JP2002223769A - Method for predicting side effect of immunosuppresant and primer used therefor - Google Patents
Method for predicting side effect of immunosuppresant and primer used thereforInfo
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- JP2002223769A JP2002223769A JP2001024723A JP2001024723A JP2002223769A JP 2002223769 A JP2002223769 A JP 2002223769A JP 2001024723 A JP2001024723 A JP 2001024723A JP 2001024723 A JP2001024723 A JP 2001024723A JP 2002223769 A JP2002223769 A JP 2002223769A
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、汎用されている免
疫抑制剤であるタクロリムス及びシクロスポリンの副作
用の判定方法及びそれに用いられるプライマーに関す
る。TECHNICAL FIELD The present invention relates to a method for determining the side effects of tacrolimus and cyclosporin, which are widely used immunosuppressants, and primers used therefor.
【０００２】[0002]
【従来の技術】タクロリムス及びシクロスポリンは、臓
器移植後の拒絶反応の抑制等に広く用いられている免疫
抑制剤である。しかし、これらの免疫抑制剤は、副作
用、特に神経毒性を発揮する場合があることが知られて
おり、これらの免疫抑制剤による副作用が起きるか否か
は、臓器移植の成否を左右する鍵となるものである。こ
れらの免疫抑制剤による副作用が起きるか否かは、免疫
抑制剤の血中濃度によっては説明できないことがわかっ
ており、現状では、副作用が起きるか否かを予測するこ
とができない。2. Description of the Related Art Tacrolimus and cyclosporin are immunosuppressants widely used for suppressing rejection after organ transplantation. However, it is known that these immunosuppressants may exert side effects, especially neurotoxicity, and whether or not these immunosuppressants cause side effects is a key to the success or failure of organ transplantation. It becomes. It has been known that whether or not these immunosuppressants cause side effects cannot be explained by the blood concentration of the immunosuppressant, and at present, it cannot be predicted whether or not side effects will occur.
【０００３】一方、MDR1(multidrug resistance 1)遺伝
子は、Ｐ糖蛋白質をコードする公知の遺伝子であり、そ
の塩基配列も公知である。Ｐ糖蛋白質は、種々の脂溶性
の生体異物を、ATPの加水分解に依存して細胞外へ排出
するポンプの役割を果たしている。生理機能として、食
物中の有害物質が体内に吸収されるのを防ぎ、脳、精
巣、造血幹細胞などに有害物質が侵入するのを防ぐ働き
をしている。MDR1遺伝子には種々の部位における点突然
変異が知られており、これらの単塩基多型(SNP)と種々
の薬物の体内動態との関係が調べられている。しかしな
がら、MDR1遺伝子のSNPと免疫抑制剤の副作用との関係
は全く報告されていない。On the other hand, the MDR1 (multidrug resistance 1) gene is a known gene encoding a P-glycoprotein, and its base sequence is also known. The P-glycoprotein plays a role of a pump that discharges various fat-soluble xenobiotics to the outside of the cell depending on ATP hydrolysis. As a physiological function, it functions to prevent harmful substances in food from being absorbed into the body and prevent harmful substances from entering the brain, testis, hematopoietic stem cells, and the like. Point mutations at various sites in the MDR1 gene are known, and the relationship between these single nucleotide polymorphisms (SNPs) and the pharmacokinetics of various drugs has been investigated. However, no relationship has been reported between the SNP of the MDR1 gene and the side effects of immunosuppressants.
【０００４】[0004]
【発明が解決しようとする課題】もし、上記免疫抑制剤
による副作用が起きるか否かを予測することができれ
ば、他の免疫抑制剤を用いたり、投与量を少なくする等
の対策を採ることができるので望ましい。If it is possible to predict whether or not a side effect due to the immunosuppressant will occur, it is possible to take measures such as using another immunosuppressant or reducing the dose. It is desirable because it can.
【０００５】従って、本発明の目的は、タクロリムス又
はシクロスポリンを投与した場合に副作用が起きるか否
かを予測する手段を提供することである。Accordingly, it is an object of the present invention to provide a means for predicting whether side effects will occur when tacrolimus or cyclosporin is administered.
【０００６】[0006]
【課題を解決するための手段】本願発明者らは、生体肝
移植後にタクロリムスを投与された患者のMDR1の単塩基
多型について鋭意研究した結果、MDR1遺伝子の第２６７
７番目の塩基は通常グアニンであるが、これがアデニン
又はチミンに点突然変異している場合にタクロリムス又
はシクロスポリンの副作用が起きる可能性が有意に高く
なることを見出し、本発明を完成した。Means for Solving the Problems The present inventors have conducted intensive studies on the single nucleotide polymorphism of MDR1 in patients who received tacrolimus after living donor liver transplantation.
The seventh base is usually guanine, and it has been found that when this is point-mutated to adenine or thymine, the possibility of side effects of tacrolimus or cyclosporine is significantly increased, and the present invention has been completed.
【０００７】すなわち、本発明は、MDR1遺伝子の、ｃＤ
ＮＡ配列のコード領域における位置で第２６７７番目の
塩基がグアニンであるか、アデニン又はチミンであるか
を調べることを含む、タクロリムス及びシクロスポリン
から成る群より選ばれる少なくとも１種の免疫抑制剤の
副作用の予測方法を提供する。また、本発明は、MDR1遺
伝子の、ｃＤＮＡ配列のコード領域における位置で第２
６７７番目の塩基よりも上流の領域又は該塩基を含む領
域とハイブリダイズするフォワード側プライマーと、前
記塩基よりも下流の領域又は該塩基を含む領域とハイブ
リダイズするリバース側のプライマー（ただし、フォワ
ード側プライマーとリバース側プライマーがハイブリダ
イズする領域は重複しない）とから成る、上記本発明の
方法に用いられるプライマーセットを提供する。[0007] That is, the present invention relates to the cDD of the MDR1 gene.
Including examining whether the 2677th base at the position in the coding region of the NA sequence is guanine, adenine or thymine, comprising the side effect of at least one immunosuppressant selected from the group consisting of tacrolimus and cyclosporin; Provide a forecasting method. In addition, the present invention relates to a method wherein the MDR1 gene has a second
A forward primer that hybridizes with a region upstream of the 677th base or a region containing the base, and a reverse primer that hybridizes with a region downstream of the base or a region containing the base (where the forward primer A primer set for use in the method of the present invention, comprising a primer and a region where the reverse primer hybridizes does not overlap).
【０００８】[0008]
【発明の実施の形態】本発明の方法では、MDR1遺伝子
の、ｃＤＮＡ配列のコード領域における位置で（以下、
特に断りがない限り塩基の位置はｃＤＮＡのコード領域
上の位置を示す）第２６７７番目の塩基（以下、「2677
nt」と示すことがある。他の位置についても同様）がグ
アニンであるか、アデニン又はチミンであるかを調べ
る。なお、2677ntはMDR1のエクソン２１中に位置する。
通常のMDR1遺伝子（本明細書において「野生型」という
ことがある）のｃＤＮＡのコード領域の塩基配列を配列
表の配列番号１に示す。この配列は、GenBank Accessio
n No. M14758に示される配列中に含まれている。なお、
MDR1遺伝子は、種々の位置にSNPを有しているので、配
列番号１に示す野生型は、正常なヒト遺伝子の典型的な
１例であり、種々の多型が存在する。MDR1遺伝子の2677
ntは、野生型ではグアニン(G)であるが、これがアデニ
ン(A)になる変異（2677ntのＧがＡになる変異を「G2677
A」と表示する。他の点突然変異についても同様に表示
する）及びＴになる変異G2677Tが存在する。2677ntがＧ
の場合には、これを含むコドンによりコードされるアミ
ノ酸はアラニンであるが、G2677Aの場合にはこれがスレ
オニンになり、G2677Tの場合にはセリンになる。そし
て、G2677A又はG2677Tの変異を有する患者において、タ
クロリムス又はシクロスポリンである免疫抑制剤の副作
用が生じる可能性が高くなる。従って、MDR1遺伝子の26
77ntがグアニンであるか、アデニン又はチミンであるか
を調べることにより、上記免疫抑制剤の副作用が生じる
か否かを予測することができる。なお、G2677Aは、この
点突然変異自体が、本願発明者らにより発見された新規
なものである。BEST MODE FOR CARRYING OUT THE INVENTION In the method of the present invention, the position of the MDR1 gene in the coding region of the cDNA sequence
Unless otherwise specified, the position of the base indicates the position on the cDNA coding region. The 2677th base (hereinafter, “2677
nt ". (Similarly for other positions) is guanine, adenine or thymine. 2677 nt is located in exon 21 of MDR1.
The nucleotide sequence of the coding region of the cDNA of the normal MDR1 gene (sometimes referred to as “wild type” in the present specification) is shown in SEQ ID NO: 1 in the sequence listing. This sequence is for GenBank Accessio
n Included in the sequence shown in No. M14758. In addition,
Since the MDR1 gene has SNPs at various positions, the wild type shown in SEQ ID NO: 1 is a typical example of a normal human gene, and various polymorphisms exist. 2677 of the MDR1 gene
The nt is guanine (G) in the wild type, but it is a mutation that becomes adenine (A) (a mutation in which G of 2677 nt becomes A is referred to as “G2677
A "is displayed. Other point mutations are similarly indicated) and there is a mutation G2677T that results in T. 2677nt is G
In the case of (1), the amino acid encoded by the codon containing it is alanine, but in the case of G2677A, this becomes threonine, and in the case of G2677T, it becomes serine. In patients with the G2677A or G2677T mutation, there is a high possibility that side effects of the immunosuppressant that is tacrolimus or cyclosporine will occur. Therefore, 26 of the MDR1 gene
By examining whether 77nt is guanine, adenine or thymine, it can be predicted whether or not the side effects of the immunosuppressant will occur. G2677A is a novel point mutation per se discovered by the present inventors.
【０００９】本発明の方法は、MDR1遺伝子の2677ntがグ
アニンであるか、アデニン又はチミンであるかを知るこ
とができるあらゆるいずれの方法によっても行うことが
でき、それらのいずれもが本発明の範囲に含まれる。[0009] The method of the present invention can be carried out by any method capable of knowing whether 2677 nt of the MDR1 gene is guanine, adenine or thymine, any of which is within the scope of the present invention. include.
【００１０】例えば、MDR1遺伝子のｃＤＮＡの塩基配列
は配列番号１に示されるように既にわかっている。ま
た、配列番号１６に示すように、2677ntが含まれるエク
ソン21の両端のイントロン配列の一部もわかっている(G
enBank Accession No. M29440。配列番号１６中、イン
トロン領域は1nt〜26nt及び231nt〜245nt）。従って、2
677ntを含むゲノミックMDR1領域を、ＰＣＲのような公
知の核酸増幅法により増幅し、その増幅断片の塩基配列
を決定したり（ダイレクトシーケンシング）、増幅断片
の制限酵素断片長多型(RFLP)を調べることにより行うこ
とができる。ＰＣＲはこの分野において周知であり、こ
れを行うための試薬キット及び装置が市販されているの
で容易に行うことができる。この場合、プライマーとし
ては、ゲノミックMDR1遺伝子の2677ntよりも上流の領域
又は2677ntを含む領域とハイブリダイズするフォワード
側プライマーと、2677ntよりも下流の領域又は2677ntを
含む領域とハイブリダイズするリバース側プライマー
（ただし、フォワード側プライマーとリバース側プライ
マーがハイブリダイズする領域は重複しない）から成る
プライマーセットを用い、患者の細胞から得られたゲノ
ミックＤＮＡを鋳型としてＰＣＲを行うことにより、MD
R1遺伝子の2677ntを含む領域を増幅することができる。
2677ntの塩基の種類に拘わらず１種類のプライマーセッ
トで確実に増幅が起きるようにするために、フォワード
側プライマーは2677ntよりも上流の領域とハイブリダイ
ズし、リバース側プライマーは2677ntよりも下流の領域
とハイブリダイズするものである場合がより好ましい。
なお、プライマーは、イントロン領域（例えば配列番号
１６に示されるエクソン21の両端のイントロン領域）、
エクソン領域（例えば配列番号１６に示されるエクソン
21内の領域や配列番号１に示される他のエクソンの領
域）及びイントロン−エクソン連結部領域（例えば配列
番号１６に示されるエクソン21の両端のイントロン−エ
クソン連結部領域）のいずれにハイブリダイズするもの
であってもよい。鋳型となる患者のゲノミックＤＮＡ
は、患者から採取した任意の細胞から調製することがで
き、末梢血に含まれるリンパ球細胞や血球細胞から調製
することが好都合である。細胞からのゲノミックＤＮＡ
の調製は常法により行うことができる。あるいは、常法
により細胞からｍＲＮＡを調製し、それを鋳型としたＲ
Ｔ−ＰＣＲにより、MDR1遺伝子のｃＤＮＡを増幅しても
よい。これらの核酸増幅に用いられるプライマーは、MD
R1遺伝子又はそのｃＤＮＡに特異的にハイブリダイズす
るサイズを有していればよく、通常、１２塩基〜５０塩
基、好ましくは１５塩基〜４０塩基程度のサイズを有す
る。増幅領域のサイズは特に限定されないが、あまり長
くする利点はなく、逆に増幅やシーケンシングに要する
時間や材料が無駄になるので、ダイレクトシーケンシン
グの場合は好ましくは１００塩基以下、さらに好ましく
は５０塩基以下であり、RFLPの場合は好ましくは３００
塩基以下、さらに好ましくは２００塩基以下である。も
っとも、これらより長くても構わない。また、プライマ
ーは、これがハイブリダイズするゲノミックMDR1遺伝子
又はそのｃＤＮＡの領域と完全に相補的なものが好まし
いが、ハイブリダイゼーション及びその後の鎖の伸長に
差し支えがないのであれば、少数の非相補的塩基を含ん
でいてもよい。また、MDR1遺伝子のSNPが存在する領域
にハイブリダイズするプライマーを用いる場合には、各
変異に相補的な塩基を有するプライマーを混合した混合
プライマーを用いることもできる。また、G2677A及びG2
677Tは、アミノ酸の変異をもたらすので、これらの変異
は、MDR1遺伝子産物のアミノ酸配列の解析、又は、該変
異部分を含む領域をエピトープとするモノクローナル抗
体を用いた免疫分析により調べることも可能である。For example, the nucleotide sequence of the cDNA of the MDR1 gene is already known as shown in SEQ ID NO: 1. In addition, as shown in SEQ ID NO: 16, part of the intron sequence at both ends of exon 21 including 2677 nt is also known (G
enBank Accession No. M29440. In SEQ ID NO: 16, the intron regions are 1 nt to 26 nt and 231 nt to 245 nt). Therefore, 2
The genomic MDR1 region containing 677 nt is amplified by a known nucleic acid amplification method such as PCR, the base sequence of the amplified fragment is determined (direct sequencing), and the restriction fragment length polymorphism (RFLP) of the amplified fragment is determined. You can do this by examining it. PCR is well known in the art and can be easily performed because reagent kits and equipment for performing this are commercially available. In this case, as a primer, a forward primer that hybridizes to a region upstream of or including 2677 nt of the genomic MDR1 gene, and a reverse primer that hybridizes to a region downstream of 2677 nt or a region including 2677 nt ( However, the region where the forward primer and the reverse primer hybridize does not overlap), and the genomic DNA obtained from the patient's cells is used as a template to perform PCR.
A region containing 2677 nt of the R1 gene can be amplified.
In order to ensure amplification with one type of primer set regardless of the type of the 2677 nt base, the forward primer hybridizes with the region upstream of 2677 nt, and the reverse primer hybridizes with the region downstream of 2677 nt. It is more preferred that they hybridize with
In addition, the primers include intron regions (for example, intron regions at both ends of exon 21 shown in SEQ ID NO: 16),
Exon region (for example, exon shown in SEQ ID NO: 16)
21 or the region of the other exon shown in SEQ ID NO: 1) and the intron-exon junction region (for example, the intron-exon junction region at both ends of exon 21 shown in SEQ ID NO: 16). It may be something. Genomic DNA of patient as template
Can be prepared from any cells collected from a patient, and is conveniently prepared from lymphocytes and blood cells contained in peripheral blood. Genomic DNA from cells
Can be prepared by a conventional method. Alternatively, mRNA is prepared from cells by a conventional method, and
The MDR1 gene cDNA may be amplified by T-PCR. Primers used for these nucleic acid amplifications are MD
It is sufficient if it has a size that specifically hybridizes to the R1 gene or its cDNA, and usually has a size of about 12 to 50 bases, preferably about 15 to 40 bases. The size of the amplification region is not particularly limited, but there is no advantage of making it too long. Conversely, the time and materials required for amplification and sequencing are wasted. Therefore, in the case of direct sequencing, preferably 100 bases or less, and more preferably 50 Base or less, and preferably 300 in the case of RFLP.
It is no more than a base, more preferably no more than 200 bases. However, it may be longer than these. The primer is preferably completely complementary to the region of the genomic MDR1 gene or its cDNA to which the primer hybridizes. May be included. When a primer that hybridizes to the region where the SNP of the MDR1 gene is present is used, a mixed primer obtained by mixing primers having bases complementary to each mutation can also be used. G2677A and G2677A
Since 677T causes amino acid mutations, these mutations can be examined by analyzing the amino acid sequence of the MDR1 gene product or by immunoassay using a monoclonal antibody having a region containing the mutated portion as an epitope. .
【００１１】増幅断片についてRFLPを行う場合には、26
77ntの塩基の種類に応じて切断が生じたり生じなかった
りするいずれの制限酵素も用いることができる。例え
ば、2677ntがＧの場合には、Nhe Iで切断が生じ、Ｔの
場合にはXba Iで切断が生じ、Ａの場合にはAfa Iで切断
が生じるので、これらの制限酵素をそれぞれ用いて増幅
断片を消化し、消化物のサイズを電気泳動等で調べるこ
とにより、2677ntがＧかＴかＡかを調べることができ
る。When RFLP is performed on an amplified fragment, 26
Any restriction enzyme that can or may not be cleaved depending on the type of 77 nt base can be used. For example, when 2677 nt is G, cleavage occurs at Nhe I, when T occurs, cleavage occurs at Xba I, and at A, cleavage occurs at Afa I. By digesting the amplified fragment and checking the size of the digest by electrophoresis or the like, it can be determined whether 2677 nt is G, T or A.
【００１２】また、フォワード側プライマー及びリバー
ス側プライマーのいずれか一方を、2677ntを含む領域と
ハイブリダイズする領域に設定し、鋳型となるMDR1遺伝
子又はそのｃＤＮＡの2677ntがそのプライマーの対応塩
基と相補的な場合にのみ増幅が起きるようにして、ＰＣ
Ｒ等の核酸増幅法を行い、増幅が起きるか否かを調べて
2677ntの塩基がＧかＴかＡかを調べることも可能であ
る。この場合には、2677ntがプライマーの対応塩基と相
補的でない場合に増幅が起きないようにするために、フ
ォワード側プライマーの３’末端が2677ntと対合するよ
うにプライマーを設定することが好ましい。このような
プライマーを用いると、2677ntがプライマーの３’末端
と相補的な場合にのみ増幅が起き、非相補的な場合には
増幅が起きないように確実にできるので、2677ntの塩基
がＧかＴかＡかを正確に調べることができる。なお、こ
の場合のプライマーも、MDR1遺伝子に特異的にハイブリ
ダイズするサイズを有していればよく、通常、１２塩基
〜５０塩基、好ましくは１５塩基〜４０塩基程度のサイ
ズを有する。[0012] Further, one of the forward primer and the reverse primer is set to a region that hybridizes with a region containing 2677 nt, and 2677 nt of the MDR1 gene or its cDNA as a template is complementary to the corresponding base of the primer. Amplification only occurs when
Perform a nucleic acid amplification method such as R, and check whether amplification occurs
It is also possible to check whether the 2677nt base is G, T or A. In this case, in order to prevent amplification when 2677nt is not complementary to the corresponding base of the primer, it is preferable to set the primer so that the 3 'end of the forward primer is paired with 2677nt. When such a primer is used, amplification occurs only when 2677 nt is complementary to the 3 ′ end of the primer, and when non-complementary, it can be ensured that amplification does not occur. T or A can be checked accurately. The primer in this case may have a size that specifically hybridizes to the MDR1 gene, and usually has a size of about 12 to 50 bases, preferably about 15 to 40 bases.
【００１３】さらに、2677ntを含む領域とハイブリダイ
ズする核酸プローブを用いて2677ntの塩基がＧかＴかＡ
かを調べることもできる。プローブとしては、オリゴヌ
クレオチドに蛍光標識、ビオチン標識、放射標識等の適
当な標識を付したものを用いることができる。プローブ
のサイズは、MDR1遺伝子に特異的にハイブリダイズする
サイズを有していればよく、通常、１２塩基〜１００塩
基、好ましくは１５塩基〜５０塩基程度のサイズを有す
る。標識プローブを用いる場合、2677ntの他の変異を有
する、標識していないオリゴヌクレオチドを混合した混
合プローブを用いることにより、2677ntの塩基と相補的
な対応塩基を有する標識プローブのみをハイブリダイズ
させることが容易になる。また、プローブを標識せず
に、固相に不動化し、鋳型ＤＮＡを増幅する際に標識し
て不動化プローブとハイブリダイズさせ、洗浄後、増幅
産物が固相に結合したか否かで2677ntの塩基を調べるこ
とも可能である。すなわち、MDR1遺伝子の2677ntの変異
を調べるためのＤＮＡチップを用いることもできる。オ
リゴヌクレオチドの固相への結合は常法により行うこと
ができる。Further, using a nucleic acid probe that hybridizes with a region containing 2677 nt, the base of 2677 nt is G or T or A
You can also find out. As the probe, an oligonucleotide having an appropriate label such as a fluorescent label, a biotin label, or a radiolabel can be used. The probe may have a size that specifically hybridizes to the MDR1 gene, and usually has a size of about 12 to 100 bases, preferably about 15 to 50 bases. When using a labeled probe, having another mutation of 2677 nt, by using a mixed probe obtained by mixing unlabeled oligonucleotides, it is possible to hybridize only a labeled probe having a corresponding base complementary to the 2677 nt base. It will be easier. In addition, without labeling the probe, immobilized on a solid phase, labeled when amplifying the template DNA, hybridized with the immobilized probe, and after washing, 2677 nt of whether or not the amplification product was bound to the solid phase. It is also possible to check the base. That is, a DNA chip for examining the 2677 nt mutation of the MDR1 gene can be used. The binding of the oligonucleotide to the solid phase can be performed by a conventional method.
【００１４】その他、例えばSSCP(single-strand confo
rmation polymorphism)のような他の方法により2677nt
の変異を調べることも可能である。要は、ゲノミックMD
R1遺伝子の2677ntの塩基がグアニンであるか、アデニン
又はチミンであるかを調べることができればいずれの方
法をも採用することができ、それらのいずれも本発明の
範囲に含まれる。In addition, for example, SSCP (single-strand confo
2677nt by other methods like rmation polymorphism)
It is also possible to examine mutations in In short, genomic MD
Any method can be adopted as long as it can be determined whether the 2677 nt base of the R1 gene is guanine, adenine or thymine, and any of them is included in the scope of the present invention.
【００１５】[0015]
【実施例】以下、本発明を実施例に基づきより具体的に
説明する。もっとも、本発明は下記実施例に限定される
ものではない。EXAMPLES Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
【００１６】実施例１ (1) 患者 生体肝移植を受け、術後にタクロリムスを投与された１
７人の患者について調査した。患者は、発作、痙攣、震
え又は脳症の履歴を持たない人達であった。全ての患者
は、移植の前日又は当日からタクロリムスの投与を受け
始めた。投与量は、最初の２週間が、血中最低濃度が15
〜20 ng/mlに、次の２週間が血中最低濃度が約10 ng/ml
になるように毎日調節した。１７人の患者のうち、６人
（１０歳から４７歳）が移植１０日以内に痙攣、震え又
は白質脳炎のような神経毒性症状を呈した。他の患者
（４歳〜６５歳）は、入院期間中神経毒性症状を示さな
かった。なお、移植の外科手術及びその後の投薬は、神
経毒性症状を呈した患者と呈さなかった患者で異ならな
かった。Example 1 (1) Patient Receiving living-donor liver transplantation and receiving tacrolimus after surgery 1
Seven patients were investigated. Patients had no history of seizures, convulsions, tremors or encephalopathy. All patients began receiving tacrolimus the day before or on the day of transplantation. Dosage should be in the first 2 weeks with a minimum blood concentration of 15
~ 20 ng / ml, next 10 weeks minimum blood concentration of about 10 ng / ml
Adjusted daily to become Of the 17 patients, 6 (10 to 47 years old) exhibited neurotoxic symptoms such as convulsions, tremors or leukoencephalitis within 10 days of transplantation. Other patients (4 to 65 years) did not show neurotoxic symptoms during the hospital stay. It should be noted that transplant surgery and subsequent medication did not differ between patients who exhibited neurotoxic symptoms and those who did not.
【００１７】(2) 点突然変異の検出 患者から静脈血１０ｍｌを採取し、GENOMIX（商品名、
イタリア国TriesteのTalent srl社製）を用いて末梢血
リンパ球からゲノミックＤＮＡを分離した。このゲノミ
ックＤＮＡを鋳型として用いて、各エクソン領域を増幅
するＰＣＲをそれぞれ行った。プライマーは、MDR1遺伝
子の各イントロン又はイントロン−エクソン連結部にハ
イブリダイズするオリゴヌクレオチドのペアであり、各
増幅領域は300 bp以下であった。各ＰＣＲは、100 ngの
ゲノミックＤＮＡ、0.25μＭの各プライマー、10xPCRバ
ッファII、1.5 mM MgCl2,0.2 mMの各dNTP及び1.25〜2.5
UのAmpliTaq Gold ＤＮＡポリメラーゼ（米国カリフ
ォルニア州Foster CityのApplied Biosystems社製）を
含む総量５０μｌの溶液を用いて行った。最初に９４℃
で５分間変性させた後、９４℃、0.5〜１分間の変性工
程、５０〜６０℃、0.5〜１分間のアニーリング工程、
７２℃、１〜２分間の伸長工程から成るサイクルを３０
〜４５回繰り返した。各増幅断片の塩基配列を決定し、
野生型の配列と比較した。その結果、表１に示す８種類
の点突然変異(A-41aG, C-145G, T-129C, T1236C, G2677
[A,T], C3435T, A4036G)を検出した。これらの突然変異
のうち、アミノ酸の変異がもたらされるのは、G2677A及
びG2677Tだけであった。なお、これら８種類の変異の検
出に用いた各プライマーの配列を示す配列番号を下記表
２に示す。(2) Detection of Point Mutation 10 ml of venous blood was collected from a patient, and GENOMIX (trade name,
Genomic DNA was isolated from peripheral blood lymphocytes using Talent srl, Trieste, Italy. Using this genomic DNA as a template, PCR was performed to amplify each exon region. Primers were oligonucleotide pairs that hybridized to each intron or intron-exon junction of the MDR1 gene, and each amplified region was 300 bp or less. Each PCR consisted of 100 ng of genomic DNA, 0.25 μM of each primer, 10 × PCR buffer II, 1.5 mM MgCl 2 , 0.2 mM of each dNTP and 1.25-2.5
A total of 50 μl of a solution containing U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, Calif., USA) was used. 94 ° C first
After denaturing for 5 minutes at 94 ° C. for 0.5 to 1 minute, annealing at 50 to 60 ° C. for 0.5 to 1 minute,
A cycle consisting of an extension step at 72 ° C for 1-2 minutes
Repeated ~ 45 times. Determine the base sequence of each amplified fragment,
Compared to the wild type sequence. As a result, eight types of point mutations shown in Table 1 (A-41aG, C-145G, T-129C, T1236C, G2677
[A, T], C3435T, A4036G) were detected. Of these mutations, only G2677A and G2677T resulted in amino acid mutations. In addition, the sequence numbers indicating the sequences of the respective primers used for detecting these eight types of mutations are shown in Table 2 below.
【００１８】[0018]
【表１】表１ [Table 1] Table 1
【００１９】[0019]
【表２】表２ [Table 2] Table 2
【００２０】(3) 解析 神経毒性症状が生じるか否かの原因を調べるために、段
階的判別関数分析(stepwise discriminant function an
alysis)を行った。各段階において、χ2試験の改良を用
いて、その段階で入れた変数が有意に判別を改善するか
否かを調べた。独立変数は、８種類の多型(A-41aG, C-1
45G, T-129C, T1236C, G2677[A,T], C3435T, A4036G)、
変異の数、年齢、移植片重量、移植片重量／患者標準肝
臓体積比、タクロリムス最低血中濃度、ヘマトクリット
値、AST値及びALT値であった。副作用は１０日以内に現
れたので、データは移植１０日後のものを用いた。各変
異は、カテゴリー値(categorical value)として扱った
（例えば、野生型アレルのホモ接合=0、変異アレルのヘ
テロ接合=1、変異アレルのホモ接合=2)。段階的プロセ
スにより選択された変数は３種類の突然変異(C-145G, G
2677[A,T], C3435T)、タクロリムス血中最低濃度、AST
値及びALT値であった（表３）。これらの変数の組合せ
により、１００％予測レベル（χ2=11.68; p<0.001)で
神経毒性を予測できた。エクソン２１内のSNPs(G2677
[A,T])は、神経毒性の発生についてポジティブな予測変
数であったが、エクソン1a中の変異(C-145G)及びエクソ
ン２６中の変異(C3435T)はネガティブに寄与した。(3) Analysis A stepwise discriminant function analysis was performed to investigate the cause of the occurrence of a neurotoxic symptom.
alysis). In each stage, using a modification of the chi 2 test, it was investigated whether a variable which takes at that stage will improve significantly discrimination. The independent variables consist of eight polymorphisms (A-41aG, C-1
45G, T-129C, T1236C, G2677 [A, T], C3435T, A4036G),
The number of mutations, age, graft weight, graft weight / patient standard liver volume ratio, minimum tacrolimus blood concentration, hematocrit value, AST value and ALT value. Since side effects appeared within 10 days, the data was used 10 days after transplantation. Each mutation was treated as a categorical value (eg, homozygous wild-type allele = 0, heterozygous mutant allele = 1, homozygous mutant allele = 2). The variables selected by the stepwise process consisted of three mutations (C-145G, G
2677 [A, T], C3435T), Tacrolimus blood minimum concentration, AST
Values and ALT values (Table 3). The combination of these variables could predict neurotoxicity at the 100% predictive level (レ ベ ル2 = 11.68; p <0.001). SNPs in exon 21 (G2677
[A, T]) was a positive predictor of the occurrence of neurotoxicity, while mutations in exon 1a (C-145G) and in exon 26 (C3435T) contributed negatively.
【００２１】[0021]
【表３】 表３ タクロリムス誘導神経毒性の段階的判別関数分析 Table 3 Stepwise discriminant function analysis of tacrolimus-induced neurotoxicity
【００２２】表３に示されるように、G2677[A,T]の点突
然変異は、係数が+38.7と高くなっている。このことか
ら、G2677[A,T]の点突然変異の有無を調べることによ
り、タクロリムスの副作用が起きるか否かを予測するこ
とが可能であることがわかった。As shown in Table 3, the point mutation of G2677 [A, T] has a high coefficient of +38.7. From this, it was found that by examining the presence or absence of the point mutation of G2677 [A, T], it was possible to predict whether or not a side effect of tacrolimus would occur.
【００２３】実施例２ 実施例１に記載した方法により増幅した、2677ntを含む
107 bpの増幅断片を、制限酵素Nhe I、Xba I又はAfa I
で常法により消化した。消化物を３％アガロースゲル電
気泳動にかけ、臭化エチジウムで染色した。その結果、
野生型アレルをNhe Iで消化した場合、G2677TをXba Iで
消化した場合、及びG2677AをAfa Iで消化した場合に
は、83 bpの断片が検出されたが、これら以外の場合に
は、107 bpの断片が検出された。従って、この方法によ
ってPCR-RFLPで2677ntの塩基がＧかＴかＡかを調べるこ
とができることがわかる。Example 2 Contains 2677 nt amplified by the method described in Example 1.
The amplified fragment of 107 bp was digested with restriction enzymes Nhe I, Xba I or Afa I.
In a conventional manner. The digest was subjected to 3% agarose gel electrophoresis and stained with ethidium bromide. as a result,
When the wild-type allele was digested with Nhe I, when G2677T was digested with Xba I, and when G2677A was digested with Afa I, a 83 bp fragment was detected. A bp fragment was detected. Therefore, it can be seen that by this method, it is possible to determine whether the 2677 nt base is G, T or A by PCR-RFLP.
【００２４】[0024]
【発明の効果】本発明の方法により、タクロリムス又は
シクロスポリンである免疫抑制剤の副作用が生じるか否
かを予測することが初めて可能になった。According to the method of the present invention, it has become possible for the first time to predict whether or not side effects of an immunosuppressant which is tacrolimus or cyclosporine will occur.
【００２５】[0025]
【配列表】 SEQUENCE LISTING <110> SRL, INC. <120> Method for predicting side effects of immunosuppressants and prim ers used therefor <130> 01692 <160> 16[Sequence List] SEQUENCE LISTING <110> SRL, INC. <120> Method for predicting side effects of immunosuppressants and primers used therefor <130> 01692 <160> 16
【００２６】 <210> 1 <211> 3843 <212> DNA <213> Homo sapience <400> 1 atg gat ctt gaa ggg gac cgc aat gga gga gca aag aag aag aac ttt 48 Met Asp Leu Glu Gly Asp Arg Asn Gly Gly Ala Lys Lys Lys Asn Phe 1 5 10 15 ttt aaa ctg aac aat aaa agt gaa aaa gat aag aag gaa aag aaa cca 96 Phe Lys Leu Asn Asn Lys Ser Glu Lys Asp Lys Lys Glu Lys Lys Pro 20 25 30 act gtc agt gta ttt tca atg ttt cgc tat tca aat tgg ctt gac aag 144 Thr Val Ser Val Phe Ser Met Phe Arg Tyr Ser Asn Trp Leu Asp Lys 35 40 45 ttg tat atg gtg gtg gga act ttg gct gcc atc atc cat ggg gct gga 192 Leu Tyr Met Val Val Gly Thr Leu Ala Ala Ile Ile His Gly Ala Gly 50 55 60 ctt cct ctc atg atg ctg gtg ttt gga gaa atg aca gat atc ttt gca 240 Leu Pro Leu Met Met Leu Val Phe Gly Glu Met Thr Asp Ile Phe Ala 65 70 75 80 aat gca gga aat tta gaa gat ctg atg tca aac atc act aat aga agt 288 Asn Ala Gly Asn Leu Glu Asp Leu Met Ser Asn Ile Thr Asn Arg Ser 85 90 95 gat atc aat gat aca ggg ttc ttc atg aat ctg gag gaa gac atg acc 336 Asp Ile Asn Asp Thr Gly Phe Phe Met Asn Leu Glu Glu Asp Met Thr 100 105 110 agg tat gcc tat tat tac agt gga att ggt gct ggg gtg ctg gtt gct 384 Arg Tyr Ala Tyr Tyr Tyr Ser Gly Ile Gly Ala Gly Val Leu Val Ala 115 120 125 gct tac att cag gtt tca ttt tgg tgc ctg gca gct gga aga caa ata 432 Ala Tyr Ile Gln Val Ser Phe Trp Cys Leu Ala Ala Gly Arg Gln Ile 130 135 140 cac aaa att aga aaa cag ttt ttt cat gct ata atg cga cag gag ata 480 His Lys Ile Arg Lys Gln Phe Phe His Ala Ile Met Arg Gln Glu Ile 145 150 155 160 ggc tgg ttt gat gtg cac gat gtt ggg gag ctt aac acc cga ctt aca 528 Gly Trp Phe Asp Val His Asp Val Gly Glu Leu Asn Thr Arg Leu Thr 165 170 175 gat gat gtc tct aag att aat gaa gtt att ggt gac aaa att gga atg 576 Asp Asp Val Ser Lys Ile Asn Glu Val Ile Gly Asp Lys Ile Gly Met 180 185 190 ttc ttt cag tca atg gca aca ttt ttc act ggg ttt ata gta gga ttt 624 Phe Phe Gln Ser Met Ala Thr Phe Phe Thr Gly Phe Ile Val Gly Phe 195 200 205 aca cgt ggt tgg aag cta acc ctt gtg att ttg gcc atc agt cct gtt 672 Thr Arg Gly Trp Lys Leu Thr Leu Val Ile Leu Ala Ile Ser Pro Val 210 215 220 ctt gga ctg tca gct gct gtc tgg gca aag ata cta tct tca ttt act 720 Leu Gly Leu Ser Ala Ala Val Trp Ala Lys Ile Leu Ser Ser Phe Thr 225 230 235 240 gat aaa gaa ctc tta gcg tat gca aaa gct gga gca gta gct gaa gag 768 Asp Lys Glu Leu Leu Ala Tyr Ala Lys Ala Gly Ala Val Ala Glu Glu 245 250 255 gtc ttg gca gca att aga act gtg att gca ttt gga gga caa aag aaa 816 Val Leu Ala Ala Ile Arg Thr Val Ile Ala Phe Gly Gly Gln Lys Lys 260 265 270 gaa ctt gaa agg tac aac aaa aat tta gaa gaa gct aaa aga att ggg 864 Glu Leu Glu Arg Tyr Asn Lys Asn Leu Glu Glu Ala Lys Arg Ile Gly 275 280 285 ata aag aaa gct att aca gcc aat att tct ata ggt gct gct ttc ctg 912 Ile Lys Lys Ala Ile Thr Ala Asn Ile Ser Ile Gly Ala Ala Phe Leu 290 295 300 ctg atc tat gca tct tat gct ctg gcc ttc tgg tat ggg acc acc ttg 960 Leu Ile Tyr Ala Ser Tyr Ala Leu Ala Phe Trp Tyr Gly Thr Thr Leu 305 310 315 320 gtc ctc tca ggg gaa tat tct att gga caa gta ctc act gta ttc ttt 1008 Val Leu Ser Gly Glu Tyr Ser Ile Gly Gln Val Leu Thr Val Phe Phe 325 330 335 tct gta tta att ggg gct ttt agt gtt gga cag gca tct cca agc att 1056 Ser Val Leu Ile Gly Ala Phe Ser Val Gly Gln Ala Ser Pro Ser Ile 340 345 350 gaa gca ttt gca aat gca aga gga gca gct tat gaa atc ttc aag ata 1104 Glu Ala Phe Ala Asn Ala Arg Gly Ala Ala Tyr Glu Ile Phe Lys Ile 355 360 365 att gat aat aag cca agt att gac agc tat tcg aag agt ggg cac aaa 1152 Ile Asp Asn Lys Pro Ser Ile Asp Ser Tyr Ser Lys Ser Gly His Lys 370 375 380 cca gat aat att aag gga aat ttg gaa ttc aga aat gtt cac ttc agt 1200 Pro Asp Asn Ile Lys Gly Asn Leu Glu Phe Arg Asn Val His Phe Ser 385 390 395 400 tac cca tct cga aaa gaa gtt aag atc ttg aag ggc ctg aac ctg aag 1248 Tyr Pro Ser Arg Lys Glu Val Lys Ile Leu Lys Gly Leu Asn Leu Lys 405 410 415 gtg cag agt ggg cag acg gtg gcc ctg gtt gga aac agt ggc tgt ggg 1296 Val Gln Ser Gly Gln Thr Val Ala Leu Val Gly Asn Ser Gly Cys Gly 420 425 430 aag agc aca aca gtc cag ctg atg cag agg ctc tat gac ccc aca gag 1344 Lys Ser Thr Thr Val Gln Leu Met Gln Arg Leu Tyr Asp Pro Thr Glu 435 440 445 ggg atg gtc agt gtt gat gga cag gat att agg acc ata aat gta agg 1392 Gly Met Val Ser Val Asp Gly Gln Asp Ile Arg Thr Ile Asn Val Arg 450 455 460 ttt cta cgg gaa atc att ggt gtg gtg agt cag gaa cct gta ttg ttt 1440 Phe Leu Arg Glu Ile Ile Gly Val Val Ser Gln Glu Pro Val Leu Phe 465 470 475 480 gcc acc acg ata gct gaa aac att cgc tat ggc cgt gaa aat gtc acc 1488 Ala Thr Thr Ile Ala Glu Asn Ile Arg Tyr Gly Arg Glu Asn Val Thr 485 490 495 atg gat gag att gag aaa gct gtc aag gaa gcc aat gcc tat gac ttt 1536 Met Asp Glu Ile Glu Lys Ala Val Lys Glu Ala Asn Ala Tyr Asp Phe 500 505 510 atc atg aaa ctg cct cat aaa ttt gac acc ctg gtt gga gag aga ggg 1584 Ile Met Lys Leu Pro His Lys Phe Asp Thr Leu Val Gly Glu Arg Gly 515 520 525 gcc cag ttg agt ggt ggg cag aag cag agg atc gcc att gca cgt gcc 1632 Ala Gln Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala 530 535 540 ctg gtt cgc aac ccc aag atc ctc ctg ctg gat gag gcc acg tca gcc 1680 Leu Val Arg Asn Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala 545 550 555 560 ttg gac aca gaa agc gaa gca gtg gtt cag gtg gct ctg gat aag gcc 1728 Leu Asp Thr Glu Ser Glu Ala Val Val Gln Val Ala Leu Asp Lys Ala 565 570 575 aga aaa ggt cgg acc acc att gtg ata gct cat cgt ttg tct aca gtt 1776 Arg Lys Gly Arg Thr Thr Ile Val Ile Ala His Arg Leu Ser Thr Val 580 585 590 cgt aat gct gac gtc atc gct ggt ttc gat gat gga gtc att gtg gag 1824 Arg Asn Ala Asp Val Ile Ala Gly Phe Asp Asp Gly Val Ile Val Glu 595 600 605 aaa gga aat cat gat gaa ctc atg aaa gag aaa ggc att tac ttc aaa 1872 Lys Gly Asn His Asp Glu Leu Met Lys Glu Lys Gly Ile Tyr Phe Lys 610 615 620 CTT GTC ACA ATG CAG ACA GCA GGA AAT GAA GTT GAA TTA GAA AAT GCA 1920 Leu Val Thr Met Gln Thr Ala Gly Asn Glu Val Glu Leu Glu Asn Ala 625 630 635 640 gct gat gaa tcc aaa agt gaa att gat gcc ttg gaa atg tct tca aat 1968 Ala Asp Glu Ser Lys Ser Glu Ile Asp Ala Leu Glu Met Ser Ser Asn 645 650 655 gat tca aga tcc agt cta ata aga aaa aga tca act cgt agg agt gtc 2016 Asp Ser Arg Ser Ser Leu Ile Arg Lys Arg Ser Thr Arg Arg Ser Val 660 665 670 cgt gga tca caa gcc caa gac aga aag ctt agt acc aaa gag gct ctg 2064 Arg Gly Ser Gln Ala Gln Asp Arg Lys Leu Ser Thr Lys Glu Ala Leu 675 680 685 gat gaa agt ata cct cca gtt tcc ttt tgg agg att atg aag cta aat 2112 Asp Glu Ser Ile Pro Pro Val Ser Phe Trp Arg Ile Met Lys Leu Asn 690 695 700 tta act gaa tgg cct tat ttt gtt gtt ggt gta ttt tgt gcc att ata 2160 Leu Thr Glu Trp Pro Tyr Phe Val Val Gly Val Phe Cys Ala Ile Ile 705 710 715 720 aat gga ggc ctg caa cca gca ttt gca ata ata ttt tca aag att ata 2208 Asn Gly Gly Leu Gln Pro Ala Phe Ala Ile Ile Phe Ser Lys Ile Ile 725 730 735 ggg gtt ttt aca aga att gat gat cct gaa aca aaa cga cag aat agt 2256 Gly Val Phe Thr Arg Ile Asp Asp Pro Glu Thr Lys Arg Gln Asn Ser 740 745 750 aac ttg ttt tca cta ttg ttt cta gcc ctt gga att att tct ttt att 2304 Asn Leu Phe Ser Leu Leu Phe Leu Ala Leu Gly Ile Ile Ser Phe Ile 755 760 765 aca ttt ttc ctt cag ggt ttc aca ttt ggc aaa gct gga gag atc ctc 2352 Thr Phe Phe Leu Gln Gly Phe Thr Phe Gly Lys Ala Gly Glu Ile Leu 770 775 780 acc aag cgg ctc cga tac atg gtt ttc cga tcc atg ctc aga cag gat 2400 Thr Lys Arg Leu Arg Tyr Met Val Phe Arg Ser Met Leu Arg Gln Asp 785 790 795 800 gtg agt tgg ttt gat gac cct aaa aac acc act gga gca ttg act acc 2448 Val Ser Trp Phe Asp Asp Pro Lys Asn Thr Thr Gly Ala Leu Thr Thr 805 810 815 agg ctc gcc aat gat gct gct caa gtt aaa ggg gct ata ggt tcc agg 2496 Arg Leu Ala Asn Asp Ala Ala Gln Val Lys Gly Ala Ile Gly Ser Arg 820 825 830 ctt gct gta att acc cag aat ata gca aat ctt ggg aca gga ata att 2544 Leu Ala Val Ile Thr Gln Asn Ile Ala Asn Leu Gly Thr Gly Ile Ile 835 840 845 ata tcc ttc atc tat ggt tgg caa cta aca ctg tta ctc tta gca att 2592 Ile Ser Phe Ile Tyr Gly Trp Gln Leu Thr Leu Leu Leu Leu Ala Ile 850 855 860 gta ccc atc att gca ata gca gga gtt gtt gaa atg aaa atg ttg tct 2640 Val Pro Ile Ile Ala Ile Ala Gly Val Val Glu Met Lys Met Leu Ser 865 870 875 880 gga caa gca ctg aaa gat aag aaa gaa cta gaa ggt gct ggg aag atc 2688 Gly Gln Ala Leu Lys Asp Lys Lys Glu Leu Glu Gly Ala Gly Lys Ile 885 890 895 gct act gaa gca ata gaa aac ttc cga acc gtt gtt tct ttg act cag 2736 Ala Thr Glu Ala Ile Glu Asn Phe Arg Thr Val Val Ser Leu Thr Gln 900 905 910 gag cag aag ttt gaa cat atg tat gct cag agt ttg cag gta cca tac 2784 Glu Gln Lys Phe Glu His Met Tyr Ala Gln Ser Leu Gln Val Pro Tyr 915 920 925 aga aac tct ttg agg aaa gca cac atc ttt gga att aca ttt tcc ttc 2832 Arg Asn Ser Leu Arg Lys Ala His Ile Phe Gly Ile Thr Phe Ser Phe 930 935 940 acc cag gca atg atg tat ttt tcc tat gct gga tgt ttc cgg ttt gga 2880 Thr Gln Ala Met Met Tyr Phe Ser Tyr Ala Gly Cys Phe Arg Phe Gly 945 950 955 960 gcc tac ttg gtg gca cat aaa ctc atg agc ttt gag gat gtt ctg tta 2928 Ala Tyr Leu Val Ala His Lys Leu Met Ser Phe Glu Asp Val Leu Leu 965 970 975 gta ttt tca gct gtt gtc ttt ggt gcc atg gcc gtg ggg caa gtc agt 2976 Val Phe Ser Ala Val Val Phe Gly Ala Met Ala Val Gly Gln Val Ser 980 985 990 tca ttt gct cct gac tat gcc aaa gcc aaa ata tca gca gcc cac atc 3024 Ser Phe Ala Pro Asp Tyr Ala Lys Ala Lys Ile Ser Ala Ala His Ile 995 1000 1005 atc atg atc att gaa aaa acc cct ttg att gac agc tac agc acg gaa 3072 Ile Met Ile Ile Glu Lys Thr Pro Leu Ile Asp Ser Tyr Ser Thr Glu 1010 1015 1020 ggc cta atg ccg aac aca ttg gaa gga aat gtc aca ttt ggt gaa gtt 3120 Gly Leu Met Pro Asn Thr Leu Glu Gly Asn Val Thr Phe Gly Glu Val 1025 1030 1035 1040 gta ttc aac tat ccc acc cga ccg gac atc cca gtg ctt cag gga ctg 3168 Val Phe Asn Tyr Pro Thr Arg Pro Asp Ile Pro Val Leu Gln Gly Leu 1045 1050 1055 agc ctg gag gtg aag aag ggc cag acg ctg gct ctg gtg ggc agc agt 3216 Ser Leu Glu Val Lys Lys Gly Gln Thr Leu Ala Leu Val Gly Ser Ser 1060 1065 1070 ggc tgt ggg aag agc aca gtg gtc cag ctc ctg gag cgg ttc tac gac 3264 Gly Cys Gly Lys Ser Thr Val Val Gln Leu Leu Glu Arg Phe Tyr Asp 1075 1080 1085 ccc ttg gca ggg aaa gtg ctg ctt gat ggc aaa gaa ata aag cga ctg 3312 Pro Leu Ala Gly Lys Val Leu Leu Asp Gly Lys Glu Ile Lys Arg Leu 1090 1095 1100 aat gtt cag tgg ctc cga gca cac ctg ggc atc gtg tcc cag gag ccc 3360 Asn Val Gln Trp Leu Arg Ala His Leu Gly Ile Val Ser Gln Glu Pro 1105 1110 1115 1120 atc ctg ttt gac tgc agc att gct gag aac att gcc tat gga gac aac 3408 Ile Leu Phe Asp Cys Ser Ile Ala Glu Asn Ile Ala Tyr Gly Asp Asn 1125 1130 1135 agc cgg gtg gtg tca cag gaa gag atc gtg agg gca gca aag gag gcc 3456 Ser Arg Val Val Ser Gln Glu Glu Ile Val Arg Ala Ala Lys Glu Ala 1140 1145 1150 aac ata cat gcc ttc atc gag tca ctg cct aat aaa tat agc act aaa 3504 Asn Ile His Ala Phe Ile Glu Ser Leu Pro Asn Lys Tyr Ser Thr Lys 1155 1160 1165 gta gga gac aaa gga act cag ctc tct ggt ggc cag aaa caa cgc att 3552 Val Gly Asp Lys Gly Thr Gln Leu Ser Gly Gly Gln Lys Gln Arg Ile 1170 1175 1180 gcc ata gct cgt gcc ctt gtt aga cag cct cat att ttg ctt ttg gat 3600 Ala Ile Ala Arg Ala Leu Val Arg Gln Pro His Ile Leu Leu Leu Asp 1185 1190 1195 1200 gaa gcc acg tca gct ctg gat aca gaa agt gaa aag gtt gtc caa gaa 3648 Glu Ala Thr Ser Ala Leu Asp Thr Glu Ser Glu Lys Val Val Gln Glu 1205 1210 1215 gcc ctg gac aaa gcc aga gaa ggc cgc acc tgc att gtg att gct cac 3696 Ala Leu Asp Lys Ala Arg Glu Gly Arg Thr Cys Ile Val Ile Ala His 1220 1225 1230 cgc ctg tcc acc atc cag aat gca gac tta ata gtg gtg ttt cag aat 3744 Arg Leu Ser Thr Ile Gln Asn Ala Asp Leu Ile Val Val Phe Gln Asn 1235 1240 1245 ggc aga gtc aag gag cat ggc acg cat cag cag ctg ctg gca cag aaa 3792 Gly Arg Val Lys Glu His Gly Thr His Gln Gln Leu Leu Ala Gln Lys 1250 1255 1260 ggc atc tat ttt tca atg gtc agt gtc cag gct gga aca aag cgc cag 3840 Gly Ile Tyr Phe Ser Met Val Ser Val Gln Ala Gly Thr Lys Arg Gln 1265 1270 1275 1280 TGA 3843<210> 1 <211> 3843 <212> DNA <213> Homo sapience <400> 1 atg gat ctt gaa ggg gac cgc aat gga gga gca aag aag aag aac ttt 48 Met Asp Leu Glu Gly Asp Arg Asn Gly Gly Ala Lys Lys Lys Asn Phe 1 5 10 15 ttt aaa ctg aac aat aaa agt gaa aaa gat aag aag gaa aag aaa cca 96 Phe Lys Leu Asn Asn Lys Ser Glu Lys Asp Lys Lys Glu Lys Lys Pro 20 25 30 act gtc agt gta ttt tca atg ttt cgc tat tca aat tgg ctt gac aag 144 Thr Val Ser Val Phe Ser Met Phe Arg Tyr Ser Asn Trp Leu Asp Lys 35 40 45 ttg tat atg gtg gtg gga act ttg gct gcc atc atc cat ggg gct gga 192 Leu Tyr Met Val Val Gly Thr Leu Ala Ala Ile Ile His Gly Ala Gly 50 55 60 ctt cct ctc atg atg ctg gtg ttt gga gaa atg aca gat atc ttt gca 240 Leu Pro Leu Met Met Leu Val Phe Gly Glu Met Thr Asp Ile Phe Ala 65 70 75 80 aat gca gga aat tta gaa gat ctg atg tca aac atc act aat aga agt 288 Asn Ala Gly Asn Leu Glu Asp Leu Met Ser Asn Ile Thr Asn Arg Ser 85 90 95 gat atc aat gat aca ggg ttc ttc atg aat ctg gag gaa gac atg acc 336 Asp Ile Asn Asp Thr Gly Phe Phe Met Asn Leu Glu Glu Asp Met Thr 100 105 110 agg tat gcc tat tat tac agt gga att ggt gct ggg gtg ctg gtt gct 384 Arg Tyr Ala Tyr Tyr Tyr Ser Gly Ile Gly Ala Gly Val Leu Val Ala 115 120 125 gct tac att cag gtt tca ttt tgg tgc ctg gca gct gga aga caa ata 432 Ala Tyr Ile Gln Val Ser Phe Trp Cys Leu Ala Ala Gly Arg Gln Ile 130 135 140 cac aaa att aga aaa cag ttt ttt cat gct ata atg cga cag gag ata 480 His Lys Ile Arg Lys Gln Phe Phe His Ala Ile Met Arg Gln Glu Ile 145 150 155 160 ggc tgg ttt gat gtg cac gat gtt ggg gag ctt aac acc cga ctt aca 528 Gly Trp Phe Asp Val His Asp Val Gly Glu Leu Asn Thr Arg Leu Thr 165 170 175 gat gat gtc tct aag att aat gaa gtt att ggt gac aaa att gga atg 576 Asp Asp Val Ser Lys Ile Asn Glu Val Ile Gly Asp Lys Ile Gly Met 180 185 190 ttc ttt cag tca atg gca aca ttt ttc act ggg ttt ata gta gga ttt 624 Phe Phe Gln Ser Met Ala Thr Phe Phe Thr Gly Phe Ile Val Gly Phe 195 200 205 aca cgt ggt tgg aag cta acc ctt gtg att ttg gcc atc agt cct gtt 672 Thr Arg Gly Trp Lys Leu Thr Leu Val Ile Leu Ala Ile Ser Pro Val 210 215 220 ctt gga ctg tca gct gct gtc tgg gca aag ata cta tct tca ttt act 720 Leu Gly Leu Ser Ala Ala Val Trp Ala Lys Ile Leu Ser Ser Phe Thr 225 230 235 240 gat aaa gaa ctc tta gcg tat gca aaa gct gga gca gta gct gaa gag 768 Asp Lys Glu Leu Leu Ala Tyr Ala Lys Ala Gly Ala Val Ala Glu Glu 245 250 255 gtc ttg gca gca attaga act gtg att gca ttt gga gga caa aag aaa 816 Val Leu Ala Ala Ile Arg Thr Val Ile Ala Phe Gly Gly Gln Lys Lys 260 265 270 gaa ctt gaa agg tac aac aaa aat tta gaa gaa gct aaa aga att ggg Glu Glu Leu Tyr Asn Lys Asn Leu Glu Glu Ala Lys Arg Ile Gly 275 280 285 ata aag aaa gct att aca gcc aat att tct ata ggt gct gct ttc ctg 912 Ile Lys Lys Ala Ile Thr Ala Asn Ile Ser Ile Gly Ala Ala Phe Leu 290 300 ctg atc tat gca tct tat gct ctg gcc ttc tgg tat ggg acc acc ttg 960 Leu Ile Tyr Ala Ser Tyr Ala Leu Ala Phe Trp Tyr Gly Thr Thr Leu 305 310 315 320 gtc ctc tca ggg gaa tat tct att gga caa gta act gta ttc ttt 1008 Val Leu Ser Gly Glu Tyr Ser Ile Gly Gln Val Leu Thr Val Phe Phe 325 330 335 tct gta tta att ggg gct ttt agt gtt gga cag gca tct cca agc att 1056 Ser Val Leu Ile Gly Ala Phe Ser Val Gly Gln Ala Ser Pro Ser Ile 340 345 350 350 gaa gca ttt gca aat gca aga gga gca gct tat gaa atc ttc aag ata 1104 Glu Ala Phe Ala Asn Ala Arg Gly Ala Ala Tyr Glu Ile Phe Lys Ile 355 360 365 att gat aat aag cca agt att gac agc tat tcg aag agt ggg cac aaa 1152 Ile Asp Asn Lys Pro Ser Ile Asp Ser Tyr Ser Lys Ser Gly His Lys 370 375 380 cca gat aat att aag gga aat ttg gaa ttc aga aat gtt cac ttc agt 1200 Asp Asn Ile Lys Gly Asn Leu Glu Phe Arg Asn Val His Phe Ser 385 390 395 400 400 tac cca tct cga aaa gaa gtt aag atc ttg aag ggc ctg aac ctg aag 1248 Tyr Pro Ser Arg Lys Glu Val Lys Ile Leu Lys Gly Leu Asn Leu Lys 405 410 415 gtg cag agt ggg cag acg gtg gcc ctg gtt gga aac agt ggc tgt ggg 1296 Val Gln Ser Gly Gln Thr Val Ala Leu Val Gly Asn Ser Gly Cys Gly 420 425 430 aag agc aca aca gtc cag ctg atg cag agg ctc tat gac ccc aca gag 1344 Lys Ser Thr Thr Val Val Gln Leu Met Gln Arg Leu Tyr Asp Pro Thr Glu 435 440 445 ggg atg gtc agt gtt gat gga cag gat att agg acc ata aat gta agg 1392 Gly Met Val Ser Val Asp Gly Gln Asp Ile Arg Thr Ile Asn Val Arg 450 455 460 ttt cta cgg gaa atc att ggt gtg gtg agt cag gaa cct gta ttg ttt 1440 Phe Leu Arg Glu Ile Ile Gly Val Val Ser Gln Glu Pro Val Leu Phe 465 470 475 480 gcc acc acg ata gct gaa aac att cgc tat ggc cgt gaa aat gtc acc 1488 Ala Thr Thr Ile Ala Glu Asn Ile Arg Tyr Gly Arg Glu Asn Val Thr 485 490 495 atg gat gag att gag aaa gct gtc aag gaa gcc tat gac ttt 1536 Met Asp Glu Ile Glu Lys Ala Val Lys Glu Ala Asn Ala Tyr Asp Phe 500 505 510 atc atg aaa ctg cct cat aaa ttt gac acc ctg gtt gga gag aga ggg 1584 Ile Met Lys Leu Pro His Lys Phe Asp Thr Leu Val Gly Glu Arg Gly 515 520 525 gcc cag ttg agt ggt ggg cag aag cag agg atc gcc att gca cgt gcc 1632 Ala Gln Leu Ser Gly Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala 530 535 535 540 ctg gtt cgc aac ccc aag atc ctc ctg ctg gat gag gcc acg tca gcc 1680 Leu Val Arg Asn Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala 545 550 555 560 ttg gac aca gaa agc gaa gca gtg gtt gag gtt gag gtt gag gtt gag gtt gag ag gcc 1728 Leu Asp Thr Glu Ser Glu Ala Val Val Gln Val Ala Leu Asp Lys Ala 565 570 575 aga aaa ggt cgg acc acc att gtg ata gct cat cgt ttg tct aca gtt 1776 Arg Lys Gly Arg Thr Thr Ile Val Ile Ala His Arg Leu Ser Thr Val 580 585 590 cgt aat gct gac gtc atc gct ggt ttc gat gat gga gtc att gtg gag 1824 Arg Asn Ala Asp Val Ile Ala Gly Phe Asp Asp Gly Val Ile Val Glu 595 600 605 aaa gga aat cat gat gaact atg aaa gag aaa ggc att tac ttc aaa 1872 Lys Gly Asn His Asp Glu Leu Met Lys Glu Lys Gly Ile Tyr Phe Lys 610 615 620 CTT GTC ACA ATG CAG ACA GCA GGA AAT GAA GTT GAA TTA GAA AAT GCA 1920 Leu Val Thr M Gln Thr Ala Gly Asn Glu Val Glu Leu Glu Asn Ala 625 630 635 640 gct gat gaa tcc aaa agt gaa att gat gcc ttg gaa atg tct tca aat 1968 Ala Asp Glu Ser Lys Ser Glu Ile Asp Ala Leu Glu Met Ser Ser A sn 645 650 655 gat tca aga tcc agt cta ata aga aaa aga tca act cgt agg agt gtc 2016 Asp Ser Arg Ser Ser Leu Ile Arg Lys Arg Ser Thr Arg Arg Ser Val 660 665 670 cgt gga tca caa gcc caa gac aga aag ctt agt acc aaa gag gct ctg 2064 Arg Gly Ser Gln Ala Gln Asp Arg Lys Leu Ser Thr Lys Glu Ala Leu 675 680 685 gat gaa agt ata cct cca gtt tcc ttt tgg agg att atg aag cta aat 2112 Asp Glu Ser Ile Pro Pro Val Ser Phe Trp Arg Ile Met Lys Leu Asn 690 695 700 tta act gaa tgg cct tat ttt gtt gtt ggt gta ttt tgt gcc att ata 2160 Leu Thr Glu Trp Pro Tyr Phe Val Val Gly Val Phe Cys Ala Ile Ile 705 710 715 715 720 aat gga ggc ctg caa cca gca ttt gca ata ata ttt tca aag att ata 2208 Asn Gly Gly Leu Gln Pro Ala Phe Ala Ile Ile Phe Ser Lys Ile Ile 725 730 735 735 ggg gtt ttt aca aga att gat gat cct gaa aca cga agt 2256 Gly Val Phe Thr Arg Ile Asp Asp Pro Glu Thr Lys Arg Gln Asn Ser 740 745 750 aac ttg ttt tca cta ttg ttt cta gcc ctt gga att att tct ttt att 2304 Asn Leu Phe Ser Leu Leu Phe Leu Ala Leu Gl y Ile Ile Ser Phe Ile 755 760 765 aca ttt ttc ctt cag ggt ttc aca ttt ggc aaa gct gga gag atc ctc 2352 Thr Phe Phe Leu Gln Gly Phe Thr Phe Gly Lys Ala Gly Glu Ile Leu 770 775 780 780 acc aag cgg ct tac atg gtt ttc cga tcc atg ctc aga cag gat 2400 Thr Lys Arg Leu Arg Tyr Met Val Phe Arg Ser Met Leu Arg Gln Asp 785 790 795 800 gtg agt tgg ttt gat gac cct aaa aac acc act gga gca ttg act acc 2448 Val Ser Trp Phe Asp Asp Pro Lys Asn Thr Thr Gly Ala Leu Thr Thr 805 810 815 agg ctc gcc aat gat gct gct caa gtt aaa ggg gct ata ggt tcc agg 2496 Arg Leu Ala Asn Asp Ala Ala Gln Val Lys Gly Ala Ile Gly Ser Arg 820 825 830 ctt gct gta att acc cag aat ata gca aat ctt ggg aca gga ata att 2544 Leu Ala Val Ile Thr Gln Asn Ile Ala Asn Leu Gly Thr Gly Ile Ile 835 840 845 ata tcc ttc atc tat ggt tgg caa ctacaca ctg tta ctc tta gca att 2592 Ile Ser Phe Ile Tyr Gly Trp Gln Leu Thr Leu Leu Leu Leu Ala Ile 850 855 860 gta ccc atc att gca ata gca gga gtt gtt gaa atg aaa atg ttg tct 2640 Val Pro Ile Ile Ala Ala Gly Val Val Glu Met Lys Met Leu Ser 865 870 875 880 gga caa gca ctg aaa gat aag aaa gaa cta gaa ggt gct ggg aag atc 2688 Gly Gln Ala Leu Lys Asp Lys Lys Glu Leu Glu Gly Ala Gly Lys 895 890 gct act gaa gca ata gaa aac ttc cga acc gtt gtt tct ttg act cag 2736 Ala Thr Glu Ala Ile Glu Asn Phe Arg Thr Val Val Ser Leu Thr Gln 900 905 910 gag cag aag ttt gaa cat atg tat gct cag agt ttg cag gta cca tac 2784 Glu Gln Lys Phe Glu His Met Tyr Ala Gln Ser Leu Gln Val Pro Tyr 915 920 925 aga aac tct ttg agg aaa gca cac atc ttt gga att aca ttt tcc ttc 2832 Arg Asn Ser Leu Arg Lys Ala His Ile Phe Gly Ile Thr Phe Ser Phe 930 935 940 acc cag gca atg atg tat ttt tcc tat gct gga tgt ttc cgg ttt gga 2880 Thr Gln Ala Met Met Tyr Phe Ser Tyr Ala Gly Cys Phe Arg Phe Gly 945 950 955 960 gcc tac ttg gtg gca cat aaa ctc atg agc ttt gag gat gtt ctg tta 2928 Ala Tyr Leu Val Ala His Lys Leu Met Ser Phe Glu Asp Val Leu Leu 965 970 975 gta ttt tca gct gtt gtc ttt ggt gcc atg gcc gtg ggg caa gtc agt29 Val Phe Ser Ala Val Val Phe Gly Ala Met Ala Val Gly Gln Val Ser 980 985 990 tca ttt gct cct gac tat gcc aaa gcc aaa ata tca gca gcc cac atc 3024 Ser Phe Ala Pro Asp Tyr Ala Lys Ala Lys Ile Ser Ala Ala His Ile 995 1000 1005 atc atg atc att gaa aaa acc cct ttg att gac agc tac agc acg gaa 3072 Ile Met Ile Ile Glu Lys Thr Pro Leu Ile Asp Ser Tyr Ser Thr Glu 1010 1015 1020 ggc cta atg ccg aac aca ga tga aat gtc aca ttt ggt gaa gtt 3120 Gly Leu Met Pro Asn Thr Leu Glu Gly Asn Val Thr Phe Gly Glu Val 1025 1030 1035 1040 gta ttc aac tat ccc acc cga ccg gac atc cca gtg ctt cag gga ctg 3168 Val Phe Asn Tyr Thr Arg Pro Asp Ile Pro Val Leu Gln Gly Leu 1045 1050 1055 agc ctg gag gtg aag aag ggc cag acg ctg gct ctg gtg ggc agc agt 3216 Ser Leu Glu Val Lys Lys Gly Gln Thr Leu Ala Leu Val Gly Ser Ser1060 1065 ggc tgt ggg aag agc aca gtg gtc cag ctc ctg gag cgg ttc tac gac 3264 Gly Cys Gly Lys Ser Thr Val Val Gln Leu Leu Glu Arg Phe Tyr Asp 1075 1080 1085 ccc ttg gca ggg aaa gtg ctg ctt gat ggc aaa gaa ata aag cga ctg 3312 Pro Leu Ala Gly Lys Val Leu Leu Asp Gly Lys Glu Ile Lys Arg Leu 1090 1095 1100 aat gtt cag tgg ctc cga gca cac ctg ggc atc gtg tcc cag gag ccc 3rp Val Asn Arg Ala His Leu Gly Ile Val Ser Gln Glu Pro 1105 1110 1115 1120 atc ctg ttt gac tgc agc att gct gag aac att gcc tat gga gac aac 3408 Ile Leu Phe Asp Cys Ser Ile Ala Glu Asn Ile Ala Tyr Gly Asp Asn 1130 1135 agc cgg gtg gtg tca cag gaa gag atc gtg agg gca gca aag gag gcc 3456 Ser Arg Val Val Ser Gln Glu Glu Ile Val Arg Ala Ala Lys Glu Ala 1140 1145 1150 aac ata cat gcc ttc atc gag tca ctg cct aat agc act aaa 3504 Asn Ile His Ala Phe Ile Glu Ser Leu Pro Asn Lys Tyr Ser Thr Lys 1155 1160 1165 gta gga gac aaa gga act cag ctc tct ggt ggc cag aaa caa cgc att 3552 Val Gly Asp Lys Gly Thr Gln Leu Ser Gly Gly Gln Lys Gln Arg Ile 1170 1175 1180 gcc ata gct cgt gcc ctt gtt aga cag cct cat att ttg ctt ttg gat 3600 Ala Ile Ala Arg Ala Leu Val Arg Gln Pro His Ile Leu Leu Leu Asp 1185 1190 1195 1200 gaa gcc acg tca gct ctg gat aca gaa agt gaa aag gtt gtc caa gaa 3648 Glu Ala Thr Ser Ala Leu Asp Thr Glu Ser Glu Lys Val Val Gln Glu 1205 1210 1215 gcc ctg gac aaa gcc aga gac gcgc att gtg att gct cac 3696 Ala Leu Asp Lys Ala Arg Glu Gly Arg Thr Cys Ile Val Ile Ala His 1220 1225 1230 cgc ctg tcc acc atc cag aat gca gac tta ata gtg gtg ttt cag aat 3744 Arg Leu Ser Thr Ile Gln Asla Asp Leu Ile Val Val Phe Gln Asn 1235 1240 1245 ggc aga gtc aag gag cat ggc acg cat cag cag ctg ctg gca cag aaa 3792 Gly Arg Val Lys Glu His Gly Thr His Gln Gln Leu Leu Ala Gln Lys 1250 1255 1260 ggc atc tat ttt tca atg gtc agt gtc cag gct gga aca aag cgc cag 3840 Gly Ile Tyr Phe Ser Met Val Ser Val Gln Ala Gly Thr Lys Arg Gln 1265 1270 1275 1280 TGA 3843
【００２７】 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as foward primer for amplifying region containi ng 41nt in MDR1 gene <400> 2 taaatgcgaa tcccgagaaa a 21<210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as foward primer for amplifying region containinng 41nt in MDR1 gene <400> 2 taaatgcgaa tcccgagaaa a 21
【００２８】 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 41nt in MDR1 gene <400> 3 tcccggcccg gattgactga a 21<210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 41nt in MDR1 gene <400> 3 tcccggcccg gattgactga a 21
【００２９】 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 145nt in MDR1 gene <400> 4 tgattggctg ggcaggaaca g 21<210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 145nt in MDR1 gene <400> 4 tgattggctg ggcaggaaca g 21
【００３０】 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 145nt in MDR1 gene <400> 5 aatcttggaa gaagatactc c 21<210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 145nt in MDR1 gene <400> 5 aatcttggaa gaagatactc c 21
【００３１】 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 1236nt in MDR1 gene <400> 6 tttttctcac ggtcctggta g 21<210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 1236nt in MDR1 gene <400> 6 tttttctcac ggtcctggta g 21
【００３２】 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 1236nt in MDR1 gene <400> 7 catcccctct gtggggtcat a 21<210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 1236nt in MDR1 gene <400> 7 catcccctct gtggggtcat a 21
【００３３】 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 2677nt in MDR1 gene <400> 8 tacccatcat tgcaatagca g 21<210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region containing 2677nt in MDR1 gene <400> 8 tacccatcat tgcaatagca g 21
【００３４】 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 2677nt in MDR1 gene <400> 9 tttagtttga ctcaccttgc tag 23<210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region containing 2677nt in MDR1 gene <400> 9 tttagtttga ctcaccttgc tag 23
【００３５】 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 2677nt in MDR1 gene <400> 10 tttagtttga ctcacctttc tag 23<210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 2677nt in MDR1 gene <400> 10 tttagtttga ctcacctttc tag 23
【００３６】 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 2677nt in MDR1 gene <400> 11 tttagtttga ctcaccttcc c 21<210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region containing 2677nt in MDR1 gene <400> 11 tttagtttga ctcaccttcc c 21
【００３７】 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 3435nt in MDR1 gene <400> 12 ttgatggcaa agaaataaag c 21<210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region containing 3435nt in MDR1 gene <400> 12 ttgatggcaa agaaataaag c 21
【００３８】 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 3435nt in MDR1 gene <400> 13 cttacattag gcagtgactc g 21<210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 3435nt in MDR1 gene <400> 13 cttacattag gcagtgactc g 21
【００３９】 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 4036nt in MDR1 gene <400> 14 tcctcagtca agttcagagt c 21<210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as forward primer for amplifying region contain ing 4036nt in MDR1 gene <400> 14 tcctcagtca agttcagagt c 21
【００４０】 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 4036nt in MDR1 gene <400> 15 gttataaaat ttataatgca gtttaacata 30<210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Used as reverse primer for amplifying region contain ing 4036nt in MDR1 gene <400> 15 gttataaaat ttataatgca gtttaacata 30
【００４１】 <210> 16 <211> 245 <212> DNA <213> Homo sapience <400> 16 tttctctaat ttgttttgtt ttgcag gct ata ggt tcc agg ctt gct gta att 53 Ala Ile Gly Ser Arg Leu Ala Val Ile 1 5 acc cag aat ata gca aat ctt ggg aca gga ata att ata tcc ttc atc 101 Thr Gln Asn Ile Ala Asn Leu Gly Thr Gly Ile Ile Ile Ser Phe Ile 10 15 20 25 tat ggt tgg caa cta aca ctg tta ctc tta gca att gta ccc atc att 149 Tyr Gly Trp Gln Leu Thr Leu Leu Leu Leu Ala Ile Val Pro Ile Ile 30 35 40 gca ata gca gga gtt gtt gaa atg aaa atg ttg tct gga caa gca ctg 197 Ala Ile Ala Gly Val Val Glu Met Lys Met Leu Ser Gly Gln Ala Leu 45 50 55 aaa gat aag aaa gaa cta gaa ggt gct ggg aag gtgagtcaaa ctaaa 245 Lys Asp Lys Lys Glu Leu Glu Gly Ala Gly Lys 60 65 <210> 16 <211> 245 <212> DNA <213> Homo sapience <400> 16 tttctctaat ttgttttgtt ttgcag gct ata ggt tcc agg ctt gct gta att 53 Ala Ile Gly Ser Arg Leu Ala Val Ile 1 5 acc cag aat ata gca aat ctt ggg aca gga ata att ata tcc ttc atc 101 Thr Gln Asn Ile Ala Asn Leu Gly Thr Gly Ile Ile Ile Ser Phe Ile 10 15 20 25 tat ggt tgg caa cta aca ctg tta ctc tta gca att gta ccc atc att 149 Tyr Gly Trp Gln Leu Thr Leu Leu Leu Leu Ala Ile Val Pro Ile Ile 30 35 40 gca ata gca gga gtt gtt gaa atg aaa atg ttg tct gga caa gca ctg 197 Ala Ile Ala Gly Val Val Glu Met Lys Met Leu Ser Gly Gln Ala Leu 45 50 55 aaa gat aag aaa gaa cta gaa ggt gct ggg aag gtgagtcaaa ctaaa 245 Lys Asp Lys Lys Glu Leu Glu Gly Ala Gly Lys 60 65
Claims (6)
域における位置で第２６７７番目の塩基がグアニンであ
るか、アデニン又はチミンであるかを調べることを含
む、タクロリムス及びシクロスポリンから成る群より選
ばれる少なくとも１種の免疫抑制剤の副作用の予測方
法。At least one selected from the group consisting of tacrolimus and cyclosporin, which comprises examining whether the 2677th base at the position in the coding region of the cDNA sequence of the MDR1 gene is guanine, adenine or thymine. A method for predicting side effects of one type of immunosuppressant.
域における位置で第２６７７番目の塩基を含む領域を核
酸増幅法により増幅し、増幅産物の塩基配列を決定する
か又は増幅産物の制限酵素断片長多型を調べることによ
り行う請求項１記載の方法。2. A region containing the 2677th base at a position in the coding region of the cDNA sequence of the MDR1 gene is amplified by a nucleic acid amplification method, and the base sequence of the amplification product is determined or the length of the restriction fragment of the amplification product is determined. The method according to claim 1, which is performed by examining a polymorphism.
及び／又はAfa Iで消化した場合の制限酵素断片長調べ
ることを含む請求項２記載の方法。3. The amplification products of the restriction products Nhe I and Xba I
3. The method according to claim 2, which comprises examining the restriction enzyme fragment length when digested with AfaI.
求項１ないし３のいずれか１項に記載の方法。4. The method according to claim 1, wherein the immunosuppressant is tacrolimus.
域における位置で第２６７７番目の塩基よりも上流の領
域又は該塩基を含む領域とハイブリダイズするフォワー
ド側プライマーと、前記塩基よりも下流の領域又は該塩
基を含む領域とハイブリダイズするリバース側プライマ
ー（ただし、フォワード側プライマーとリバース側プラ
イマーがハイブリダイズする領域は重複しない）とから
成る、請求項２ないし４のいずれか１項に記載の方法に
用いられるプライマーセット。5. A forward primer that hybridizes with a region upstream of the 2677th base or a region containing the base at the position in the coding region of the cDNA sequence of the MDR1 gene, and a region downstream of the base or The method according to any one of claims 2 to 4, comprising a reverse primer that hybridizes with the region containing the base (however, a region where the forward primer and the reverse primer hybridize does not overlap). Primer set used.
域における位置で第２６７７番目の塩基よりも上流の領
域とハイブリダイズするフォワード側プライマーと、前
記塩基よりも下流の領域とハイブリダイズするリバース
側プライマーとから成る、請求項５記載のプライマーセ
ット。6. A forward primer that hybridizes with a region upstream of the 2677th base at a position in the coding region of the cDNA sequence of the MDR1 gene, and a reverse primer that hybridizes with a region downstream of the base. The primer set according to claim 5, comprising:
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Non-Patent Citations (3)
| Title |
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| JPN6010058268, Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，２０００，９７（７），ｐ．３４７３−８ * |
| JPN6010058269, Ｂｌｏｏｄ，１９９８，９１（５），ｐ．１７４９−５６ * |
| JPN6010058270, Ｃｌｉｎ．Ｐｈａｒｍａｃｏｌ．Ｔｈｅｒ．，２０００，６８（１），ｐ．９８−１０３ * |
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| JP2010263810A (en) * | 2009-05-13 | 2010-11-25 | Kumamoto Univ | Method for determining side effect or drug action of erlotinib |
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