JP2004283049A - Method for judging poor methabolizer in japanese - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and highly accurately judging the poor methabolizer of human cytochrome P450 2D6 in Japanese. <P>SOLUTION: This method for judging the poor methabolizer of human cytochrome P450 2D6 in Japanese in vitro comprises a process for detecting four to ten kinds of genetic polymorphisms including at least 2D6*36, preferably five to ten kinds of genetic polymorphisms including 2D6*5, 2D6*14, 2D6*18, 2D6*21 and 2D6*36, more preferably six to ten kinds of genetic polymorphisms including 2D6*5, 2D6*14, 2D6*4, 2D6*18, 2D6*21 and 2D6*36, among the human cytochrome P450 2D6. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００６８】
２Ｄ６^＊３ の検出
１^ｓｔ−ＰＣＲ反応として、１試料当たりミリＱ水３７．６μｌに２ｍＭｄＮＴＰｓ ４μｌ、ＰＣＲ Ｂｕｆｆｅｒ ５μｌ、Ｐｒｉｍｅｒ各２ｐｍｏｌ、ＴａｑＤＮＡポリメラーゼ２ｕ、Ｍｇを終濃度１．５ｍＭとなるように最終液量４９μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９４℃・１分、６６℃・１．５分、７２℃・１．５分の反応を３０回繰り返し行い、増幅産物を得た。次いで、得られた増幅産物０．１μｌを用いて２ｎｄＰＣＲ反応を行った。すなわち、ミリＱ水３８．５μｌに２ｍＭｄＮＴＰｓ ４μｌ、ＰＣＲ Ｂｕｆｆｅｒ ５μｌ、Ｍｇ終濃度１．５ｍＭ、Ｐｒｉｍｅｒ各２ｐｍｏｌ、ＴａｑＤＮＡポリメラーゼ２ｕを最終液量４９．９μｌとなるように調製し９４℃・１分、５５℃・１分、７２℃・１分の反応を１５回繰り返し行い、増幅産物を得た。２^ｎｄＰＣＲ反応は野生型を検出するＰｒｉｍｅｒ（ｗ）と変異型を検出するｐｒｉｍｅｒ（ｍ）を用いたアリル特異的ＰＣＲ法を用いた。
１^ｓｔＰＣＲで用いたＰｒｉｍｅｒ配列を以下に示す。
Ｆｏｒｗａｒｄ ：ＴＣＴ ｇＴＡ ＣＣＴ ＣＣＴ ＡＴＣ ＣＡＣ ｇＴＣ Ａ
Ｒｅｖｅｒｓｅ ： ＣＣＴ ｇｇｇ ＣＣＣ ＣＴｇ ＣＡＣ ＴｇＴ ＴＴ
２^ｎｄＰＣＲで用いたＰｒｉｍｅｒ配列を以下に示す。
野生型検出用：ｇＣＴ ＡＡＣ ＴｇＡ ｇＣＡ ＣＡｇ
変異型検出用：ｇＣＴ ＡＡＣ ＴｇＡ ｇＣＡ Ｃｇ
Ｒｅｖｅｒｅｓｅ Ｐｒｉｍｅｒ ：ＣＣｇ ｇＡＴ ｇＴＡ ｇｇＡ ＴＣＡ ＴｇＡ ｇＣ
【００６９】
２Ｄ６^＊４ の検出
１試料当たりミリＱ水１６．３μｌに２ｍＭｄＮＴＰｓ ２μｌ、ＰＣＲ Ｂｕｆｆｅｒ ２．５μｌ、Ｐｒｉｍｅｒ各１０ｐｍｏｌ、ＴａｑＤＮＡポリメラーゼ１ｕ、Ｍｇを終濃度２．０ｍＭとなるように最終液量２４μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９４℃・３０秒、６１℃・１０秒、７２℃・１．分の反応を４０回繰り返し行った後、７２℃で７ｍｉｎ保持し、増幅産物を得た。得られた増幅産物１０μｌにＭｖａＩ（１０ｕ／μｌ）１μｌとｂｕｆｆｅｒ２μｌおよびミリＱ水７μｌ添加後、３７℃にて２時間以上インキュベートした。その後電気泳動にて得られた泳動像よりタイピングを行った。
用いたＰｒｉｍｅｒ配列を以下に示す。
Ｆｏｒｗａｒｄ ：ＣＣｇ ＣＣＴ ＴＣｇ ＣＣＡ ＡＣＣ ＡＣＴ
Ｒｅｖｅｒｒｓｅ ：ｇＡＡ ＡＣＣ ＴＡＡ ＡＡＴ ＣｇＡ ＡＡＴ ＣＴＣ Ｔｇ
【００７０】
２Ｄ６^＊５ の検出
１試料当たりミリＱ水１７．９μｌに２ｍＭｄＮＴＰｓ ２μｌ、ＰＣＲ Ｂｕｆｆｅｒ ２．５μｌ、Ｐｒｉｍｅｒ各１２．５ｐｍｏｌ、ＬＡ−ＴａｑＤＮＡポリメラーゼ１．２５ｕ、Ｍｇを終濃度１．１ｍＭとなるように最終液量２４μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９３℃・１分、６５℃・３０秒、６８℃・５．分の反応を３５回繰り返し行った後、７２℃で１０ｍｉｎ保持し、増幅産物を得た。得られた増幅産物を電気泳動にて検出しタイピングを行った。
用いたＰｒｉｍｅｒ 配列を以下に示す。
Ｆｏｒｗａｒｄ：ＡＣＣ ｇｇｇ ＣＡＣ ＣＴｇ ＴＡＣ ＴＣＣ ＴＣＡ
Ｒｅｖｅｒｓｅ：ｇＣＡ ＴｇＡ ｇＣＴ ＡＡｇ ｇＣＡ ＣＣＣ ＡｇＡ Ｃ
【００７１】
２Ｄ６^＊ １０の検出
１試料当たりミリＱ水１７．１μｌに２ｍＭｄＮＴＰｓ ２．５μｌ、ＰＣＲ Ｂｕｆｆｅｒ ２．５μｌ、Ｐｒｉｍｅｒ各１５ｐｍｏｌ、ＴａｑＤＮＡポリメラーゼ１．２５ｕ、Ｍｇを終濃度１．２ｍＭとなるように最終液量２５μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９５℃・３０秒、６６℃・３０秒、７２℃・３０秒の反応を３５回繰り返し行い、増幅産物を得た。ＰＣＲ反応には野生型を検出するＰｒｉｍｅｒ（ｗ）と変異型を検出するｐｒｉｍｅｒ（ｍ）を用いたアリル特異的ＰＣＲ法を用いた。
用いたＰｒｉｍｅｒ配列を以下に示す。

【００７２】
２Ｄ６^＊１８ の検出
１試料当たりミリＱ水１６．８μｌに２ｍＭｄＮＴＰｓ ２．５μｌ、ＰＣＲ Ｂｕｆｆｅｒ ２．５μｌ、Ｐｒｉｍｅｒ各１０ｐｍｏｌ、ａｍｐｌｉＴａｑＤＮＡポリメラーゼ１ｕ、Ｍｇを終濃度１ｍＭとなるように最終液量２４μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９６℃・１分、５９℃・１分、７２℃・１．分の反応を３５回繰り返し行い、増幅産物を得た。得られた増幅産物を電気泳動にて検出しタイピングを行った。
用いたＰｒｉｍｅｒ配列を以下に示す。
Ｆｏｒｗａｒｄ：ＡｇＣ ＴＣＴ ＴＣＣ ＴＣＴ ＴＣＴ ＴＣＡ ＣＣ
Ｒｅｖｅｒｓｅ：ＣＡｇ ｇＡＡ ＡｇＣ ＡＡＡ ｇＡＣ ＡＣＣ ＡＴｇ
【００７３】
２Ｄ６^＊２１ の検出
１試料当たりミリＱ水５．１μｌに２ｍＭｄＮＴＰｓ １．６μｌ、ＰＣＲ Ｂｕｆｆｅｒ １．０μｌ、Ｐｒｉｍｅｒ各２ｐｍｏｌ、ａｍｐｌｉＴａｑＤＮＡポリメラーゼ０．５ｕ、Ｍｇを終濃度２．５ｍＭとなるように最終液量９μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９８℃・２０秒、６８℃・４分、７２℃・１０．分の反応を３０回繰り返し行い、増幅産物を得た。次に得られた増幅産物をミリＱ水にて５倍に希釈した溶液１μｌにミリＱ水１７．５５μｌと２．５ｍＭｄＮＴＰｓ ２．０μｌ、ＰＣＲ Ｂｕｆｆｅｒ ２．５μｌ、Ｐｒｉｍｅｒ各１０ｐｍｏｌ、ａｍｐｌｉＴａｑＧｏｌｄ１ｕ、Ｍｇを終濃度０．７５ｍＭとなるように最終液量２４μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９６℃・１分、６８℃・１分、７２℃・１分の反応を２０回繰り返し行い、増幅産物を得た（２^ｎｄＰＣＲ）。２^ｎｄＰＣＲ反応は野生型を検出するＰｒｉｍｅｒ（ｗ）と変異型を検出するｐｒｉｍｅｒ（ｍ）を用いたアリル特異的ＰＣＲ法を用いた。
得られた増幅産物を電気泳動にて検出しタイピングを行った。
１ｓｔＰＣＲで用いたＰｒｉｍｅｒ配列を以下に示す。
Ｆｏｒｗａｒｄ：ＣＣｇ ＣＣＴ ＴＣｇ ＣＣＡ ＡＣＣ ＡＣＴ
Ｒｅｖｅｒｓｅ：ＴＣＡ ｇＣＣ ＴＣＡ ＡＣｇ ＴＡＣ ＣＣＣ Ｔ
２^ｎｄＰＣＲで用いたＰｒｉｍｅｒ配列を以下に示す。
Ｆｏｒｗａｒｄ：ｇＡＣ ＣＣＣ ｇＴＴ ＣＴｇ ＴＣＴ ｇｇＣ ｇＴ

【００７４】
２Ｄ６^＊３６ の検出
１試料当たりミリＱ水１７．６５μｌに２．５ｍＭｄＮＴＰｓ ２μｌ、ＰＣＲ Ｂｕｆｆｅｒ ２．５μｌ、Ｐｒｉｍｅｒ各５ｐｍｏｌ、ＬＡ−ＴａｑＤＮＡポリメラーゼ１．２５ｕ、Ｍｇを終濃度１．１ｍＭとなるように最終液量２４μｌを調製し１００ｎｇ／μｌのゲノムを１μｌ加え９４℃・１０秒、６８℃・５分、７２℃・１０．分の反応を３０回繰り返し行い、増幅産物を得た。得られた増幅産物を電気泳動にて検出しタイピングを行った。
用いたＰｒｉｍｅｒ配列を以下に示す。
Ｆｏｒｗａｒｄ：ｇＣＣ ＡＣＴ ＣＴＣ ｇＴｇ ＴＣｇ ＴＣＡ ｇＣＴ ＴＴ
Ｒｅｖｅｒｓｅ：ｇｇＣ ＡＴｇ ＡｇＣ ＴＡＡ ｇｇＣ ＡＣＣ
【００７５】
上記検出により得られた結果を表１に示す。表１に示されるように、日本人の健常成人１６２名において、２Ｄ６＊３６及び２Ｄ６＊２１が存在することが確認された。また、ＣＹＰ２Ｄ６の変異遺伝子として、２Ｄ６＊４、２Ｄ６＊１８が日本に存在することが報告されているものの、今回の対象１６２名には確認されなかった。
【００７６】
これら検出結果に基づいて、ＣＹＰ２Ｄ６変異遺伝子の頻度を算出した（表１）。
【００７７】
【表１】

【００７８】
得られた頻度情報をもとに、Ｈａｒｄｙ−Ｗｅｉｎｂｅｒｇの法則より貧メタボライザ−になりうる遺伝的多型の頻度を算出した。ＣＹＰ２Ｄ６は常染色体上に存在する遺伝子であり、貧メタボライザ−の原因遺伝子は劣性遺伝することから、Ｈａｒｄｙ−Ｗｅｉｎｂｅｒｇの平衡に当てはめて考えることが可能である。
【００７９】
算出した結果を表２に示す。
【００８０】
【表２】

【００８１】
表２に示されるように、上記ジェノタイプにより得られた結果から算出されるＣＹＰ２Ｄ６の貧メタボライザーの予測頻度は０．９２％であった。この予測頻度０．９２％は、従来の予測頻度と比較して、フェノタイピングにより得られた実測値０．８４％をより説明し得るものであり、日本人におけるＣＹＰ２Ｄ６貧メタボライザーの予測精度が格段に高まることが明らかとなった。
【００８２】
【発明の効果】
上述したように、本発明により、日本人におけるＣＹＰ２Ｄ６の貧メタボライザ−を高い精度で且つ簡便に検出できる方法が提供される。ＣＹＰ２Ｄ６の遺伝子多型としては、約５０種類が知られているが、本発明を用いれば、４〜１０種、好ましくは５〜１０種、より好ましくは６〜１０種という少数の遺伝子多型を検出することによって、簡便かつ精度の高い方法で日本人における貧メタボライザーを判定することが可能になる。
【００８３】
具体的には、２Ｄ６＊３６を含む４〜１０種、好ましくは２Ｄ６＊５、２Ｄ６＊１４、２Ｄ６＊１８、２Ｄ６＊２１及び２Ｄ６＊３６の５〜１０種、より好ましくは、２Ｄ６＊５、２Ｄ６＊１４、２Ｄ６＊４、２Ｄ６＊１８、２Ｄ６＊２１及び２Ｄ６＊３６を含む６〜１０種の遺伝子多型を重点的に検出することによって、日本人におけるＣＹＰ ２Ｄ６貧メタボライザーを高い精度でかつ簡便に判定することが可能となる。
【００８４】
また、検出した遺伝子多型の種類に基づいて、ＣＹＰ２Ｄ６代謝活性が低下しているか、又は完全に消失しているのかなどの判定も行うことができる。更には、ＣＹＰ２Ｄ６が代謝に関与する薬物に対する感受性、具体的には、投与薬物の薬効や副作用の有無などの判定も行うことができる。
【００８５】
本発明は、薬効や副作用の有無等の予測や予知に有効であって、個々人の体質に応じた適切な医療を可能にするものであり、医療の質を向上させる、優れた手段を提供するものである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for simply and appropriately determining a poor metabolizer in Japanese by detecting a polymorphism in the CYP2D6 gene.
[0002]
[Prior art]
Genes may contain point mutations or mutations such as partial or total deletion and insertion or substitution of the gene. It is known that, when the gene of the drug metabolizing enzyme has these mutations, the protein metabolizing enzyme which is a protein may be deficient due to lack of synthesis, or the metabolic ability may be reduced. This means that some people may be more effective than others and may be more susceptible to side effects when given the same amount of drug.
[0003]
Of the family of cytochrome P450s (CYPs), which are important drug metabolizing enzymes, CYP2D6 is the metabolism of more than 65 kinds of clinically important drugs such as aminotriptyline and flecainide, such as antidepressants, antiarrhythmics, and antipsychotics. And is one of the most important molecular species. For example, when metoprolol is administered, if the administered patient is a poor metabolizer of CYP2D6, the β-receptor blocking effect becomes stronger (see Non-Patent Document 1).
[0004]
As described above, the responsiveness of a drug differs depending on the individual gene, and determining the CYP2D6 gene polymorphism in a patient to be administered is of clinical significance from the viewpoint of determining the dose of the drug, preventing side effects, and the like. Is considered to be extremely high.
[0005]
The frequency of CYP2D6 poor metabolizers in Japanese has been reported to be 0.84% by phenotyping with debrison, sparteine and metoprolol. On the other hand, nearly 50 CYP2D6 gene polymorphisms have been reported so far, but the gene polymorphisms that explain poor metabolizers in Japan have not yet been sufficiently identified.
[0006]
Although there have been reported cases in which the frequency of CYP2D6 poor metabolizers has been examined using several mutant genes (see Non-Patent Documents 2 and 3), the estimated frequency is about 0.29% or 0.39%. And the values obtained by phenotyping were significantly different, and could not fully explain poor metabolizers in Japanese.
[0007]
[Non-patent document 1]
Gene Medicine, vol. 5, no. 1, 2001, p. 16-19
[0008]
[Non-patent document 2]
Michihiro Chida et al. , Pharmacogenetics 1999, 9: 601-605.
[0009]
[Non-Patent Document 3]
Michihiro Chida et al. , Pharmacogenetics 1999, 9: 287-293.
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for easily and accurately determining a CYP2D6 poor metabolizer in Japanese.
[0011]
[Means for Solving the Problems]
The present inventors have made intensive studies in view of the above situation. As a result, it has been found that by detecting a specific type of the CYP2D6 gene polymorphism, it is possible to almost explain the poor metabolizer of CYP2D6 in Japanese people. Reached.
[0012]
That is, the present invention relates to the following matters.
[0013]
1. In vitro determination method of poor metabolizer in Japanese, characterized by detecting poor metabolizer of CYP2D6 by detecting 4 to 10 gene polymorphisms including at least 2D6 * 36 among CYP2D6 gene polymorphisms .
[0014]
2. A method for in vitro determination of CYP2D6 enzyme activity in Japanese, comprising determining the degree of CYP2D6 enzyme activity by detecting at least 4 to 10 gene polymorphisms including at least 2D6 * 36 among the CYP2D6 gene polymorphisms. .
[0015]
3. A method for determining the sensitivity of a CYP2D6 to a drug involved in metabolism by detecting 4 to 10 gene polymorphisms including at least 2D6 * 36 among the CYP2D6 gene polymorphisms. In vitro determination method.
[0016]
4. Item 5. In any one of Items 1 to 3, wherein 5 to 10 types of polymorphisms including at least 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36 among the CYP2D6 gene polymorphisms are detected. In vitro determination method.
[0017]
Preferably, among the CYP2D6 gene polymorphisms, in vitro according to any one of Items 1 to 3, wherein 5 gene polymorphisms of 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36 are detected. This is a determination method.
[0018]
5. Any one of Items 1 to 3 for detecting at least 6 to 10 gene polymorphisms including at least 2D6 * 4, 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36 among the CYP2D6 gene polymorphisms The in vitro determination method described in 1.
[0019]
Preferably, any one of items 1 to 3, which detects six gene polymorphisms of 2D6 * 4, 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21, and 2D6 * 36 among CYP2D6 gene polymorphisms. It is an in vitro determination method described.
[0020]
6. Item 6. The in vitro determination method according to any one of Items 1 to 5, wherein a gene polymorphism is detected using one or more nucleic acid amplification methods selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA.
[0021]
7. Item 6. The in vitro determination method according to any one of Items 1 to 5, wherein the gene polymorphism is detected using an invader method.
[0022]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0023]
Judgment method
The present invention detects 4 to 10 types of polymorphisms in human cytochrome P450 2D6 (CYP2D6) that require the detection of 2D6 * 36 to determine the poor metabolizer of CYP2D6 in Japanese. It is characterized by. By mainly detecting 4 to 10 types of gene polymorphisms including 2D6 * 36, it becomes possible to easily and easily detect Japanese CYP2D6 poor metabolizers with high accuracy.
[0024]
In the present invention, a poor metabolizer refers to a person who has no or extremely low specific drug metabolizing activity.
[0025]
The gene polymorphism refers to a gene mutation in which 1% or more of a mutant gene (allele) other than a wild type (W; wild type) is present in a population.
[0026]
Examples of gene polymorphisms to be detected other than 2D6 * 36 include, among the polymorphisms of human cytochrome P450 2D6, the decrease in metabolic activity of human cytochrome P450 2D6 or the disappearance thereof. It is preferable to select them in descending order.
[0027]
Genetic polymorphisms in which the metabolic activity of human cytochrome P450 2D6 is reduced or eliminated include, for example, 2D6 * 3, 2D6 * 4, 2D6 * 5, 2D6 * 6, 2D6 * 7, 2D6 * 8, 2D6 * 9, 2D6 * 10, 2D6 * 11, 2D6 * 12, 2D6 * 13, 2D6 * 14, 2D6 * 15, 2D6 * 16, 2D6 * 17, 2D6 * 18, 2D6 * 19, 2D6 * 20, 2D6 * 21 , 2D6 * 38, 2D6 * 40, 2D6 * 41 and the like.
[0028]
Among them, it is preferable that 2D6 * 5, 2D6 * 14, and 2D6 * 21, which frequently occur in Japanese, are essential. Further, it is preferable to select 5 to 10 kinds by adding 2D6 * 18. In addition, when adding other than the above five types, it is preferable to select a base polymorphism that loses the activity, specifically, 2D6 * 3, 2D6 * 4, 2D6 * 6, 2D6 * 7, 2D6 * 8, It is preferable to select 2D6 * 11 or the like.
[0029]
In addition, 6 kinds including 2D6 * 4, which is considered to be common in Anglo-Saxon systems, were added, that is, 6 kinds of 2D6 * 5, 2D6 * 14, 2D6 * 4, 2D6 * 18, 2D6 * 36 and 2D6 * 21 were required. It is most preferred to detect about 10, preferably up to 10 gene polymorphisms. When further adding other than these six types, it is preferable to select a gene polymorphism in which the above-mentioned activity is lost. Since 2D6 * 4 is considered to be very rare in Japanese, it is considered that 2D6 * 3 is replaced with 2D6 * 3, 2D6 * 6, 2D6 * 7, 2D6 * 8, or 2D6 * 11. It is good also considering the six types exchanged with the heel.
[0030]
Comprehensive detection of nearly 50 reported CYP2D6 gene polymorphisms is inefficient and difficult in practice. On the other hand, in the determination based on the detection of only 2D6 * 5 or 2D6 * 4, it is not possible to accurately determine the poor metabolizer in Japanese with a sufficient probability.
[0031]
In the present invention, 4 to 10 species including 2D6 * 36, particularly 5 to 10 species including 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36, preferably 2D6 * 5, 2D6 * Six to ten genetic polymorphisms, including 14, 2D6 * 4, 2D6 * 18, 2D6 * 21, and 2D6 * 36, were found to be major polymorphisms in Japanese.
[0032]
Thus, 4-10 species, including 2D6 * 36, preferably 4-10 species, including 2D6 * 5, 2D6 * 14, 2D6 * 21 and 2D6 * 36, especially 2D6 * 5, 2D6 * 14, 2D6 * 18, 5 to 10 kinds of 2D6 * 21 and 2D6 * 36, preferably 5 kinds of 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36, further 2D6 * 5, 2D6 * 14, 2D6 * 4, 6 to 10 kinds including 2D6 * 18, 2D6 * 21 and 2D6 * 36, preferably 6 including 2D6 * 5, 2D6 * 14, 2D6 * 4, 2D6 * 18, 2D6 * 21 and 2D6 * 36 By focusing on species polymorphisms, it becomes possible to detect CYP 2D6 poor metabolizers in Japanese with higher accuracy and more easily. Note that the upper limit is set to 10 types in order to set the range to a preferable range from the viewpoint of detection efficiency. If necessary, 10 or more gene polymorphisms may be detected.
[0033]
The major polymorphisms to be detected in the present invention are as follows.
[0034]
2D6 * 4 is a polymorphism having a single base substitution at each of the following sites in the CYP2D6 gene, and has no activity.
100C> T, 974C> A, 984A> G, 997C> G, 1039C> T, 1661G> C, 1846G> A, 1858C> T, 2850C> T, 2938C> T, 3887T> C, 4180G> C
2D6 * 5 is a polymorphism in which all CYP2D6 genes are deleted, and has no activity.
[0035]
2D6 * 14 is a polymorphism having a single base substitution at each of the following sites in the CYP2D6 gene, and has no activity.
100C> T, 1661G> C, 1758G> A, 2850C> T, 4180G> C
2D6 * 18 is a polymorphism having an insertion of 9 bases (GTGCCCACT) at 4125-4133 in the CYP2D6 gene and has no activity.
[0036]
2D6 * 21 is a polymorphism having a single nucleotide substitution at the following sites in the CYP2D6 gene, and has no activity. 740C> T, 678G> A, 629A> G, 214G> C, 221C> A, 223C> G, 227T> C, 310G> T, 601delC, 1235A> G, 1426C> T, 1584C> G, 1661G> C, 2573insC, 2850C> T, 3584G> A, 4180G> C, 6272-6274delACA
2D6 * 36 is a polymorphism having a single base substitution at the following sites in the CYP2D6 gene, and has a reduced activity.
100C> T, 1039C> T, 1661G> C, 4180G> C, gene conversion to CYP2D7 in exon9
By mainly detecting these gene polymorphisms, it becomes possible to determine the CYP 2D6 poor metabolizer in Japanese with higher accuracy and more easily. Further, by examining the types of the detected gene polymorphisms, it is possible to determine the degree of the enzyme activity of CYP2D6, specifically, whether the metabolic activity of CYP2D6 is normal, decreased, or completely eliminated. . Furthermore, the sensitivity of CYP2D6 to a drug involved in metabolism can be determined based on the detection result of the gene polymorphism. Specifically, it is possible to determine the strength of the action of the drug, the presence or absence of a side effect of the drug, and the like.
[0037]
Drugs metabolized by CYP2D6 include various drugs in which CYP2D6 is involved in metabolism, and include, for example, alprenolol, carvedilol, flecainide, metoprolol, metoprolol, Mexiletine, propafenone, propanolol, timolol, timolol, amitriptyline, clomipramine, fluvoxamine, fluvoxamine, fluvoxamine, fluvoxamine, fluvoxamine e), paroxetine, perphenazine, risperidone, thioridazine, codeine, dextromethorphan, methoxyphenamine, methoxyphenamine, etc. No.
[0038]
When administering these drugs, determining the gene polymorphism is important in performing individual drug therapy in which the most appropriate drug is administered to the patient in an appropriate manner, and the CYP2D6 has a drug effect or side effect of a drug involved in metabolism. It is also effective for prediction and prediction of clinical evaluation such as.
[0039]
Gene polymorphism detection
In the present invention, in a gene sample prepared from a Japanese subject, whether or not the nucleic acid has several expected mutations is examined in vitro to detect the type of CYP2D6 gene polymorphism in the subject. By doing so, the above determination is made. As a method for detecting a gene polymorphism, a generally used method can be appropriately used.
[0040]
For example, a nucleic acid sequencing method (sequencing method), a nucleic acid amplification method (gene amplification method) such as a PCR (polymerase chain reaction) method, a Southern blot method in which an amplified nucleic acid is immobilized on a nylon membrane and detected using a labeled probe, A sandwich hybridization method (ASO method) in which a detection probe is acted upon after capturing with a supplementary probe immobilized on an individual carrier, a site containing a mutation site is amplified using a pair of oligonucleotides, and then amplified as a single-stranded nucleic acid. By electrophoresis, a method (PCR-SSCP method) utilizing the fact that the migration distance of electrophoresis varies depending on the presence or absence of a mutation, an Invader method, or the like can be used.
[0041]
The nucleic acid sequencing method is a method for detecting and identifying a base sequence contained in a nucleic acid sequence, and reads a base sequence of a sample nucleic acid using an automatic sequencer or the like, and detects a polymorphism by examining a sequence at a mutation site. Is the way.
[0042]
Examples of the nucleic acid amplification method (gene amplification method) include NASBA, LCR, SDA, RCR, TMA and the like in addition to the PCR.
[0043]
In the PCR, a single-stranded target nucleic acid is reacted with an oligonucleotide, four types of deoxynucleoside triphosphates (dNTPs) and a DNA polymerase, whereby an oligonucleotide extension reaction using the target nucleic acid as a template occurs to generate a nucleic acid sequence. Is a method of amplifying a target nucleic acid by repeatedly performing a reaction for synthesizing a complementary strand of the target nucleic acid.
[0044]
NASBA and TMA are methods for synthesizing single-stranded DNA from RNA by reverse transcription, and then using the DNA as a template to act on the oligonucleotide and RNA polymerase to repeat the reverse transcription and RNA synthesis to amplify the target nucleic acid. It is.
[0045]
LCR is a method for amplifying a target nucleic acid by repeating a reaction in which two kinds of oligonucleotides, deoxynucleoside triphosphate (dNTP), and a ligase are allowed to act on a target nucleic acid to bind the two kinds of oligonucleotides on the target nucleic acid. .
[0046]
SDA is a method of amplifying a nucleic acid by repeating an extension reaction and a treatment with a restriction enzyme using an oligonucleotide that is complementary to a template having a restriction enzyme site in its sequence.
[0047]
RCR is a method in which a complementary oligonucleotide and a DNA polymerase are allowed to act on a circular target nucleic acid so that a newly generated oligonucleotide is continuously separated from a template to amplify the target nucleic acid.
[0048]
In addition, among PCR, various methods such as a PCR-RFLP method, an Allele Specific PCR method, and a Long PCR method can be appropriately used.
[0049]
The PCR-RFLP method is a method of detecting a fragment having a different size depending on the presence or absence of a mutation by amplifying a site including a mutation site using a pair of oligonucleotides, treating the site with a restriction enzyme, and separating by electrophoresis. is there.
[0050]
The Allele Specific PCR method uses a wild-type oligonucleotide that is completely complementary to the end region of the amplification region of the wild-type gene as one of a pair of oligonucleotides used for the gene amplification method. And a mutant oligonucleotide completely complementary to the end region of the amplification region. The mutant oligonucleotide has a nucleotide whose 3 'end is complementary to the nucleotide having the expected point mutation. The sample gene is subjected to a gene amplification method using the oligonucleotides for wild type and mutant type separately. If the sample gene is wild-type, nucleic acid amplification occurs when using the wild-type oligonucleotide, but when using the mutant-type oligonucleotide, the 3 ′ end of the oligonucleotide corresponds to the sample gene. Since it is not complementary to the nucleotide (mismatch), no elongation reaction occurs and no amplification of the nucleic acid occurs. On the other hand, if the sample gene is mutant, amplification does not occur when the wild-type oligonucleotide is used, and amplification occurs when the mutant oligonucleotide is used. Therefore, by examining whether or not amplification occurs when each oligonucleotide is used, it is possible to determine whether the sample gene is a wild type or a mutant type, thereby identifying a point mutation in the sample gene. Can be. At this time, as a method for checking whether amplification has occurred, the amplified product is subjected to agarose gel electrophoresis, stained with a nucleic acid-specific binding fluorescent reagent such as ethidium bromide, and then irradiated with UV to detect the presence of the amplified nucleic acid. How to
[0051]
The Long PCR method is a PCR method in which the amplification region is 0.5 Kb or more, preferably 1 Kb or more, although the method and principle are not different from the PCR method.
[0052]
In the invader method, an invader oligo having a sequence complementary to a DNA target fragment and a complementary oligo (signal probe) having a 5 'flap structure and detecting SNP are used. First, an invader oligo and a signal probe are hybridized to a target DNA. At this time, the invader oligo and the probe have a structure in which one base overlaps (invasive structure). Cleavase acts on this portion, and when the base of the signal probe at the SNP site and the base of the target are complementary (no SNP), the 5 'flip of the signal probe is cleaved. The cleaved 5 'flip hybridizes to a FRET probe (Fluorescence resonance energy transfer probe). The fluorescent dye and the quencher (Quencher) are close to each other on the FRET probe, and the fluorescence is suppressed. However, when the 5 'flip DNA is bound, the fluorescent dye portion is cleaved by Cleavase, and the fluorescent signal is detected. It is possible.
[0053]
Another detection method is to use a 5 ′ exonuclease of DNA polymerase, to use a pair of oligonucleotides and oligonucleotides that are fluorescently labeled at both ends to complementarily bind to the mutation site, and to detect the fluorescence released by the PCR reaction. It is released by a method for detecting a substance, or by binding a sample DNA previously single-stranded to a carrier on which an oligonucleotide that complementarily binds to a mutation site is immobilized and gradually raising the temperature of the bound carrier. And other methods for detecting DNA.
[0054]
In the present invention, these various methods can be appropriately used and used. In particular, a nucleic acid amplification method such as PCR and a detection method using an invader method are preferable.
[0055]
As a specific embodiment, for example, when a gene polymorphism is detected using a nucleic acid amplification method, a wild-type oligonucleotide and one or two types of mutant-type detection oligonucleotides are separately or simultaneously added to a sample. Let it work.
In the present invention, the wild-type detection oligonucleotide is an oligonucleotide having a sequence complementary to a chromosome containing a polymorphic site having a normal phenotype or a fragment thereof. The mutant type detection oligonucleotide is an oligonucleotide having a sequence complementary to a chromosome or a fragment thereof including a polymorphic site having a sequence different from that of the wild type. Preferably, the oligonucleotide is designed such that the second nucleotide from the 3 'end of the oligonucleotide for detecting wild type and the mutant type is complementary to the nucleotide of the wild type or mutant sequence. More preferably, the second nucleotide from the 3 ′ end of the oligonucleotide for wild-type detection and the mutant-type detection complementarily corresponds to the nucleotide of the wild-type or mutant-type sequence, and is complementary to both the wild-type and the mutant-type. Oligonucleotides designed to have non-bases 3 to 7 from the 3 'end. In the case of such a design, in the combination of the oligonucleotide for wild type / wild type nucleic acid and the oligonucleotide for mutant type / mutant nucleic acid, each oligonucleotide matches at least from the 3 ′ end to the second sequence, so that the extension reaction is performed. Happens. On the other hand, in the combination of the oligonucleotide for wild-type / mutated nucleic acid and the oligonucleotide for mutant / wild-type nucleic acid, an artificial mismatch of the 3 ′ end of the oligonucleotide or the second base from the 3 ′ end, and further another base is caused. Therefore, no elongation reaction occurs.
[0056]
The length of the oligonucleotide is 13 to 35 bases, preferably 16 to 30 bases. There is at least one mismatch site in the oligonucleotide. The position is not particularly limited as long as it is anywhere from the 3 'end of the 3' end to the 5 'end, but is preferably a position close to the 3' end of the 3 'end, more preferably the 3' end of the 3 'end. Is preferred.
[0057]
Third, when an artificial mismatch is used, when a nucleic acid sequence that is not expected to undergo an extension reaction is used as a template, a mismatch of two bases is formed together with the nucleotide polymorphism site, and the extension reaction is strongly inhibited.
In the present invention, the method for extending an oligonucleotide can be basically performed using a conventional method. Usually, a single-stranded denatured chromosome or a fragment thereof containing a specific nucleotide polymorphism site is combined with four types of deoxynucleoside triphosphates (dNTPs) and a DNA polymerase together with a wild-type detection oligonucleotide and one or more types. The oligonucleotides are extended using the target nucleic acid as a template by simultaneously or separately using the two mutant-type detection oligonucleotides.
[0058]
The extension reaction can be performed according to the method described in Molecular Cloning, A Laboratory Manual (Sambrook et al., 1989). In the present invention, a method for amplifying a chromosome or a fragment containing a specific nucleotide polymorphism site can also be basically performed using a conventional method, and is usually a specific nucleotide polymorphism modified into a single strand. A chromosome containing the site or a fragment thereof, together with four types of deoxynucleoside triphosphates (dNTPs), a DNA polymerase and a reverse primer, an oligonucleotide for detecting wild type and one or two types of oligonucleotides for detecting mutant type (for (Corresponding to a word primer) simultaneously or separately, and the amplification is performed between the forward oligonucleotide and the reverse oligonucleotide using the target nucleic acid as a template. As the nucleic acid amplification method, the above-mentioned PCR, NASBA, LCR, SDA, RCR, TMA and the like can be used.
[0059]
In the method as described above, a nucleic acid amplification method is performed using a wild-type detection oligonucleotide capable of amplifying a wild-type nucleic acid and a mutant-type detection oligonucleotide capable of amplifying a mutant nucleic acid separately or simultaneously.
[0060]
When a sample nucleic acid is extended or amplified using a wild-type detection oligonucleotide, a reaction occurs when the sample nucleic acid is wild-type, but does not occur when a mutant nucleic acid is used. Conversely, when the sample nucleic acid is extended or amplified using the mutant-type detection oligonucleotide, the reaction occurs if the sample nucleic acid is a mutant type, but does not occur if the sample nucleic acid is a wild-type. Therefore, one sample is divided into two, and one performs the reaction using the wild-type detection oligonucleotide, and the other performs the reaction using the mutant-type detection oligonucleotide, and examines whether the reaction has occurred. This makes it possible to clearly know whether the sample nucleic acid is a wild type or a mutant type. In particular, higher organisms, including humans, have one father-derived gene and one maternal-derived gene for one type of gene. It is also possible to distinguish between homozygous mutants or both heterozygotes. That is, in the case of heterozygous reaction, since both the wild-type gene and the mutant gene are present, a reaction occurs both when the wild-type detection oligonucleotide is used and when the mutant-type detection oligonucleotide is used.
[0061]
Further, in order to detect whether or not the oligonucleotide has reacted, a nucleic acid sample containing a chromosome or a fragment containing a specific nucleotide polymorphism site, a certain amount of a wild-type detection oligonucleotide and one or two kinds of After performing the reaction using the mutant type detection oligonucleotide (corresponding to a forward primer) simultaneously or separately, the amount of the oligonucleotide generated by the amplification reaction is caused by reacting with the double-stranded nucleic acid-specific binding substance. Can be detected.
[0062]
Upon detection, a labeled oligonucleotide can be used. For example, each of the oligonucleotides is previously labeled with an enzyme, a double-stranded nucleic acid-specific binding substance, biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, a luminophore, or the like, and the reaction is performed after the extension or amplification reaction. By detecting the labeled oligonucleotide, the gene polymorphism can be detected. Examples of the enzyme include alkaline phosphatase, peroxidase and the like. Examples of the fluorescent substance include FITC, 6-FAM, HEX, TET, TAMRA, Texas Red, Cy3, Cy5, and the like. Examples of the double-stranded nucleic acid-specific binding substance include ethylene bromide and SYBR GreenI. Haptens include biotin, digoxigenin and the like. As radioactive materials,³²P,³⁵S and the like.
Examples of the luminophore include ruthenium. The label may be attached to any position of the oligonucleotide as long as it does not affect the extension reaction of the oligonucleotide. Preferably, it is a 5 'site.
[0063]
When different labels are used for the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide, detection can be performed in one reaction tank.
[0064]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.
[0065]
Example
In order to identify the causative gene of human cytochrome P450 2D6 poor metabolizer in Japanese, * 2, * 3, * 4, * 5, * 10, * 18, * 21, reported as polymorphisms of CYP2D6, * 36 was examined to determine its frequency.
[0066]
First, genomic DNA was extracted from 162 healthy Japanese adults and used as a sample. Then, using the obtained genomic DNA as a template, typing by PCR was performed as follows.
[0067]
2D6^*Detection of 2
2.5 μl of 2 mM dNTPs, 2.5 μl of PCR Buffer, 2.5 μl of PCR Buffer, 15 pmol of each of Primer, 1.25 u of Taq DNA polymerase, 1.25 u of Mg, and 25 μl of final solution to give a final concentration of 1.5 mM were prepared for each sample in 17.1 μl of Milli-Q water and 100 ng. The reaction at 95 ° C. for 30 seconds, 66 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times, and an amplification product was obtained. For the PCR reaction, an allele-specific PCR method using Primer (w) for detecting a wild type and Primer (m) for detecting a mutant type was used.
The Primer sequence used is shown below.

[0068]
2D6^*Detection of 3
1^stAs a PCR reaction, 4 μl of 2 mM dNTPs, 5 μl of PCR Buffer, 2 μmol of each of Primer, 2 pmol of each Primer, 2 umol of Taq DNA polymerase, 2 u of Mg, and 3 μl of Milli-Q water per sample were prepared in a final volume of 49 μl to a final concentration of 1.5 mM. The reaction was repeated 30 times at 94 ° C. for 1 minute, at 66 ° C. for 1.5 minutes, and at 72 ° C. for 1.5 minutes, to obtain an amplification product. Next, a 2nd PCR reaction was performed using 0.1 μl of the obtained amplification product. That is, 4 μl of 2 mM dNTPs, 5 μl of PCR Buffer, 5 μl of Mg buffer, 1.5 mM of each of Primer, 2 pmol of each Primer, and 2 u of Taq DNA polymerase were prepared in 38.5 μl of Milli-Q water to a final volume of 49.9 μl, and 94 ° C., 1 minute, 55 The reaction was repeated 15 times at 1 ° C. for 1 minute and at 72 ° C. for 1 minute to obtain an amplification product. 2^ndThe PCR reaction used an allele-specific PCR method using Primer (w) for detecting a wild type and Primer (m) for detecting a mutant type.
1^stThe Primer sequence used in the PCR is shown below.
Forward: TCT gTA CCT CCT ATC CAC gTC A
Reverse: CCT ggg CCC CTg CAC TgT TT
2^ndThe Primer sequence used in the PCR is shown below.
For wild type detection: gCT AAC TgA gCA CAg
For mutation detection: gCT AAC TgA gCA Cg
Reverse Primer: CCg gAT gTA ggA TCA TgA gC
[0069]
2D6^*Detection of 4
2 μl of 2 mM dNTPs, 1 μl of 2 mM dNTPs, 2.5 μl of PCR Buffer, 10 pmol of each of Primer, 1 u of Taq DNA polymerase, 1 u of Mg, and 1 μl of Mg per sample were prepared in a final volume of 24 μl to a final concentration of 2.0 mM, and a genome of 100 ng / μl was prepared. Was added at 94 ° C for 30 seconds, 61 ° C for 10 seconds, and 72 ° C for 1 second. After repeating the reaction for 40 minutes for 40 minutes, the mixture was kept at 72 ° C. for 7 minutes to obtain an amplification product. After adding 1 μl of MvaI (10 u / μl), 2 μl of buffer and 7 μl of Milli-Q water to 10 μl of the obtained amplification product, the mixture was incubated at 37 ° C. for 2 hours or more. Thereafter, typing was performed from the electrophoresis image obtained by electrophoresis.
The Primer sequence used is shown below.
Forward: CCg CCT TCg CCA ACC ACT
Reverser: gAA ACC TAA AAT CgA AAT CTC Tg
[0070]
2D6^*Detection of 5
Prepare 2 μl of 2 mM dNTPs, 1 μl of 2 mM dNTPs, 2.5 μl of PCR Buffer, 12.5 pmol of each Primer, 1.25 uL of LA-Taq DNA polymerase, and 1.25 u of Mg in a final concentration of 1.1 mM in 17.9 μl of Milli-Q water per sample. Then, 1 μl of 100 ng / μl genome was added, 93 ° C. for 1 minute, 65 ° C. for 30 seconds, 68 ° C. for 5. After repeating the reaction for 35 minutes for 35 minutes, the mixture was kept at 72 ° C. for 10 minutes to obtain an amplification product. The obtained amplification product was detected by electrophoresis and typing was performed.
The Primer sequence used is shown below.
Forward: ACC ggg CAC CTg TAC TCC TCA
Reverse: gCA TgA gCT AAg gCA CCC AgA C
[0071]
2D6^*  10 detections
2.5 μl of 2 mM dNTPs, 2.5 μl of PCR Buffer, 15 μmol of each of Primer, 1.25 u of Taq DNA polymerase, 1.25 u of Taq DNA polymerase, and 25 μl of final solution were prepared in a final concentration of 1.2 mM for each sample in 17.1 μl of Milli-Q water and 100 ng. The reaction at 95 ° C. for 30 seconds, 66 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times, and an amplification product was obtained. For the PCR reaction, an allele-specific PCR method using Primer (w) for detecting a wild type and Primer (m) for detecting a mutant type was used.
The Primer sequence used is shown below.

[0072]
2D6^*Detection of 18
16.8 μl of Milli-Q water / 2.5 μl of 2 mM dNTPs, 2.5 μl of PCR Buffer, 2.5 μl of Primer, 10 pmol each of Primer, 1 u of ampliTaq DNA polymerase, 1 u of Mg, 1 μg of Mg, and a final volume of 24 μl to give a final concentration of 1 mM per sample, and a genome of 100 ng / μl Is added at 96 ° C for 1 minute, 59 ° C for 1 minute, 72 ° C for 1 minute. The reaction was repeated 35 times to obtain an amplification product. The obtained amplification product was detected by electrophoresis and typing was performed.
The Primer sequence used is shown below.
Forward: AgC TCT TCC TCT TCT TCA CC
Reverse: CAg gAA AgC AAA gAC ACC ATg
[0073]
2D6^*Detection of 21
1.6 μl of 2 mM dNTPs, 1.0 μl of PCR Buffer, 2 pmol of each Primer, 0.5 μl of ampliTaq DNA polymerase, 0.5 μl of AmpliTaq DNA polymerase, and 5 μl of final solution in 9 μl of Mg were prepared for each sample in 5.1 μl of Milli-Q water and 100 ng. / Μl of the genome is added at 1 μl, at 98 ° C. for 20 seconds, at 68 ° C. for 4 minutes, and at 72 ° C. for 10 minutes. The reaction was repeated 30 times to obtain an amplification product. Next, 17.55 μl of Milli-Q water, 2.0 μl of 2.5 mM dNTPs, 2.5 μl of PCR Buffer, 2.5 μl of PCR Buffer, 10 pmol of each Primer, ampliTaqGold1u, and Mg were added to 1 μl of a solution obtained by diluting the obtained amplification product 5 times with Milli-Q water. A final volume of 24 μl was prepared at a concentration of 0.75 mM, and 1 μl of 100 ng / μl genome was added, and the reaction was repeated 20 times at 96 ° C. for 1 minute, at 68 ° C. for 1 minute, and at 72 ° C. for 1 minute. (2^ndPCR). 2^ndThe PCR reaction used an allele-specific PCR method using Primer (w) for detecting a wild type and Primer (m) for detecting a mutant type.
The obtained amplification product was detected by electrophoresis and typing was performed.
The Primer sequence used in the first PCR is shown below.
Forward: CCg CCT TCg CCA ACC ACT
Reverse: TCA gCC TCA ACg TAC CCC T
2^ndThe Primer sequence used in the PCR is shown below.
Forward: gAC CCC gTT CTg TCT ggC gT

[0074]
2D6^*36 detections
Prepare 2 μl of 2.5 mM dNTPs, 2.5 μl of PCR Buffer, 5 pmol of each Primer, 1.25 uL of LA-Taq DNA polymerase, 1.25 u of LA-Taq DNA polymerase, and final volume of 24 μl so that the final concentration is 1.1 mM in 17.65 μl of Milli-Q water per sample Then, 1 μl of 100 ng / μl genome was added, 94 ° C. for 10 seconds, 68 ° C. for 5 minutes, 72 ° C. for 10 minutes. The reaction was repeated 30 times to obtain an amplification product. The obtained amplification product was detected by electrophoresis and typing was performed.
The Primer sequence used is shown below.
Forward: gCC ACT CTC gTg TCg TCA gCT TT
Reverse: ggC ATg AgC TAA ggC ACC
[0075]
Table 1 shows the results obtained by the above detection. As shown in Table 1, it was confirmed that 2D6 * 36 and 2D6 * 21 were present in 162 healthy Japanese adults. In addition, although it was reported that 2D6 * 4 and 2D6 * 18 exist in Japan as CYP2D6 mutant genes, they were not confirmed in the present 162 subjects.
[0076]
Based on these detection results, the frequency of the CYP2D6 mutant gene was calculated (Table 1).
[0077]
[Table 1]

[0078]
Based on the obtained frequency information, the frequency of a genetic polymorphism that could become a poor metabolizer was calculated from Hardy-Weinberg's law. CYP2D6 is a gene present on an autosomal chromosome, and the causative gene of a poor metabolizer is recessive, so it can be applied to Hardy-Weinberg equilibrium.
[0079]
Table 2 shows the calculated results.
[0080]
[Table 2]

[0081]
As shown in Table 2, the prediction frequency of the poor metabolizer of CYP2D6 calculated from the result obtained by the genotype was 0.92%. This prediction frequency of 0.92% can better explain the actually measured value of 0.84% obtained by phenotyping, as compared with the conventional prediction frequency, and the prediction accuracy of the CYP2D6 poor metabolizer in Japanese is low. It became clear that it would increase significantly.
[0082]
【The invention's effect】
As described above, the present invention provides a method for easily and accurately detecting a poor metabolizer of CYP2D6 in Japanese. Approximately 50 types of CYP2D6 gene polymorphisms are known. However, according to the present invention, as few as 4 to 10, preferably 5 to 10, and more preferably 6 to 10 polymorphisms are reduced. The detection makes it possible to determine a poor metabolizer in Japanese by a simple and accurate method.
[0083]
Specifically, 4 to 10 kinds including 2D6 * 36, preferably 5 to 10 kinds of 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36, more preferably 2D6 * 5, By focusing on 6 to 10 gene polymorphisms including 2D6 * 14, 2D6 * 4, 2D6 * 18, 2D6 * 21 and 2D6 * 36, CYP 2D6 poor metabolizer in Japanese can be detected with high accuracy. Moreover, it is possible to easily determine.
[0084]
In addition, it is possible to determine whether the CYP2D6 metabolic activity is reduced or completely eliminated based on the type of the detected gene polymorphism. Furthermore, it is also possible to determine the sensitivity of CYP2D6 to a drug involved in metabolism, specifically, the drug effect of the administered drug and the presence or absence of side effects.
[0085]
INDUSTRIAL APPLICABILITY The present invention is effective in predicting and predicting the presence or absence of a medicinal effect and side effects, enables appropriate medical care according to the constitution of an individual, and provides excellent means for improving the quality of medical care. Things.

Claims (7)

In vitro determination method of poor metabolizer in Japanese, characterized by detecting poor metabolizer of CYP2D6 by detecting 4 to 10 gene polymorphisms including at least 2D6 * 36 among CYP2D6 gene polymorphisms . A method for in vitro determination of CYP2D6 enzyme activity in Japanese, comprising determining the degree of CYP2D6 enzyme activity by detecting at least 4 to 10 gene polymorphisms including at least 2D6 * 36 among the CYP2D6 gene polymorphisms. . A method for determining the sensitivity of a CYP2D6 to a drug involved in metabolism by detecting 4 to 10 gene polymorphisms including at least 2D6 * 36 among the CYP2D6 gene polymorphisms. In vitro determination method. The method according to any one of claims 1 to 3, wherein 5 to 10 gene polymorphisms including at least 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21, and 2D6 * 36 among the CYP2D6 gene polymorphisms are detected. In vitro determination method. 4. The method according to any one of claims 1 to 3, wherein 6 to 10 gene polymorphisms including at least 2D6 * 4, 2D6 * 5, 2D6 * 14, 2D6 * 18, 2D6 * 21 and 2D6 * 36 among the CYP2D6 gene polymorphisms are detected. The in vitro determination method according to any of the above. The in vitro determination method according to any one of claims 1 to 5, wherein the gene polymorphism is detected using one or more nucleic acid amplification methods selected from the group consisting of PCR, NASBA, LCR, SDA, RCR, and TMA. The in vitro determination method according to any one of claims 1 to 5, wherein the gene polymorphism is detected using an invader method.