigma, USA, final concentration of 0.1-25. Mu.M). After preincubation at 37 ℃ for 5min, 1pmol of the recombinant microsomes constructed in example 2 was added to initiate the reaction. After incubation at 37 ℃ for 40min, 200. Mu.L of 0.1M ACN and 30. Mu.L of 200 ng/. Mu.L internal standard midazolam (purchased from Sigma, USA) were added and centrifuged at 10,000 Xg for 10min at 4 ℃ with vortexing. Taking 2 mu L to be detected by a Waters XEVO TQD liquid chromatography-mass spectrometer.

The results of the Michaelis-Menten data analysis of this example are shown in FIG. 5. Further pharmacokinetic analysis was performed and the results are shown in table 4:

table 4: results of pharmacokinetic analysis of losartan metabolized by each microsome

As can be seen from fig. 5 and table 4, the overall enzymatic activity of I213V of the present invention was much higher than the wild type x 1 type, and also higher than the mutant type x 2 and x 3 types.

As can be seen from the above examples, the metabolic activity of the I213V mutant enzyme of the present invention is much higher than that of the wild type x 1 type. Therefore, in practice, it is considered that the individual carrying the genotype is appropriately adjusted in the dosage of the drug, such as increasing the amount of the drug to be used. This gene-directed drug modulation is more important for fast-metabolizing drugs.

The sequence is as follows:

SEQ ID NO.1: genomic DNA sequence

TGGAGGAACCTGTCTCTACTAAATATACAAAATTAGCTGGATGTGGTGGCACATG CCTGTAATCCCAGCTACCTGGGAAGCTCAGGTGGAGAATCACTTGAACCTAGGAAGC AGAGGTTGTGGTGAGCTGAGATCGTGCCATTACACTCCAGCCCAGGCAACAAGAGTG AAACTCCATCTCAAAACAAACAAATAAACAAAAAACAACAACAACAAAAAACAACA ACAAAGTTTTCATTTAATTTTTTAACAAACTAATCTATGTTAATTTGATATTAGTTTGTTA ACATAACAAACTAATAATATGTTAGGCATATTATTCATTTATATTTGTTTTCCTTCTCATA AAACACATTCTGCTCTTACATTTATTTTCTTAAATACATGAATTCACCAACAGTCTCTTT GGTTCCTCTCTACTGGTTCAACACATGACTCTTTCAGCTATTCATTATATATAAAAAGCT TTGAAATCTCCAACTATTCTTGCCCTTTCCATCTCAGTGCCTTGCTGTCTACTGACTTTG CAGACTGATGTGATTCCCTCTGAAACATGAATTATTAGGTTTTTAGAAAATGCCTTTTT GTTCTTTCCAAAGTAAAAGACAAATAGGCTGGGAATGTAAATTTAGCATTTGAACAAC CATTATTTAACCAGCTAGGTTGTAATGGTCAACTCAGGATTAATGTAAAAGTGAAGTGT TGATTTTATGCATGCCGAACTCTTTTTTGCTGTTAAGGGAATTTGTAGGTAAGATAATTT CTAAACTACTATTATCTGTTAACAAATACAGTGTTTTATATCTAAAGTTTAATAGTATTTT AAATTGTTTCTAATTATTTAGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCTC CCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAAT TTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGGT CCAGGTAAGGCCAAGATTTTTGCTTCCTGAGAAACCACTTATTCTCTTTTTTTTCTGAC AAATCCAAAATTCTACATGGATCAAGCTCTGAAGTGCATTTTTGAATACTACAGTCTTG CCCAGACAGCCTTGGGGTGAATATCTGGGAAAGACGGCCAAGCCCTTTATTTTATGCA TGGGAAATAAATGCCTCAATATAGGCCTGATTTCTAAGCCCATTAGCTCCCTCATCAAT GTTTTTTCTGCTAAACTCCAAAGCCCTGTTTCTGTAAAGTACTTTGTTGACAGCCCTAA AGCGTGCTCATAGCACTCCATGGATATCCAGGCACTTTGGAGGCTCCCATTACTCACA AGACTTGTCCTTCAATTAACACTTTGTCGTATTATGTGGCAGAAATATCCTAATTTAAAA GACATTATCTCCTTCTAGTAGGGAGAATATTTGGGACCATAGAAGCTGCCAAGAAACA CTGAATAGGGCAGGGGTGTTTGATATCTCAGTTGGGATCCTAGCTGATGAGATAGCTG GGTTAGGAATGACAAAATTATTGGTTTTATGGTGTATGAACCATAAACAGACATCACAC TTATACCCTGTGCTGAGCTGGCATGTTTTATTCTCTGCCTAAATAATAATTGTGTGATTTT ATAGAAGTCATTTAACTGCTCTGGTGCACAGTTGGAATTTGAAGTATCTTTGAGCCCCT CCCACTTCTAAAATACTAATTCCATTTCAGAGGCTGCTTGATAGAAATCAATATAGTAG GGACTAGCTTTGTACTATCAATCAGGTTGTCCAAATTCTTTTAACCTATGCTGTCATCTA CAAAACGTGAATGTAGTAATTCATGCCATCTTATATTTCAAGATTATAGAGAAGAATTGT AAAAAGTAACAGAATTAACATAAAGATGCTTTTATACTATAAAAAGGAGGTCTGAGTC TAGGAAATGATTATCATCTGGTTAGAATTGATCCTCTGGTCAGAATTTTCTTTCT

SEQ ID NO.2: coding sequence

ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTC TGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTG ATTGGAAATATCCTACAGATAGGTATTAAGGACATCAGCAAATCCTTAACCAATCTCTC AAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGC ATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGCAAG AGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAAT GGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGG ATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGA GGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCT CCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAA TTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGG TCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAAT TACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCA AGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATG GAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACT GCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCT CTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAA CGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTAC ACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGC CCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAAC CATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGA TGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTC ATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAG CTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCC AAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTAC CAGCTGTGCTTCATTCCTGTCTGA

SEQ ID NO.3: protein sequences

MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSK VYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSARGIFPLAERANRGFGIVFSNGKKW KEIRRFSLMTLRNFGMGKRSIEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICSII FHKRFDYKDQQFLNLMEKLNENIKILSSPWVQICNNFSPIIDYFPGTHNKLLKNVAFMKS YILEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETT STTLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLL PTSLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKY FMPFSAGKRICVGEALAGMELFLFLTSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQL CFIPV

Reference documents:

1.Aquilante CA.Sulfonylurea pharmacogenomics in Type 2diabetes:the influence of drug target and diabetes risk polymorphisms.Expert Rev Cardiovasc Ther.2010；8(3): 359–372.

2.Xu HM,Murray M,Mclachlan AJ.Influence of genetic polymorphisms on the pharmacokinetics and pharmacodynamics of sulfonylurea drugs.Current Drug Metabolism. 2009；10(6):643-658.

3.Wang B,Wang J,Huang SQ,et al.Genetic polymorphism of the human cytochrome P450 2C9 gene and its clinical significance.Current Drug Metabolism.2009；10(7):781-834。

4. the polymorphism of the plum, the royal jelly, the primary and secondary nucleotides CYP2C9 gene and the research progress of the functional significance thereof, chinese clinical pharmacology and therapeutics 2008;13 (6):601-609.

5.Zhou Sh.F,Liu J.P.Chowbay B.Polymorphism of human cytochrome P450 enzymes and its clinical impact.Drug Metab Rev.2009；41(2):89-295。

Sequence listing

<110> Chua Jianping

<120> CYP2C9 gene segment containing 637A >

<130> DSP1F192374YJ

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2001

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

tggaggaacc tgtctctact aaatatacaa aattagctgg atgtggtggc acatgcctgt 60

aatcccagct acctgggaag ctcaggtgga gaatcacttg aacctaggaa gcagaggttg 120

tggtgagctg agatcgtgcc attacactcc agcccaggca acaagagtga aactccatct 180

caaaacaaac aaataaacaa aaaacaacaa caacaaaaaa caacaacaaa gttttcattt 240

aattttttaa caaactaatc tatgttaatt tgatattagt ttgttaacat aacaaactaa 300

taatatgtta ggcatattat tcatttatat ttgttttcct tctcataaaa cacattctgc 360

tcttacattt attttcttaa atacatgaat tcaccaacag tctctttggt tcctctctac 420

tggttcaaca catgactctt tcagctattc attatatata aaaagctttg aaatctccaa 480

ctattcttgc cctttccatc tcagtgcctt gctgtctact gactttgcag actgatgtga 540

ttccctctga aacatgaatt attaggtttt tagaaaatgc ctttttgttc tttccaaagt 600

aaaagacaaa taggctggga atgtaaattt agcatttgaa caaccattat ttaaccagct 660

aggttgtaat ggtcaactca ggattaatgt aaaagtgaag tgttgatttt atgcatgccg 720

aactcttttt tgctgttaag ggaatttgta ggtaagataa tttctaaact actattatct 780

gttaacaaat acagtgtttt atatctaaag tttaatagta ttttaaattg tttctaatta 840

tttagcctca ccctgtgatc ccactttcat cctgggctgt gctccctgca atgtgatctg 900

ctccattatt ttccataaac gttttgatta taaagatcag caatttctta acttaatgga 960

aaagttgaat gaaaacatca agattttgag cagcccctgg gtccaggtaa ggccaagatt 1020

tttgcttcct gagaaaccac ttattctctt ttttttctga caaatccaaa attctacatg 1080

gatcaagctc tgaagtgcat ttttgaatac tacagtcttg cccagacagc cttggggtga 1140

atatctggga aagacggcca agccctttat tttatgcatg ggaaataaat gcctcaatat 1200

aggcctgatt tctaagccca ttagctccct catcaatgtt ttttctgcta aactccaaag 1260

ccctgtttct gtaaagtact ttgttgacag ccctaaagcg tgctcatagc actccatgga 1320

tatccaggca ctttggaggc tcccattact cacaagactt gtccttcaat taacactttg 1380

tcgtattatg tggcagaaat atcctaattt aaaagacatt atctccttct agtagggaga 1440

atatttggga ccatagaagc tgccaagaaa cactgaatag ggcaggggtg tttgatatct 1500

cagttgggat cctagctgat gagatagctg ggttaggaat gacaaaatta ttggttttat 1560

ggtgtatgaa ccataaacag acatcacact tataccctgt gctgagctgg catgttttat 1620

tctctgccta aataataatt gtgtgatttt atagaagtca tttaactgct ctggtgcaca 1680

gttggaattt gaagtatctt tgagcccctc ccacttctaa aatactaatt ccatttcaga 1740

ggctgcttga tagaaatcaa tatagtaggg actagctttg tactatcaat caggttgtcc 1800

aaattctttt aacctatgct gtcatctaca aaacgtgaat gtagtaattc atgccatctt 1860

atatttcaag attatagaga agaattgtaa aaagtaacag aattaacata aagatgcttt 1920

tatactataa aaaggaggtc tgagtctagg aaatgattat catctggtta gaattgatcc 1980

tctggtcaga attttctttc t 2001

<210> 2

<211> 1473

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 2

atggattctc ttgtggtcct tgtgctctgt ctctcatgtt tgcttctcct ttcactctgg 60

agacagagct ctgggagagg aaaactccct cctggcccca ctcctctccc agtgattgga 120

aatatcctac agataggtat taaggacatc agcaaatcct taaccaatct ctcaaaggtc 180

tatggccctg tgttcactct gtattttggc ctgaaaccca tagtggtgct gcatggatat 240

gaagcagtga aggaagccct gattgatctt ggagaggagt tttctgcaag aggcattttc 300

ccactggctg aaagagctaa cagaggattt ggaattgttt tcagcaatgg aaagaaatgg 360

aaggagatcc ggcgtttctc cctcatgacg ctgcggaatt ttgggatggg gaagaggagc 420

attgaggacc gtgttcaaga ggaagcccgc tgccttgtgg aggagttgag aaaaaccaag 480

gcctcaccct gtgatcccac tttcatcctg ggctgtgctc cctgcaatgt gatctgctcc 540

attattttcc ataaacgttt tgattataaa gatcagcaat ttcttaactt aatggaaaag 600

ttgaatgaaa acatcaagat tttgagcagc ccctgggtcc agatctgcaa taatttttct 660

cctatcattg attacttccc gggaactcac aacaaattac ttaaaaacgt tgcttttatg 720

aaaagttata ttttggaaaa agtaaaagaa caccaagaat caatggacat gaacaaccct 780

caggacttta ttgattgctt cctgatgaaa atggagaagg aaaagcacaa ccaaccatct 840

gaatttacta ttgaaagctt ggaaaacact gcagttgact tgtttggagc tgggacagag 900

acgacaagca caaccctgag atatgctctc cttctcctgc tgaagcaccc agaggtcaca 960

gctaaagtcc aggaagagat tgaacgtgtg attggcagaa accggagccc ctgcatgcaa 1020

gacaggagcc acatgcccta cacagatgct gtggtgcacg aggtccagag atacattgac 1080

cttctcccca ccagcctgcc ccatgcagtg acctgtgaca ttaaattcag aaactatctc 1140

attcccaagg gcacaaccat attaatttcc ctgacttctg tgctacatga caacaaagaa 1200

tttcccaacc cagagatgtt tgaccctcat cactttctgg atgaaggtgg caattttaag 1260

aaaagtaaat acttcatgcc tttctcagca ggaaaacgga tttgtgtggg agaagccctg 1320

gccggcatgg agctgttttt attcctgacc tccattttac agaactttaa cctgaaatct 1380

ctggttgacc caaagaacct tgacaccact ccagttgtca atggatttgc ctctgtgccg 1440

cccttctacc agctgtgctt cattcctgtc tga 1473

<210> 3

<211> 490

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 3

Met Asp Ser Leu Val Val Leu Val Leu Cys Leu Ser Cys Leu Leu Leu

1 5 10 15

Leu Ser Leu Trp Arg Gln Ser Ser Gly Arg Gly Lys Leu Pro Pro Gly

20 25 30

Pro Thr Pro Leu Pro Val Ile Gly Asn Ile Leu Gln Ile Gly Ile Lys

35 40 45

Asp Ile Ser Lys Ser Leu Thr Asn Leu Ser Lys Val Tyr Gly Pro Val

50 55 60

Phe Thr Leu Tyr Phe Gly Leu Lys Pro Ile Val Val Leu His Gly Tyr

65 70 75 80

Glu Ala Val Lys Glu Ala Leu Ile Asp Leu Gly Glu Glu Phe Ser Ala

85 90 95

Arg Gly Ile Phe Pro Leu Ala Glu Arg Ala Asn Arg Gly Phe Gly Ile

100 105 110

Val Phe Ser Asn Gly Lys Lys Trp Lys Glu Ile Arg Arg Phe Ser Leu

115 120 125

Met Thr Leu Arg Asn Phe Gly Met Gly Lys Arg Ser Ile Glu Asp Arg

130 135 140

Val Gln Glu Glu Ala Arg Cys Leu Val Glu Glu Leu Arg Lys Thr Lys

145 150 155 160

Ala Ser Pro Cys Asp Pro Thr Phe Ile Leu Gly Cys Ala Pro Cys Asn

165 170 175

Val Ile Cys Ser Ile Ile Phe His Lys Arg Phe Asp Tyr Lys Asp Gln

180 185 190

Gln Phe Leu Asn Leu Met Glu Lys Leu Asn Glu Asn Ile Lys Ile Leu

195 200 205

Ser Ser Pro Trp Val Gln Ile Cys Asn Asn Phe Ser Pro Ile Ile Asp

210 215 220

Tyr Phe Pro Gly Thr His Asn Lys Leu Leu Lys Asn Val Ala Phe Met

225 230 235 240

Lys Ser Tyr Ile Leu Glu Lys Val Lys Glu His Gln Glu Ser Met Asp

245 250 255

Met Asn Asn Pro Gln Asp Phe Ile Asp Cys Phe Leu Met Lys Met Glu

260 265 270

Lys Glu Lys His Asn Gln Pro Ser Glu Phe Thr Ile Glu Ser Leu Glu

275 280 285

Asn Thr Ala Val Asp Leu Phe Gly Ala Gly Thr Glu Thr Thr Ser Thr

290 295 300

Thr Leu Arg Tyr Ala Leu Leu Leu Leu Leu Lys His Pro Glu Val Thr

305 310 315 320

Ala Lys Val Gln Glu Glu Ile Glu Arg Val Ile Gly Arg Asn Arg Ser

325 330 335

Pro Cys Met Gln Asp Arg Ser His Met Pro Tyr Thr Asp Ala Val Val

340 345 350

His Glu Val Gln Arg Tyr Ile Asp Leu Leu Pro Thr Ser Leu Pro His

355 360 365

Ala Val Thr Cys Asp Ile Lys Phe Arg Asn Tyr Leu Ile Pro Lys Gly

370 375 380

Thr Thr Ile Leu Ile Ser Leu Thr Ser Val Leu His Asp Asn Lys Glu

385 390 395 400

Phe Pro Asn Pro Glu Met Phe Asp Pro His His Phe Leu Asp Glu Gly

405 410 415

Gly Asn Phe Lys Lys Ser Lys Tyr Phe Met Pro Phe Ser Ala Gly Lys

420 425 430

Arg Ile Cys Val Gly Glu Ala Leu Ala Gly Met Glu Leu Phe Leu Phe

435 440 445

Leu Thr Ser Ile Leu Gln Asn Phe Asn Leu Lys Ser Leu Val Asp Pro

450 455 460

Lys Asn Leu Asp Thr Thr Pro Val Val Asn Gly Phe Ala Ser Val Pro

465 470 475 480

Pro Phe Tyr Gln Leu Cys Phe Ile Pro Val

485 490

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacaatggaa cgaaggagaa caagaccaaa ggac 34

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtttcattc cactatttct gacactgaca 30

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tacaaataca atgaaaatat catg 24

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctaacaacca ggactcataa t 21

<210> 8

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctattcttgc cctttccatc tcagtgcctt g 31

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cttgttattg gtctattcag ggatttgact 30

<210> 10

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

taggcaagca tggaataagg gagtagg 27

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aatcaccatt agtttgaaac agattacagc 30

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cccctgaatt gctacaacaa a 21

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acccggtgat ggtagaggtt 20

<210> 14

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cttctttgga acgggatttc ctcatctgc 29

<210> 15

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tctgtcctta tcattttgag aaccagcat 29

<210> 16

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tacctctagg gatacac 17

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ctaacaacca ggactcataa t 21

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttgctgttaa gggaatttgt aggtaagata 30

<210> 19

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tagtggtcta ttttgttatt cattcat 27

<210> 20

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ttccagtttc tatgttg 17

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

acccggtgat ggtagaggtt 20

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acgggatttc ctcatctg 18

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cgatacactg aacagttatt gc 22

Claims (3)

1. The sequence of the nucleic acid fragment is shown as SEQ ID NO.1 or SEQ ID NO.2, or is a complementary sequence of the sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. Use of the nucleic acid fragment of claim 1 for the preparation of a test marker for detecting mutations in the CYP2C9 gene.

CYP2C9 protein, wherein the protein sequence is shown as SEQ ID NO. 3.

Images