JPH1014585A - New oligonucleotide primer and examination of point mutation in exon 4 of human cytochrome p450 2c19 gene using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new primer having each specific base sequences, capable of detecting the point mutation m2 in exon 4 of human cytochrome P450 2C19 gene, and useful for dysbolism examination of (S)-mephenytoin as an anticonvulsant. SOLUTION: This primer is a new oligonucleotide primer having base sequences of respective formulas I and II and useful for examining the point mutation m2 in exon 4 of human cytochrome P450 2C19 gene; thus being useful in the clinicat examination of the dysbolism of (S)-mephenytoin as an anticonvulsant and capable of classifying the genotype (genotyping). This primer is obtained by synthesizing a base sequence hybridizable with the domain of 610-629nt (on a cDNA basis) in exon 4 of human cytochrome P450 2C19 gene and another base sequence hybridizable with the domain of 67-86nt (on a cDNA basis) in intron 4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human cytochrome.
Oligonucleotide primers used for examining point mutations in exon 4 of P450 2C19 gene (hereinafter sometimes referred to as “CYP2C19”) and method for examining mutations in exon 4 of human cytochrome P450 2C19 gene using the same About.

[0002]

BACKGROUND ART Genetic polymorphism is observed in the metabolic ability of (S) -mephenytoin, an anticonvulsant. This genetic polymorphism
Not only the metabolism of (S) -mephenytoin but also the genetically important diazepam, imipramine, omeprazole and propranolol, and the selective serotonin reuptake inhibitor (serotonin reuptake inhibitor) It is clinically important to investigate metabolic abnormalities of (S) -mephenytoin.

[0003] Conventionally, metabolic abnormalities of (S) -mephenytoin are as follows:
The test was performed by orally administering (S) -mephenytoin to a subject and quantifying 4′-hydroxymephenytoin contained in urine excreted 8 hours later. However,
This method is time consuming and requires administration of unnecessary medication to the subject.

[0004] The genetic cause of metabolic abnormalities of (S) -mephenytoin has been elucidated, at least for the Japanese. That is, a point mutation in which G in exon 5 of CYP2C19 becomes A (the site is 681 nt of CYP2C19 cDNA,
This point mutation is referred to herein as "m1")
(Sonia MF de Morais et al., The Journal of Biol
ogical Chemistry Vol.269, No. 22, pp.15419-15422,
1994 (hereinafter referred to as “Reference 1”)) and CYP2
A point mutation in which G in exon 4 of C19 becomes A (the site is 636 nt of CYP2C19 cDNA, and this point mutation is referred to as “m2” in the present specification) (Sonia MF de Mo
rais et al., Molecular Pharmaoclogy, 46: 594-598,19
94 (hereinafter, this document is referred to as “document 2”)), it is known that metabolic abnormality of (S) -mephenytoin occurs. Therefore, by examining the above m1 and m2 in exons 5 and 4 of CYP2C19,
(S) -mephenytoin metabolic abnormality can be tested,
Furthermore, the genotype can be classified (genotyping).

[0005] References 1 and 2 disclose PCR,
-A method for detecting m1 and m2 by the RFLP method is described. That is, PCR is performed for each of exon 5 and exon 4 using each pair of primers, and the amplified product is digested with a restriction enzyme and separated by electrophoresis, whereby fragments having different sizes depending on the presence or absence of the mutation are obtained. Is detected.

[0006]

In general, when conducting a clinical test at a clinical test center or the like, it is necessary to test a very large number of samples within a limited time. is important. When performing a genetic test for metabolic abnormality of (S) -mephenytoin, it is necessary to test both m1 in exon 5 and m2 in exon 4 as described above. If the tests for m1 and m2 can be performed under the same PCR conditions, both tests can be performed simultaneously using the same apparatus, which is very efficient. The present inventors used the primers described in the above References 1 and 2 to perform PCR for detecting m1.
In order to carry out PCR for the detection of m2 under the same conditions as above, intensive research was carried out, but there was a problem that many nonspecific bands would appear in the detection of m2. When processing a large number of samples within a limited time, it is not preferable that a large number of nonspecific bands cause misdiagnosis.

Accordingly, an object of the present invention is to detect m1 in exon 5 and m2 in exon 4 of CYP2C19 using the same P.
An object of the present invention is to provide a means for performing under a CR condition without the appearance of a non-specific band.

[0008]

Means for Solving the Problems As a result of intensive studies, the inventors of the present invention have found that by using an oligonucleotide primer different from that described in Reference 2 as an oligonucleotide primer for detecting m2, The present inventors have found that the same PCR can be performed under the same conditions as the PCR for detecting m2, and the present invention has been completed.

[0009] That is, the present invention relates to SEQ ID NO: 1 in the sequence listing.
And having the base sequence shown in SEQ ID NO: 2, respectively.
An oligonucleotide primer for testing a point mutation m2 in exon 4 of the human cytochrome P450 2C19 gene is provided. Furthermore, the present invention provides a step of performing PCR using the above-described two kinds of oligonucleotide primers of the present invention as primers and using the human cytochrome P450 2C19 gene as a template, and a step of analyzing the obtained PCR product by a restriction fragment length polymorphism method. Including, human cytochrome P450 2C19
A method for testing a point mutation m2 in exon 4 of a gene is provided.

[0010]

DETAILED DESCRIPTION OF THE INVENTION As described above, the oligonucleotide primer of the present invention is for detecting the above-mentioned point mutation m2 in exon 4 of CYP2C19. forwar
The d-side primer (hereinafter, sometimes referred to as “primer F”) has a base sequence represented by SEQ ID NO: 1 in the sequence listing, and the reverse-side primer (hereinafter, sometimes referred to as “primer R”) has It has the base sequence shown by 2. Primer F is 61 in exon 4 of CYP2C19
The primer R hybridizes with a region of 67 to 86 nt in the intron 4 of CYP2C19 (the first base of the intron 4 is 1 nt).

When PCR is performed using the primers F and R of the present invention, annealing is performed at 53 to 63 ° C.
For 1-2 minutes at 72 ° C. for 1-2 minutes. The denaturation condition is not particularly limited as long as the denaturation is performed, but it is usually performed at 94 ° C. for 1 minute to 2 minutes. Although the number of cycles is not particularly limited, 20 to 40 cycles are preferable. Further, the concentration of magnesium chloride in the PCR buffer is preferably 1.0 to 2.0 mM. The PCR method itself is well known in this field, and kits and devices for necessary reagents are commercially available, so that the PCR method can be easily carried out using them.

[0012] By using the primer of the present invention,
PCR for detecting m1 in exon 5 of CYP2C19
PCR can now be performed under the same thermal conditions as described above. Therefore, PCR for testing m1 and PCR for testing m2 can be performed simultaneously in the same apparatus. The primer for detecting m1 is described in Reference 1, and the primers for forward and reverse
The nucleotide sequences of the primers on the side are shown in SEQ ID NOs: 3 and 4 in the sequence listing, respectively. When the primer of the present invention is used, m1
Not only are the thermal conditions the same as PCR for the detection of
Since the same PCR buffer can be used, the testing efficiency is further improved. In addition, even when the PCR for the test of m2 is performed simultaneously with the PCR for the test of m1 in the same apparatus, the reaction is carried out in a separate tube.

After PCR, the obtained PCR product is subjected to restriction fragment length polymorphism analysis (RFLP). RFLP itself is a well-known method. That is, by digesting the PCR product with a restriction enzyme, the PCR product is digested with a restriction enzyme that generates fragments having different lengths depending on the presence or absence of a point mutation, separated by electrophoresis, and the band size is measured. How to When m2 is examined by performing PCR using the primers F and R of the present invention, the restriction enzyme BamHI can be used. That is, when the PCR product is digested with BamHI, if m2 is present in the gene, a band of 119 bp appears when separated by gel electrophoresis, and a wild-type (hereinafter, wild-type is referred to as “wt”) In some cases, a band of 93 bp appears, so that it can be examined whether m2 exists in the sample gene. Since there is one pair of homologous chromosomes,
In the case of wt homozygosity, only a 93 bp band appears, in the case of m2 homozygosity, only a 119 bp band appears, and in the case of wt and m2 heterozygosity, both bands appear. Therefore, the genotype can also be tested by the method of the present invention. The restriction enzymes that can be used for RFLP are not limited to BamHI,
Other restriction enzymes that generate fragments of different sizes depending on the presence or absence of m2 can be used.

The RFLP for the examination of m1 can be performed using SmaI as described in Reference 1, or can be performed using MspI as described in Examples below. When MspI was used, a band of 120 bp appeared in wt and 169 bp in m1.
Band appears.

[0015]

The present invention will be described more specifically below with reference to examples. However, the present invention is not limited to the following examples.

(1) Genotyping From 186 Japanese, 7 ml of venous blood was collected and DNA was extracted from peripheral lymphocytes using a commercially available extraction kit. PCR was performed using the genomic DNA obtained using the primers F and R of the present invention as a template. Further, having the nucleotide sequence shown in SEQ ID NO: 3 or 4,
PCR was performed simultaneously in a separate tube using the primers described in Reference 1, but in the same apparatus. 200 μM deoxyribonucleoside triphosphate (dNTP) mixture (dATP,
PCR buffer containing dCTP, dGTP and dTTP (Takara Shuzo), 0.2 μM of each primer, 1.25 units of AmpliTaq DNA polymerase (Hoffmann-La Roche) and 1.5 mM magnesium chloride (10 mM Tris-HCl, pH 8.3,
200 ng of the genomic DNA was amplified in 50 mM KCl, 0.01% gelatin). Amplification was performed using a thermocycler manufactured by Perkin Elmer. After first denaturation at 94 ° C for 5 minutes, a denaturation step of 94 ° C for 1 minute, 57 ° C,
A cycle consisting of a 1 minute annealing step, a 72 ° C., 2 minute elongation step was repeated 40 times, and finally a 5 minute
Extension was performed for minutes. Next, 25 units of MspI were added to the tube for the m1 test, and 25 units were added to the tube for the m2 test.
A unit of BamHI was added and incubated at 37 ° C. for 1 hour to perform a restriction enzyme cleavage step. The digested PCR amplification product was electrophoresed on a 3% agarose gel and stained with ethidium bromide.

Typical results are shown in FIG. As expected, the MspI digest had a 169 bp band or 120 b
The p band was observed, and both bands were observed for the heterozygous one. As expected, a band of 119 bp or 93 bp was observed in the BamHI digest, and both bands were observed in the heterozygous one. Also, no non-specific band was observed.

The genotyping results obtained by the above method are shown in Table 1 below.

[0019]

[Table 1]

(2) Relationship between genotype and phenotype The phenotype of 46 of the 186 individuals was examined.
The phenotype was determined by quantifying 4'-hydroxymephenytoin in urine excreted 8 hours after oral administration of 100 mg of racemic mephenytoin to each individual. This test demonstrates the ability to hydroxylate the 4 'position of (S) -mephenytoin. The ratio of the excreted 4'-hydroxymephenytoin to the dose on a molar basis (%) is defined as a metabolic disorder (poor metabolizers,
PM), and the rest of the people (extensive metabolizers,
EM).

FIG. 2 shows the relationship between the measured phenotype and the genotype examined previously. As is evident from FIG. 2, none of the metabolic abnormal persons has wt, while the normal person always has at least one gene in wt. Therefore, by the method of the present invention, without examining the phenotype (S)-
It was confirmed that diagnosis of metabolic abnormality of mephenytoin can be performed.

Comparative Examples 1 and 2 Known m2 detection primers (forw
The nucleotide sequences of the ard-side and reverse-side primers are shown in SEQ ID NOS: 5 and 6, respectively.
1 PCR was performed under PCR conditions for detection (denaturation was 94 ° C for 1 minute, annealing was 57 ° C for 30 seconds, and extension was 72 ° C for 1 minute 30 times). In Comparative Example 1, the concentration of magnesium chloride in the buffer was adjusted to 1.0 mM described in Reference 1.
In Comparative Example 2, the concentration was 3.0 mM described in Reference 2.
As a result, amplification did not occur in Comparative Example 1 and amplification occurred in Comparative Example 2, and although bands having the expected size were observed, many nonspecific bands also appeared.

Comparative Example 3 The same PC as in the above-mentioned Example of the present invention was prepared by using the primers described in Reference 2 shown in SEQ ID NOs: 5 and 6 in the sequence listing.
PCR was performed under the R condition. As a result, amplification occurred, and although bands of the expected size were observed, many nonspecific bands also appeared.

[0024]

Industrial Applicability According to the present invention, an oligonucleotide primer for detecting m2 and an m2 using the same, which enable PCR for testing m1 and m2 to be performed under the same conditions.
For the first time was offered. According to the present invention,
Since the tests for m1 and m2 can be performed at the same time using the same PCR device, there is an effect that the efficiency of the test is greatly improved.

[0025]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Sequence AACATCAGGA TTGTAAGCAC 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid sequence TCAGGGCTTG GTCAATATAG 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: nucleic acid sequence AATTACAACC AGAGCTTGGC 20

SEQ ID NO: 4 Sequence length: 22 Sequence type: nucleic acid sequence TATCACTTTC CATAAAAGCA AG 22

SEQ ID NO: 5 Sequence length: 25 Sequence type: nucleic acid sequence TATTATTATC TGTTAACTAA TATGA 25

SEQ ID NO: 6 Sequence length: 20 Sequence type: nucleic acid sequence ACTTCAGGGC TTGGTCAATA 20

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an electrophoretogram showing the result of genotyping by the method of the present invention.

FIG. 2 is a diagram showing the relationship between genotypes and phenotypes determined by the method of the present invention.

Claims (6)

[Claims] 1. An oligonucleotide primer for testing a point mutation m2 in exon 4 of the human cytochrome P450 2C19 gene, which has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. 2. An oligonucleotide primer for testing a point mutation m2 in exon 4 of the human cytochrome P450 2C19 gene, which has the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 3. The method according to claim 1, wherein the oligonucleotide primer according to claim 1 and the oligonucleotide primer according to claim 2 are used as primers to obtain human cytochrome P450 2C1.
9 A step of performing PCR using the gene as a template,
A method for testing a point mutation m2 in exon 4 of the human cytochrome P450 2C19 gene, comprising a step of analyzing a CR product by a restriction fragment length polymorphism method. 4. The method according to claim 3, wherein BamHI is used as a restriction enzyme.
The described method. 5. The PCR is carried out using a pair of oligonucleotide primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4 in the Sequence Listing, respectively.
Human cytochrome P450 2C1 using C19 gene as template
9. The method according to claim 3, wherein the PCR is carried out simultaneously with the PCR for the examination of the point mutation m1 in exon 5 of the 9 gene under the same thermal conditions.
The method according to any one of claims 1 to 4. 6. The method according to claim 5, wherein the two PCRs are performed using PCR buffers of the same composition.