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WO2007055261A1 - Method for determination of ugt1a1 gene polymorphism - Google Patents

Method for determination of ugt1a1 gene polymorphism Download PDF

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Publication number
WO2007055261A1
WO2007055261A1 PCT/JP2006/322337 JP2006322337W WO2007055261A1 WO 2007055261 A1 WO2007055261 A1 WO 2007055261A1 JP 2006322337 W JP2006322337 W JP 2006322337W WO 2007055261 A1 WO2007055261 A1 WO 2007055261A1
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Prior art keywords
ugt1a1
gene
polymorphisms
polymorphism
single nucleotide
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PCT/JP2006/322337
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French (fr)
Japanese (ja)
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Junichi Azuma
Yasushi Fujio
Tsuyoshi Fukuda
Masako Ohno
Tomoko Tanabe
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The New Industry Research Organization
Osaka University
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Priority to JP2007544171A priority Critical patent/JPWO2007055261A1/en
Publication of WO2007055261A1 publication Critical patent/WO2007055261A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for simultaneously testing a plurality of polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, and more particularly, to a clinically important polymorphism related to UGT 1A1 * 28. Together, it relates to a simple and quick method for testing single nucleotide polymorphism (SNP), which is another important polymorphism.
  • SNP single nucleotide polymorphism
  • the present invention provides a technique useful for, for example, a test for examining the efficacy or side effect of a drug metabolized by UGT1A1.
  • UDP-glucuronosyltransferase 1A1 (hereinafter referred to as “UGT 1A1”), an in vivo conjugating enzyme, is an enzyme involved in the metabolism of pyrilvin and foreign drugs.
  • UGT 1A1 an enzyme involved in the metabolism of pyrilvin and foreign drugs.
  • Non-patent Document 14 There have been several reports on the presence of genetic polymorphisms affecting the expression level and enzyme activity of this enzyme.
  • some UGT1A1 gene polymorphisms have been reported to be related to side effects of irinotecan, an anticancer drug, or to neonatal jaundice, which is relatively common in Japanese (see Non-Patent Document 5 below). 7)
  • UGT1A1 gene polymorphisms that are considered clinically important.
  • UGT1A1 * 28 existing in the promoter region upstream of the UGT1A1 gene.
  • the UGT1A1 * 28 allele has 7 repeats, compared to 6 in the wild type allele (UGT1A1 * 1 allele), which occupies the majority of the TA sequence repeatability in the promoter region TATA box. Since it is an allele with the TA sequence inserted in the 40th to 39th genome, it is also written as “-40_-39insTA”. (The translation start point of the UGT1A1 gene is “+1”.)
  • the other five gene polymorphisms are polymorphisms related to UGT1A1 * 6 UGT1A1 * 27 UGT1A1 * 29 UG T1A1 * 7, and UGT1A1 * 60. These five gene polymorphisms are all single nucleotide polymorphisms (SNPs), except for polymorphisms related to UGT1A1 * 60. Present, causing an amino acid substitution in the translation product.
  • the UGT1A1 * 6 allele has adenine (A) at position 211 on exon 1, which is a guanine (G) in the wild type, and its polymorphic site is denoted as “G211A”.
  • the 686th base on exon 1, which is cytosine (C) in the wild type, is adenine (A), and the polymorphic site is denoted as “C686A”.
  • the UGT1A1 * 29 allele is cytosine (C) in the wild type.
  • the 1099th base on exon 4 is guanine (G), and the polymorphic site is expressed as “C10 99G”.
  • the 1456th base on exon 5 which is thymine (T) in the wild type is guanine (G), and the polymorphic site is expressed as “T1456G”.
  • the UGT1A1 * 60 allele has guanine (G) as the 3279th base, which is thymine (T) in the wild type, and its polymorphic site is denoted as "T-3279G”. (In some cases, “T-3263G” is displayed.)
  • T thymine
  • T-3263G the polymorphic site of UGT1A1 * 60 allele is located further upstream than the polymorphic site of UGT1A1 * 28 in the promoter region. It is.
  • Melting curve method is a method to detect polymorphism by analyzing melting temperature (Tm value) of PCR product by melting curve analysis using the fact that melting temperature of DNA molecule depends on its nucleic acid composition. is there. This method is used in a light cycler manufactured by Roche, and the time required for polymorphism detection is short, but the equipment costs are initially high.
  • Tm value melting temperature
  • PCR products are directly sequenced by a sequencer to detect polymorphisms. This method is accurate and accurate, but it is costly, time consuming, and laborious.
  • the pie-mouth sequence method is a method for detecting a polymorphism by observing chemiluminescence, detecting the complementary strand synthesis from the luminescence, and sequencing the DNA molecule. This method is used in Biotage equipment, and it takes a short time, but requires an initial equipment cost.
  • each of the above methods is a method for detecting each polymorphism individually.
  • other clinically important single nucleotide polymorphisms can be easily and quickly produced at low cost.
  • the method of detecting with high accuracy has not yet been established!
  • Non-Patent Document 1 N Engl J Med 1995; 333: 1171-5
  • Non-Patent Document 2 Biochim Biophys Acta 1998; 1406: 267-73
  • Non-Patent Document 3 Drug Metab Dispos 2003; 31: 108-13
  • Non-Patent Document 4 Biochem Biophys Res Commun 2002; 292: 492-7
  • Non-Patent Document 5 Cancer Res 2000; 60: 6921-6
  • Non-Patent Document 6 Pharmacogenet Genomics 2005; 15: 35-41
  • Non-Patent Document 7 Pediatrics 1999; 103: 1224- 7
  • the present invention has been made paying attention to the above-mentioned problems, and its first problem is to provide a method for simultaneously testing a plurality of polymorphisms related to the UGT 1A1 gene.
  • the UGT1A1 * 28 polymorphism it is intended to provide a simple, rapid and accurate method for testing other clinically important single nucleotide polymorphisms at low cost.
  • a second problem of the present invention is that the above method is applied to other gene polymorphism testing methods, and a plurality of polymorphisms related to the target gene, that is, an insertion type in which a certain base sequence is inserted, and this To provide a method for easily and quickly inspecting a plurality of polymorphisms, which are a combination force of a polymorphism between a lacking form and a single nucleotide polymorphism, at low cost.
  • the polymorphism related to UGT1A1 * 28 is a difference in the length of 2 bases of force, and this needs to be detected visually. Therefore, the PCR product is shortened to less than lOObp and the difference in length of two bases is detected by using high-resolution capillary electrophoresis. Tried to do.
  • mismatch primers were used to amplify the gene region containing each single nucleotide polymorphism, and a restriction enzyme cleavage site that was cleaved according to the base of the polymorphic site was created.
  • the present invention includes the following inventions A) to K) as industrially and medically and medically useful inventions.
  • the method comprises a step of amplifying at least one gene region in which an amino acid is present by the same gene amplification reaction, a step of digesting the obtained amplified fragment with a restriction enzyme, and subjecting the resultant fragment to a capillary electrophoresis.
  • a gene region including a polymorphic site related to UGT1A1 * 28 is amplified by PCR using a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4. The inspection method described in A) above.
  • a method for examining multiple polymorphisms related to the target gene simultaneously includes at least one polymorphism (SNP) in addition to the gene region containing the polymorphism due to the presence or absence of the insertion site.
  • SNP polymorphism
  • the base of the single nucleotide polymorphism is amplified.
  • digestion is performed with restriction enzymes. After that, the DNA fragments in each sample are separated and detected according to the fragment length by capillary electrophoresis.
  • capillary electrophoresis has high resolution, it is possible to detect the difference in length of two bases in the UGT1A1 * 28 polymorphism, and single nucleotide polymorphism typing from the length of other DNA fragments detected at the same time. Yes, it is possible.
  • amplification reactions such as PCR, digestion reactions with restriction enzymes
  • a series of simple and rapid methods of single electrophoresis can test and determine multiple clinically important polymorphisms of the UGT1A1 gene with high accuracy.
  • the single-electrophoresis does not require a large facility and is a multiplex PCR that simultaneously amplifies a plurality of PCR products, so that the present invention can realize a low-cost test.
  • UGT1A1 By conducting a polymorphism test of the UGT1A1 gene according to the present invention, it is possible to provide a method for predicting the efficacy or side effect of a drug metabolized by UGT1A1. For example, many studies have reported that there is a correlation between the side effect of the anticancer drug irinotecan and the UGT1A1 gene polymorphism (Non-Patent Documents 5, 6, etc.). In addition, the degree of side effects when irinotecan is administered can be predicted from the test results according to the present invention.
  • the present invention has the ability to test multiple gene polymorphisms simultaneously. Predicting side effects and drug efficacy based on multiple gene polymorphisms is an appropriate drug suitable for individual patients based on preferred genomic information. It contributes to the realization of treatment and can contribute to the improvement of medical quality, the safety of drugs and the efficiency of treatment.
  • UGT1A1 gene polymorphism By testing UGT1A1 gene polymorphism, it is possible to determine whether or not it has a genetic predisposition affecting high serum pyrilbin levels, that is, without abnormal liver function test values such as AST and ALT It is possible to scientifically explain high serum pyrilrubin levels.
  • the inspection method of the present invention can also be used for such inspection.
  • a method for simply and quickly and accurately testing a plurality of polymorphisms related to a target gene at low cost by applying the testing method to other genetic polymorphism testing methods. can do.
  • FIG. 1 is a gel image showing the results of a capillary electrophoresis in the method for testing a UGT1A1 gene polymorphism according to an example of the present invention.
  • FIG. 2] (a) to (c) are graphs showing the results of the above-mentioned capillary electrophoresis.
  • NC National Center for Biotechnology Information
  • test method of the present invention a plurality of polymorphisms related to the UGT1A1 gene were simultaneously tested and determined for 155 samples from Japan.
  • three polymorphisms related to UGT1A1 * 60, UGTIAI * 28, and UGTIAI * 6 were examined, and PCR was used to amplify the gene region containing each polymorphism.
  • One of the features of this test method is that three sets of primers are used, and three PCR products are obtained simultaneously by the same gene amplification reaction under the same PCR conditions. The sequences of the three sets of primers used are listed in Table 1 below.
  • the PCR reaction was performed by putting the six primers shown in Table 1 in one tube.
  • the reaction (20 / z L) consists of 60 ng genomic DNA, 1.0 unit AmpliTaq DNA polymerase, 0.25 U UGT1A1 * 60 primer, 0.2 ⁇ UGT1A1 * 28 primer, 075 0 UGT1A1 * 6 primer and 0.2 mM deoxynucleotide triphosphate each.
  • the PCR conditions are (1) 94 ° C for 5 minutes, and (2) 94 ° C for 30 seconds, 55 ° C for 40 seconds, and 72 ° C for 40 seconds. The reaction was performed.
  • each reaction solution is subjected to electrophoresis using a simple multi-sample capillary electrophoresis device “H DA GT-12 TM (eGene)”, and the DNA fragments in each reaction solution are subjected to electrophoresis. Separated and detected according to size (fragment length).
  • the polymorphic site of UGT1A1 * 60 when the wild-type allele “W” is used, the polymorphic site is not cleaved by the restriction enzyme Hindi. Therefore, as shown in Table 1, the fragment length is 115 bp and 93 bp. (Because there is one restriction enzyme cleavage site in the amplified fragment). On the other hand, in the case of the mutant allele “M”, the polymorphic site is cleaved by the restriction enzyme Hindi, so that the fragment length becomes 115 bp, 67 bp and 26 bp.
  • the polymorphic site of UGT1A1 * 28 is not cleaved by a restriction enzyme, and the double nucleotide repeat sequence (TA repeat) is a mutant allele “M” that is one more than the wild type allele “W” As shown in Table 1, the fragment length is 85 bp, and when the wild type allele “W”, the fragment length is It becomes 83bp.
  • TA repeat double nucleotide repeat sequence
  • UGT1A1 * 28 was determined by PCR-RFLP method. UGT1A1 * 28 was determined by a melting curve method using a Roche light cycler according to a previously reported method (Hepatology 2000; 32: 792-5). If the result is not clear by this method, The genotype of UGT1A1 * 28 was determined using the Tato Sequence method.
  • the UGT1A1 * 6 allele was determined using a method slightly improved from the previously reported method (Cancer Res 2000; 60: 6 921-6). In this determination, as described above, a mismatch primer having an Mspl recognition site was designed only when the wild type allele was “W” (see Table 2). Similarly, for the UGT1A1 * 29 allele and the UGT1A1 * 60 allele, mismatch primers were designed to have Cfrl3I and Dral recognition sites only when the wild type allele was “W”. The UGT1A1 * 7 allele and the UGT1A1 * 27 allele were determined by the PCR-RFLP method using the restriction enzyme Bsrl according to the method already reported (Cancer Res 2000; 60: 6921-6).
  • UGT1A1 * Allele specific PCR method (Allele specific PCR method) was performed on 6 or UGT1A1 * 27 as follows. For the UGT1A1 * 28 allele, each PCR product was purified and subjected to direct sequencing to confirm the polymorphism of UGT1A1 * 28.
  • PCR was performed using 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2, 150 ng genomic DNA.
  • the reaction was performed in a 50 L PCR reaction solution containing 2.5 units of AmpliTaq DNA polymerase, 200 nM of each primer, and 0.2 mM of dioxynucleotide triphosphate.
  • the primers used and the PCR conditions are shown in Table 3 below.
  • Fig. 1 is a gel image showing a part of the result of capillary electrophoresis by the test method of the present invention
  • Fig. 2 (a) to (c) are graphs showing a part of the result of the capillary electrophoresis.
  • the wild type allele is indicated as “* 1”.
  • the difference between the two bases of the polymorphic site of UGT1A1 * 28 can be visually detected by dividing the amplified fragment into 85 bp and 83 bp by using single electrophoresis. It was. At the same time, the monobasic polymorphism related to UGT1A1 * 60 and UGT1A1 * 6 could be detected with high accuracy.
  • the inspection method of the present invention is not limited to the above method, and various modifications can be made within the scope of the present invention.
  • the above method is a force that simultaneously inspects three polymorphisms of UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6.
  • the present invention is limited to the inspection of these three polymorphisms. It is not a thing.
  • the method of the above-described embodiment has the power to inspect three polymorphisms at the same time.
  • the present invention may inspect two polymorphisms at the same time.
  • other clinically important single nucleotide polymorphisms are examined.
  • it is possible to simultaneously examine four or more polymorphisms it is necessary to pay careful attention to the design of the primers so that different amplified fragments do not overlap and become difficult to detect.
  • the amplified fragment in designing the primer, (1) in order to detect a difference of 110 ⁇ 1 18 1 * 28, 21 ⁇ , the amplified fragment should be less than lOObp, and (2) Make sure that DNA fragments after digestion with restriction enzymes do not overlap each other and make it difficult to detect. (3) Consider the relative distance and positional relationship of each DNA fragment so that polymorphism can be easily detected. did. Specifically, in order to detect the polymorphism of UGT1A1 * 28, primers were designed so that at least one (preferably a plurality) of other amplified fragments appeared within about 30 bp before or after the amplified fragment.
  • each primer may be slightly shorter or longer than those listed in Table 1, but care must be taken to amplify the three gene regions under the same reaction conditions.
  • the gene sample was prepared by extracting genomic DNA from the peripheral blood of each subject.
  • the gene sample to be used for the test is the subject's arbitrary.
  • DNA can be purified and extracted from organs / tissues / cells (including blood, cells in amniotic fluid, cells cultured from collected tissues, etc.) according to a conventional method. As long as gene amplification by PCR (or other gene amplification) is possible, DNA purification and extraction steps are omitted or May be simplified.
  • the reaction conditions in the PCR and the reagents used are not particularly limited to the methods of the above examples, but it is advisable to pay attention to the reaction conditions so that the entire target gene region is amplified.
  • the PCR method is a simple method and has good accuracy. Therefore, it is preferable to use the PCR method for the gene amplification method, but other amplification methods (eg, RCA method) other than the PCR method are used. Good (see “Post-Sequence Genomic Sciences (1) Strategies of SNP gene polymorphism” (Nakayama Shoten)).
  • the type of buffer, reaction temperature, reaction time, etc. may be determined according to the type of restriction enzyme used. As in the method of the above example, it is preferable to use two kinds of restriction enzymes at the same time and detect two kinds of SNPs by the PCR-RFLP method.
  • DNA is separated and detected at higher resolution than normal gel electrophoresis by electrophoresis of DNA in the capillary, and can be performed using a commercially available apparatus.
  • Any device that can detect a 2 bp difference in the UGT1A1 * 28 polymorphism may be used.
  • any kit including primers designed for testing a plurality of polymorphisms related to the UGT1A1 gene may be used.
  • the primer included in the test kit is exemplified by the three sets of primers listed in Table 1. The length may be 1 to 5 bases longer or shorter than this.
  • the kit configuration may include one or more primers selected from the primer group listed in Table 2.
  • “a plurality of polymorphisms related to the UGT1A1 gene” are simultaneously examined.
  • the polymorphisms related to the UGT1A1 gene are, in other words, the UGT1A1 gene or its vicinity in the genome.
  • An existing polymorphism, exon of UGT1A1 genomic gene The present invention is not limited to testing for polymorphisms existing in the region or intron region, but may be those for testing polymorphisms present in transcriptional regulatory regions such as the promoter region and control regions.
  • the “gene polymorphism” includes a polymorphism existing in such a promoter region and the like, and has a broad meaning of a polymorphism related to the gene.
  • UGT1A1 By conducting a polymorphism test of the UGT1A1 gene according to the present invention, the efficacy or side effect of a drug metabolized by UGT1A1 can be predicted. For example, the presence of UGT1A1 * 28 and UGT1A1 * 60 alone and UGT1A1 * 6 together with UGT1A1 * 28 has been reported to increase the risk of irinotecan side effects. By doing so, it is possible to predict patients who are likely to cause serious side effects of irinotecan.
  • the test method of the present invention can also be applied to the test of gene polymorphisms other than the UGT1A1 gene, and can provide a method for quickly and accurately testing a plurality of polymorphisms related to the target gene at low cost. it can.
  • the present invention relates to a plurality of polymorphisms related to genes other than the UGT1A1 gene, which are polymorphisms depending on the presence or absence of an insertion portion (an insertion type in which a certain base sequence of 2 or more bases is inserted and the base) It can also be applied to the case where multiple polymorphisms consisting of a combination of single nucleotide polymorphism (SNP) and a polymorphism between deletion type lacking sequence) and single nucleotide polymorphism (SNP) are simultaneously examined. Examples of such genes having a plurality of polymorphisms include the following. (Table 4 below shows the names of molecules encoded by each gene.)
  • Multi-polymorphic polymorphic polymorphic type polymorphic location type 2 S 3 SNP type with or without SNP (repeat name abbreviation NP but one minute s: s :: ⁇ > -H>
  • Each molecule in the table consists of 1: serotonin transporter, 2: monoamine oxidase-8, 3: androgen receptor, 4: tyrosine hydroxylase, 5: serotonin receptor 5-HT2C 6: aroma Amino acid decarboxylase (AADC), 7: donomin D2 receptor DRD2, 8: serotonin receptor 5-HTR3B, 9: serotonin receptor 5-HTR6, 10: GABA type A receptor oc 5 subunit (GABRA5) 11: Dopamine transporter (SLC6A3 (DAT)), 12: Dopamine D4 receptor DRD4, 13: Dopamine D5 receptor DRD5, 14: Phosphatidylinosito 4-phosphate-5-kinase type 2 a, 15 denylate cyclase 9 ( ADCY9), 16: GABA receptor j83 subunit (GABRB3), 17: Adrenergic receptor alpha 2B (ADRA2B) ⁇ 18: Adrenergic receptor al pha
  • the single nucleotide polymorphisms (second and third polymorphisms) shown in Table 4 can also be examined by PCR-RFLP using restriction enzymes, as in the above-described methods. If necessary, use a mismatch primer to create a restriction enzyme cleavage site.
  • the test method of the present invention detects a difference of 2 bp in the polymorphic site with high accuracy, and therefore, especially when the difference is 10 bp or less (more preferably 5 bp or less) for the polymorphism due to the presence or absence of the insertion site. It can be described as a highly superior inspection method.
  • both polymorphisms may not be polymorphisms related to the same gene.
  • the present invention may be applied to examination of a plurality of polymorphisms related to different genes.
  • the present invention relates to a method for testing a UGT1A1 gene polymorphism and the like, As mentioned above, it can be used for multiple quick and simple polymorphism tests, and it can be used for tests, diagnoses, etc. to check the efficacy or side effects of drugs metabolized by UGT1A1.

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Abstract

Disclosed is a method for simultaneous determination of two or more polymorphisms associated with UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The method comprises the steps of: amplifying both of a gene region having a polymorphic site associated with UGT1A1*28 and at least one gene region having a single nucleotide polymorphism (SNP) in a gene amplification reaction; and digesting the resulting amplification fragments with a restriction enzyme and then subjecting the digestion products to capillary electrophoresis, wherein two or more polymorphisms are determined simultaneously based on the results of the capillary electrophoresis. Similarly, the polymorphism of a gene other than UGT1A1 gene can also be determined rapidly, in a simple manner and with good accuracy.

Description

明 細 書  Specification
UGT1A1遺伝子多型の検査法  UGT1A1 gene polymorphism testing method
技術分野  Technical field
[0001] 本発明は、 UDP-グルクロノシルトランスフェラーゼ 1A1 (UGT1A1)遺伝子に関連 する複数の多型を同時に検査する方法に関し、より詳細には、臨床的に重要な UGT 1A1 * 28に関する多型と一緒に、他の重要な多型である一塩基多型（SNP)を簡易 迅速かつ精度良く検査する方法に関する。本発明は、例えば、 UGT1A1で代謝さ れる薬剤の薬効あるいは副作用を調べる検査などに有用な技術を提供するものであ る。  [0001] The present invention relates to a method for simultaneously testing a plurality of polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, and more particularly, to a clinically important polymorphism related to UGT 1A1 * 28. Together, it relates to a simple and quick method for testing single nucleotide polymorphism (SNP), which is another important polymorphism. The present invention provides a technique useful for, for example, a test for examining the efficacy or side effect of a drug metabolized by UGT1A1.
背景技術  Background art
[0002] 生体内抱合酵素である UDP-グルクロノシルトランスフェラーゼ 1A1 (以下、「UGT 1A1」という。）は、ピリルビンおよび外来薬物の代謝に関与する酵素である。この酵 素には、その発現量や酵素活性に影響を及ぼす遺伝子多型の存在が幾つか報告さ れている（下記非特許文献 1 4)。さらに、幾つかの UGT1A1遺伝子多型は、抗が ん剤であるイリノテカンの副作用に関係し、あるいは日本人に比較的多い新生児黄 疽に関係することが報告されて 、る（下記非特許文献 5 7)  [0002] UDP-glucuronosyltransferase 1A1 (hereinafter referred to as “UGT 1A1”), an in vivo conjugating enzyme, is an enzyme involved in the metabolism of pyrilvin and foreign drugs. There have been several reports on the presence of genetic polymorphisms affecting the expression level and enzyme activity of this enzyme (Non-patent Document 14 below). Furthermore, some UGT1A1 gene polymorphisms have been reported to be related to side effects of irinotecan, an anticancer drug, or to neonatal jaundice, which is relatively common in Japanese (see Non-Patent Document 5 below). 7)
[0003] これまで UGT1A1遺伝子には 30種類以上の遺伝子多型が報告されている力 こ こでは臨床的に重要と考えられている 6つの遺伝子多型について説明する。 1つは、 UGT1A1遺伝子の上流、プロモーター領域に存在する UGT1A1 * 28に関する多 型である。 UGT1A1 * 28アレルは、プロモーター領域 TATAボックスにおける TA 配列の繰り返し力 多数を占める野生型アレル (UGT1A1 * 1アレル）では 6回であ るのに対し、 7回の繰り返し配列になっている。ゲノム上、—40 39番目に TA配 列が挿入されたアレルであるので、 「-40_-39insTA」とも表記される。（UGT1A1遺伝 子の翻訳開始点を「 + 1」とする。 )  [0003] To date, more than 30 gene polymorphisms have been reported in the UGT1A1 gene. Here we describe six gene polymorphisms that are considered clinically important. One is a polymorphism related to UGT1A1 * 28 existing in the promoter region upstream of the UGT1A1 gene. The UGT1A1 * 28 allele has 7 repeats, compared to 6 in the wild type allele (UGT1A1 * 1 allele), which occupies the majority of the TA sequence repeatability in the promoter region TATA box. Since it is an allele with the TA sequence inserted in the 40th to 39th genome, it is also written as “-40_-39insTA”. (The translation start point of the UGT1A1 gene is “+1”.)
[0004] 他の 5つの遺伝子多型は、 UGT1A1 * 6 UGT1A1 * 27 UGT1A1 * 29 UG T1A1 * 7、及び UGT1A1 * 60に関する多型である。これら 5つの遺伝子多型はす ベて一塩基多型（SNP)であり、 UGT1A1 * 60に関する多型を除 、てェキソンに存 在し、翻訳産物にアミノ酸置換を生じさせる。 UGT1A1 * 6アレルは、野生型ではグ 了ニン (G)であるェキソン 1上の 211番目の塩基がアデニン (A)になっており、その 多型部位は「G211A」と表記される。 UGT1A1 * 27アレルは、野生型ではシトシン（ C)であるェキソン 1上の 686番目の塩基がアデニン (A)になっており、その多型部位 は「C686A」と表記される。 UGT1A1 * 29アレルは、野生型ではシトシン（C)である ェキソン 4上の 1099番目の塩基がグァニン（G)になっており、その多型部位は「C10 99G」と表記される。 UGT1A1 * 7アレルは、野生型ではチミン (T)であるェキソン 5 上の 1456番目の塩基がグァニン (G)になっており、その多型部位は「T1456G」と表 記される。 [0004] The other five gene polymorphisms are polymorphisms related to UGT1A1 * 6 UGT1A1 * 27 UGT1A1 * 29 UG T1A1 * 7, and UGT1A1 * 60. These five gene polymorphisms are all single nucleotide polymorphisms (SNPs), except for polymorphisms related to UGT1A1 * 60. Present, causing an amino acid substitution in the translation product. The UGT1A1 * 6 allele has adenine (A) at position 211 on exon 1, which is a guanine (G) in the wild type, and its polymorphic site is denoted as “G211A”. In the UGT1A1 * 27 allele, the 686th base on exon 1, which is cytosine (C) in the wild type, is adenine (A), and the polymorphic site is denoted as “C686A”. The UGT1A1 * 29 allele is cytosine (C) in the wild type. The 1099th base on exon 4 is guanine (G), and the polymorphic site is expressed as “C10 99G”. In the UGT1A1 * 7 allele, the 1456th base on exon 5 which is thymine (T) in the wild type is guanine (G), and the polymorphic site is expressed as “T1456G”.
[0005] UGT1A1 * 60アレルは、野生型ではチミン (T)である一 3279番目の塩基がグァ ニン（G)になっており、その多型部位は「T-3279G」と表記される。 （「T-3263G」と表 記されることもある。）このように、 UGT1A1 * 60アレルの多型部位は、プロモーター 領域に存在する UGT1A1 * 28の多型部位よりも更に上流に位置するものとなって いる。  [0005] The UGT1A1 * 60 allele has guanine (G) as the 3279th base, which is thymine (T) in the wild type, and its polymorphic site is denoted as "T-3279G". (In some cases, “T-3263G” is displayed.) Thus, the polymorphic site of UGT1A1 * 60 allele is located further upstream than the polymorphic site of UGT1A1 * 28 in the promoter region. It is.
[0006] 上記 6種の遺伝子多型はいずれも臨床的に重要と考えられている力 日本人に存 在が確認され、臨床的に特に重要なものして、 UGT1A1 * 28、 UGT1A1 * 6、及 び UGT1A1 * 60の 3種の多型を挙げることができる。なかでも、 UGT1A1 * 28をも つ患者力 リノテカンの投与を受けた場合、副作用の危険性が高くなるといわれてい る力 上記のように、 UGT1A1 * 28は TATAボックスにおいて TA配列の繰り返しが 1回多い多型であり、野生型との長さの差異がわず力 2塩基であるため、通常の PCR -RFLP法などでは多型の検出が困難であった。  [0006] The above six polymorphisms are all considered to be clinically important. The presence of Japanese polymorphisms has been confirmed and is of particular clinical importance. UGT1A1 * 28, UGT1A1 * 6, And three polymorphisms UGT1A1 * 60. Among them, the strength of patients with UGT1A1 * 28 It is said that the risk of side effects is increased when rinotecan is administered. As described above, UGT1A1 * 28 has one TA sequence repeat in the TATA box. Because it is a polymorphism and the difference in length from the wild type is only 2 bases, it was difficult to detect the polymorphism by ordinary PCR-RFLP method.
[0007] UGT1A1 * 28の多型を検出する方法として、これまでにメルティングカーブ法、ダ ィレクトシークェンス法、ノイロシークェンス法による検出例が報告されている。メルテ イングカーブ法は、 DNA分子の融解温度がその核酸組成に依存することを利用し、 メルティングカーブ分析によって PCR産物の融解温度 (Tm値)を解析することで、多 型を検出する方法である。同方法は、ロシュ社製のライトサイクラ一機器において採 用されており、多型検出に要する時間は短いが、初期に設備コストがかかる。ダイレク トシークェンス法は、 PCR産物をシークェンサ一により直接配列決定し、多型を検出 する方法であり、判定は正確だが、コストと時間と手間がかかる。パイ口シークェンス 法は、化学発光を観測し、その発光カゝら相補鎖合成を検知し、 DNA分子を配列決 定することで、多型を検出する方法である。同方法は、 Biotage社製の機器において 採用されており、所要時間は短いものの、初期に設備コストがかかる。 [0007] As a method for detecting a polymorphism of UGT1A1 * 28, detection examples by the melting curve method, direct sequence method, and neurosequence method have been reported so far. Melting curve method is a method to detect polymorphism by analyzing melting temperature (Tm value) of PCR product by melting curve analysis using the fact that melting temperature of DNA molecule depends on its nucleic acid composition. is there. This method is used in a light cycler manufactured by Roche, and the time required for polymorphism detection is short, but the equipment costs are initially high. In the direct sequence method, PCR products are directly sequenced by a sequencer to detect polymorphisms. This method is accurate and accurate, but it is costly, time consuming, and laborious. The pie-mouth sequence method is a method for detecting a polymorphism by observing chemiluminescence, detecting the complementary strand synthesis from the luminescence, and sequencing the DNA molecule. This method is used in Biotage equipment, and it takes a short time, but requires an initial equipment cost.
[0008] また、上記いずれの方法も各多型を個別に検出する方法であり、 UGT1A1 * 28 の多型と一緒に、臨床的に重要な他の一塩基多型を、低コストで簡易迅速かつ精度 良く検出する方法は未だ確立されて!、な 、のが現状である。 [0008] In addition, each of the above methods is a method for detecting each polymorphism individually. In addition to the UGT1A1 * 28 polymorphism, other clinically important single nucleotide polymorphisms can be easily and quickly produced at low cost. In addition, the method of detecting with high accuracy has not yet been established!
非特許文献 1 : N Engl J Med 1995;333: 1171-5  Non-Patent Document 1: N Engl J Med 1995; 333: 1171-5
非特許文献 2 : Biochim Biophys Acta 1998;1406:267-73  Non-Patent Document 2: Biochim Biophys Acta 1998; 1406: 267-73
非特許文献 3 : Drug Metab Dispos 2003;31:108-13  Non-Patent Document 3: Drug Metab Dispos 2003; 31: 108-13
非特許文献 4 : Biochem Biophys Res Commun 2002;292:492-7  Non-Patent Document 4: Biochem Biophys Res Commun 2002; 292: 492-7
非特許文献 5 : Cancer Res 2000;60:6921-6  Non-Patent Document 5: Cancer Res 2000; 60: 6921-6
非特許文献 6 : Pharmacogenet Genomics 2005;15:35-41  Non-Patent Document 6: Pharmacogenet Genomics 2005; 15: 35-41
非特許文献 7： Pediatrics 1999 ;103: 1224- 7  Non-Patent Document 7: Pediatrics 1999; 103: 1224- 7
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] 本発明は、上記の問題点に着目してなされたものであり、その第 1の課題は、 UGT 1A1遺伝子に関連する複数の多型を同時に検査する方法を提供すること、より詳細 には、 UGT1A1 * 28の多型と一緒に、臨床的に重要な他の一塩基多型を、低コス トで簡易迅速かつ精度良く検査する方法を提供することである。  [0009] The present invention has been made paying attention to the above-mentioned problems, and its first problem is to provide a method for simultaneously testing a plurality of polymorphisms related to the UGT 1A1 gene. In addition to the UGT1A1 * 28 polymorphism, it is intended to provide a simple, rapid and accurate method for testing other clinically important single nucleotide polymorphisms at low cost.
[0010] 本発明の第 2の課題は、上記方法を他の遺伝子多型検査法に応用し、目的遺伝 子に関連する複数の多型、即ち、ある塩基配列が挿入された挿入型とこれを欠く欠 失型との間の多型と一塩基多型との組み合わせ力 なる複数の多型を、低コストで簡 易迅速かつ精度良く検査する方法を提供することである。  [0010] A second problem of the present invention is that the above method is applied to other gene polymorphism testing methods, and a plurality of polymorphisms related to the target gene, that is, an insertion type in which a certain base sequence is inserted, and this To provide a method for easily and quickly inspecting a plurality of polymorphisms, which are a combination force of a polymorphism between a lacking form and a single nucleotide polymorphism, at low cost.
課題を解決するための手段  Means for solving the problem
[0011] 上記のように、 UGT1A1 * 28に関する多型はわず力 2塩基の長さの差異であり、 これを視覚的に検出する必要がある。そのため、 PCR産物を lOObp以下に短くし、 分解能の高いキヤピラリー電気泳動を用いることによって 2塩基の長さの差異を検出 することを試みた。さらに、同一の遺伝子増幅反応および同一の電気泳動によって、 臨床的に重要な他の 2箇所の一塩基多型（UGT1A1 * 6と UGT1A1 * 60の多型） を同時検査することを試みた。そのため、各一塩基多型を含む遺伝子領域の増幅に 際してミスマッチプライマーを使用し、多型部位の塩基に応じて切断される制限酵素 切断部位を作り出した。また、 3組のプライマーによって 3箇所の遺伝子領域を同時 に増幅するための条件を検討した。その結果、 3箇所の遺伝子領域を同一の PCR反 応で増幅し、制限酵素による消化反応後、キヤピラリー電気泳動することによって、簡 易迅速かつ高精度に複数の多型を同時検査できること等を見出し、本発明を完成さ せるに至った。 [0011] As described above, the polymorphism related to UGT1A1 * 28 is a difference in the length of 2 bases of force, and this needs to be detected visually. Therefore, the PCR product is shortened to less than lOObp and the difference in length of two bases is detected by using high-resolution capillary electrophoresis. Tried to do. In addition, we attempted to simultaneously examine two other single nucleotide polymorphisms (UGT1A1 * 6 and UGT1A1 * 60 polymorphisms) of clinical importance by the same gene amplification reaction and the same electrophoresis. Therefore, mismatch primers were used to amplify the gene region containing each single nucleotide polymorphism, and a restriction enzyme cleavage site that was cleaved according to the base of the polymorphic site was created. We also examined the conditions for amplifying three gene regions simultaneously using three sets of primers. As a result, we found that multiple polymorphisms can be simultaneously and easily tested with high precision by amplifying three gene regions with the same PCR reaction, digesting with restriction enzymes, and then performing capillary electrophoresis. The present invention has been completed.
即ち、本発明は、産業上並びに医学 ·医療上有用な発明として、下記 A)〜K)の発 明を包含するものである。  That is, the present invention includes the following inventions A) to K) as industrially and medically and medically useful inventions.
A) UDP-グルクロノシルトランスフェラーゼ 1A1 (UGT1A1)遺伝子に関連する 複数の多型を同時に検査する方法であって、 UGT1A1 * 28に関する多型部位を 含む遺伝子領域のほか、一塩基多型（SNP)が存在する少なくとも 1つの遺伝子領 域を同一の遺伝子増幅反応によって増幅する工程と、得られた増幅断片を制限酵 素によって消化した後、キヤピラリー電気泳動に供する工程とを含み、キヤピラリー電 気泳動の結果に基づいて複数の多型を同時に判定する方法。  A) A method for examining multiple polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene simultaneously, including a gene region containing a polymorphic site for UGT1A1 * 28, as well as a single nucleotide polymorphism (SNP) The method comprises a step of amplifying at least one gene region in which an amino acid is present by the same gene amplification reaction, a step of digesting the obtained amplified fragment with a restriction enzyme, and subjecting the resultant fragment to a capillary electrophoresis. A method of simultaneously determining a plurality of polymorphisms based on a result.
B) 一塩基多型が存在する遺伝子領域として、 UGT1A1 * 60及び Z又は UGT1 A1 * 6に関する一塩基多型が存在する遺伝子領域を増幅する、上記 A)記載の検 查方法。  B) The detection method according to A), wherein a gene region in which single nucleotide polymorphisms relating to UGT1A1 * 60 and Z or UGT1 A1 * 6 are present is amplified as a gene region in which single nucleotide polymorphisms exist.
C) ミスマッチプライマーを使用して一塩基多型が存在する遺伝子領域を増幅す る、上記 B)記載の検査方法。  C) The testing method according to B) above, wherein a mismatched primer is used to amplify a gene region in which a single nucleotide polymorphism exists.
D) UGT1A1 * 60に関する一塩基多型が存在する遺伝子領域について、配列 番号 1の塩基配列を有するフォワードプライマー及び配列番号 2の塩基配列を有す るリバースプライマーを用いた PCR法による増幅を行うと共に、制限酵素に Hindiを 用いることを特徴とする、上記 C)記載の検査方法。  D) Amplification by PCR using a forward primer having the nucleotide sequence of SEQ ID NO: 1 and a reverse primer having the nucleotide sequence of SEQ ID NO: 2 for the gene region where a single nucleotide polymorphism related to UGT1A1 * 60 exists The test method according to C) above, wherein Hindi is used as a restriction enzyme.
E) UGT1A1 * 6に関する一塩基多型が存在する遺伝子領域について、配列番 号 5の塩基配列を有するフォワードプライマー及び配列番号 6の塩基配列を有するリ バースプライマーを用いた PCR法による増幅を行うと共に、制限酵素に Msplを用い ることを特徴とする、上記 C)記載の検査方法。 E) For a gene region in which a single nucleotide polymorphism relating to UGT1A1 * 6 exists, a forward primer having the nucleotide sequence of SEQ ID NO: 5 and a primer having the nucleotide sequence of SEQ ID NO: 6 The test method according to C) above, wherein amplification is performed by PCR using a verse primer and Mspl is used as a restriction enzyme.
F) UGT1A1 * 28に関する多型部位を含む遺伝子領域について、配列番号 3の 塩基配列を有するフォワードプライマー及び配列番号 4の塩基配列を有するリバース プライマーを用いた PCR法による増幅を行うことを特徴とする、上記 A)記載の検査 方法。  F) A gene region including a polymorphic site related to UGT1A1 * 28 is amplified by PCR using a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4. The inspection method described in A) above.
G) UGT1A1 * 28、 UGT1A1 * 60及び UGT1A1 * 6に関する 3箇所の多型 部位を同時に検査することを特徴とする、上記 B)記載の検査方法。  G) The inspection method according to B) above, wherein three polymorphic sites relating to UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6 are simultaneously examined.
H) 目的遺伝子に関連する複数の多型を同時に検査する方法であって、挿入部 分の有無による多型を含む遺伝子領域のほか、一塩基多型（SNP)が存在する少な くとも 1つの遺伝子領域を同一の遺伝子増幅反応によって増幅する工程と、得られた 増幅断片を制限酵素によって消化した後、キヤビラリ一電気泳動に供する工程とを含 み、キヤピラリー電気泳動の結果に基づいて複数の多型を同時に判定する方法。  H) A method for examining multiple polymorphisms related to the target gene simultaneously, and includes at least one polymorphism (SNP) in addition to the gene region containing the polymorphism due to the presence or absence of the insertion site. Including a step of amplifying a gene region by the same gene amplification reaction, and a step of digesting the obtained amplified fragment with a restriction enzyme and then subjecting it to a capillary electrophoresis. A method to determine the type at the same time.
I) 上記 A)〜H)のいずれかに記載の検査方法を用いて、被検者の薬剤に対する 反応性またはその副作用の程度を予測する方法。  I) A method for predicting the reactivity of a subject to a drug or the degree of its side effects using the test method according to any one of A) to H) above.
J) 上記 A)〜H)のいずれかに記載の検査方法を用いて、被検者の杭がん剤に対 する反応性またはその副作用の程度を予測する方法。  J) A method for predicting the reactivity of a test subject to a pile cancer drug or the degree of its side effects using the test method described in any of A) to H) above.
K) 上記 A)〜H)の 、ずれかに記載の検査方法を用いた、遺伝子多型検査キット  K) Genetic polymorphism test kit using the test method according to any one of A) to H) above
発明の効果 The invention's effect
本発明によれば、 UGT1A1 * 28に関する多型部位を含む遺伝子領域と、一塩基 多型が存在する少なくとも 1つの遺伝子領域とを同一の遺伝子増幅反応によって増 幅後、一塩基多型における塩基の相違に応じて異なる長さの DNA断片が得られる ように、制限酵素によって消化反応を行う。その後、キヤピラリー電気泳動によって各 試料中の DNA断片を断片長にしたがって分離'検出する。キヤピラリー電気泳動は 高分解能であるため、 UGT1A1 * 28の多型における 2塩基の長さの差異を検出可 能であり、同時に検出された他の DNA断片の長さから一塩基多型のタイピングが可 能である。このように、 PCR等による増幅反応、制限酵素による消化反応、キヤビラリ 一電気泳動という一連の簡便かつ迅速な方法によって、 UGT1A1遺伝子の臨床的 に重要な複数の多型を高精度に検査 ·判定することができる。また、キヤビラリ一電気 泳動は大型の設備が不要であり、複数の PCR産物を同時に増幅するマルチプレック ス PCRのため、本発明により低コストでの検査を実現できる。 According to the present invention, after a gene region containing a polymorphic site for UGT1A1 * 28 and at least one gene region where a single nucleotide polymorphism exists are amplified by the same gene amplification reaction, the base of the single nucleotide polymorphism is amplified. In order to obtain DNA fragments of different lengths depending on the difference, digestion is performed with restriction enzymes. After that, the DNA fragments in each sample are separated and detected according to the fragment length by capillary electrophoresis. Because capillary electrophoresis has high resolution, it is possible to detect the difference in length of two bases in the UGT1A1 * 28 polymorphism, and single nucleotide polymorphism typing from the length of other DNA fragments detected at the same time. Yes, it is possible. Thus, amplification reactions such as PCR, digestion reactions with restriction enzymes, A series of simple and rapid methods of single electrophoresis can test and determine multiple clinically important polymorphisms of the UGT1A1 gene with high accuracy. In addition, the single-electrophoresis does not require a large facility and is a multiplex PCR that simultaneously amplifies a plurality of PCR products, so that the present invention can realize a low-cost test.
[0014] 本発明によって UGT1A1遺伝子の多型検査を行うことで、 UGT1A1で代謝される 薬剤の薬効または副作用の程度を予測する方法を提供することができる。例えば、 抗がん剤イリノテカンの副作用と UGT1A1遺伝子多型との間に相関が認められる旨 の研究結果が多数報告されており（非特許文献 5、 6等）、このような研究結果に基づ き、本発明による検査結果からイリノテカンを投与した場合の副作用の程度を予測す ることができる。本発明は複数の遺伝子多型を同時に検査するものである力 複数の 遺伝子多型に基づいて薬の副作用予測、薬効予測することは好ましぐゲノム情報 に基づく個々の患者に適した適正な薬物治療の実現に資し、医療の質の向上、薬の 安全性並びに治療効率の向上に寄与することができる。 [0014] By conducting a polymorphism test of the UGT1A1 gene according to the present invention, it is possible to provide a method for predicting the efficacy or side effect of a drug metabolized by UGT1A1. For example, many studies have reported that there is a correlation between the side effect of the anticancer drug irinotecan and the UGT1A1 gene polymorphism (Non-Patent Documents 5, 6, etc.). In addition, the degree of side effects when irinotecan is administered can be predicted from the test results according to the present invention. The present invention has the ability to test multiple gene polymorphisms simultaneously. Predicting side effects and drug efficacy based on multiple gene polymorphisms is an appropriate drug suitable for individual patients based on preferred genomic information. It contributes to the realization of treatment and can contribute to the improvement of medical quality, the safety of drugs and the efficiency of treatment.
[0015] UGT1A1遺伝子多型の検査によって、血清ピリルビン高値に影響する遺伝的素 因を有するカゝ否かを判断することができ、即ち、 AST, ALTなどの肝機能検査値の 異常を伴わない血清ピリルビン高値を科学的に説明することが可能となる。本発明の 検査方法は、このような検査にも利用可能である。  [0015] By testing UGT1A1 gene polymorphism, it is possible to determine whether or not it has a genetic predisposition affecting high serum pyrilbin levels, that is, without abnormal liver function test values such as AST and ALT It is possible to scientifically explain high serum pyrilrubin levels. The inspection method of the present invention can also be used for such inspection.
[0016] さらに、本発明によれば、上記検査方法を他の遺伝子多型検査法に応用し、目的 遺伝子に関連する複数の多型を、低コストで簡易迅速かつ精度良く検査する方法を 提供することができる。  [0016] Further, according to the present invention, there is provided a method for simply and quickly and accurately testing a plurality of polymorphisms related to a target gene at low cost by applying the testing method to other genetic polymorphism testing methods. can do.
図面の簡単な説明  Brief Description of Drawings
[0017] [図 1]本発明の実施例に係る UGT1A1遺伝子多型の検査法における、キヤビラリ一 電気泳動の結果を示すゲルイメージである。  [0017] FIG. 1 is a gel image showing the results of a capillary electrophoresis in the method for testing a UGT1A1 gene polymorphism according to an example of the present invention.
[図 2] (a)〜（c)は、上記キヤピラリー電気泳動の結果を示すグラフである。  [FIG. 2] (a) to (c) are graphs showing the results of the above-mentioned capillary electrophoresis.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 以下、本発明の好ましい態様について説明する。なお、本明細書および図面にお いて、塩基 ·アミノ酸等を略号で表記する場合、その表記は IUPAC-IUB Commission on Biochemical Nomenclatureによる略号あるいは当該分野における慣用略号に基 づくものであり、たとえば DNAの塩基の表記については以下のとおりである。 [0018] Hereinafter, preferred embodiments of the present invention will be described. In this specification and drawings, when bases, amino acids, etc. are abbreviated, the notation is based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in this field. For example, the notation of DNA bases is as follows.
[0019] Aまたは a:アデニン、 Gまたは g :グァニン、 Cまたは c :シトシン、 Tまたは t:チミン、 R または r:アデニンまたはグァニン、 Mまたは m:アデニンまたはシトシン、 Wまたは w: アデニンまたはチミン、 Sまたは s :グァニンまたはシトシン、 Kまたは k :グァニンまたは チミン、 Yまたは y:シトシンまたはチミン、である。 [0019] A or a: adenine, G or g: guanine, C or c: cytosine, T or t: thymine, R or r: adenine or guanine, M or m: adenine or cytosine, W or w: adenine or thymine , S or s: guanine or cytosine, K or k: guanine or thymine, Y or y: cytosine or thymine.
[0020] また、遺伝子多型、遺伝子配列等に関する番号、数値その他の情報は、米国国立 ノィォテクノロジー情報センター（National Center for Biotechnology Information : NC[0020] In addition, numbers, numerical values and other information related to gene polymorphisms, gene sequences, etc. are available from the National Center for Biotechnology Information (NC).
BI)のウェブページなどで利用可能な主要な遺伝子データベースにお!/、て使用され ている番号、数値等を参酌して解釈されるものとする。 It is to be interpreted in consideration of the numbers and numerical values used in major gene databases available on the BI) web page.
[0021] [1]UGT1A1遺伝子多型の検査法 [1] UGT1A1 gene polymorphism testing method
まず、本発明者が実際に行った UGT1A1遺伝子多型の検査法 (実施例）につい て説明する。  First, the UGT1A1 gene polymorphism testing method (Example) actually carried out by the inventor will be described.
[本発明の UGT1A1遺伝子多型の検査法]  [Method of testing UGT1A1 gene polymorphism of the present invention]
日本人由来の 155サンプルについて、本発明の検査法を用いて UGT1A1遺伝子 に関連する複数の多型を同時に検査 '判定した。本実施例では、 UGT1A1 * 60、 UGTIAI * 28、及び UGTIAI * 6に関する 3箇所の多型を検査対象とし、各多型 を含む遺伝子領域の増幅には PCR法を使用した。本検査法の特徴の 1つは、 3組の プライマーを使用し、同一の PCR条件による同一の遺伝子増幅反応によって、同時 に 3種の PCR産物を得る点である。使用した 3組のプライマーの配列は下記表 1に記 載のとおりである。  Using the test method of the present invention, a plurality of polymorphisms related to the UGT1A1 gene were simultaneously tested and determined for 155 samples from Japan. In this example, three polymorphisms related to UGT1A1 * 60, UGTIAI * 28, and UGTIAI * 6 were examined, and PCR was used to amplify the gene region containing each polymorphism. One of the features of this test method is that three sets of primers are used, and three PCR products are obtained simultaneously by the same gene amplification reaction under the same PCR conditions. The sequences of the three sets of primers used are listed in Table 1 below.
[0022] [表 1] ライマ—向き 配列 PCR条件 ― 制限酵素 —断片長（bp>[0022] [Table 1] Lima—Sequence Sequence PCR Conditions — Restriction Enzymes —Fragment Length (bp>
UGT1AI*60 Forward 5'- TTAACCAAAGAACATTCTAACGG-3 ' (1) W 115+93 UGT1AI * 60 Forward 5'- TTAACCAAAGAACATTCTAACGG-3 '(1) W 115 + 93
Hindi  Hindi
Reverse 5 ' -TTG ATGTTCTC AAATTG CTTTGTTG A- 3 ' (2) M 115+67+26 Reverse 5 '-TTG ATGTTCTC AAATTG CTTTGTTG A- 3' (2) M 115 + 67 + 26
UGTIAI *28 Forward 5'-CAAACATTAACTTGGTGTATCG-3' (3) 94 30sec W 83 UGTIAI * 28 Forward 5'-CAAACATTAACTTGGTGTATCG-3 '(3) 94 30sec W 83
― Reverse― S'-TTTGCTCCTGCCAGAGGm (4) 55。C 40 sec 85 ― Reverse― S'-TTTGCTCCTGCCAGAGGm (4) 55. C 40 sec 85
UGTIAI *6 Forward 5'-CTAGCACCTGACGCCTCGTTGTACATCAGA 72で 40 sec UGTIAI * 6 Forward 5'-CTAGCACCTGACGCCTCGTTGTACATCAGA 72 for 40 sec
W 203+32 G C-3' (5) Mspl  W 203 + 32 G C-3 '(5) Mspl
M 235 M 235
Reverse 5'-CCATGAGCTCCTTGT GTGC-3' (6) Reverse 5'-CCATGAGCTCCTTGT GTGC-3 '(6)
[0023] 表 1の「配列」の欄において、カツコ内の数字は、同プライマー配列が記載される配 列表の配列番号を示す。また、下線部の塩基は、制限酵素切断部位^ |ij出するため 、人為的に異なる塩基を挿入した箇所である。このように、 UGT1A1 * 60用、及び UGT1A1 * 6用の一方のプライマーについては変異プライマー（ミスマッチプライマ 一）を作製し、これを使用して PCR反応を行った。 [0023] In the column of "Sequence" in Table 1, the numbers in Katsuko indicate the SEQ ID NOs of the Sequence Listing in which the primer sequences are described. In addition, underlined bases are used to generate restriction enzyme cleavage sites ^ | ij This is the place where an artificially different base was inserted. Thus, for one of the primers for UGT1A1 * 60 and UGT1A1 * 6, a mutant primer (mismatch primer) was prepared, and PCR reaction was performed using this.
[0024] 上記 PCR反応は、表 1に示す 6本のプライマーを 1つのチューブに入れて行った。 [0024] The PCR reaction was performed by putting the six primers shown in Table 1 in one tube.
反応液（20 /z L)は、 60ngのゲノム DNA、 1. 0ユニットの AmpliTaq DNAポリメラーゼ 、 0. 25 Μの UGT1A1 * 60用プライマー、 0. 2 μ Μの UGT1A1 * 28用プライマ 一、 0. 075 Μの UGT1A1 * 6用プライマー、各 0. 2mMのデォキシヌクレオチド 三リン酸、を含む組成とした。 PCR条件は、（1) 94°C5分の後、（2) 94°C30秒、 55°C 40秒、 72°C40秒を 1サイクル (表 1参照）として、これを 40サイクルに設定して反応を 行った。  The reaction (20 / z L) consists of 60 ng genomic DNA, 1.0 unit AmpliTaq DNA polymerase, 0.25 U UGT1A1 * 60 primer, 0.2 μΜ UGT1A1 * 28 primer, 075 0 UGT1A1 * 6 primer and 0.2 mM deoxynucleotide triphosphate each. The PCR conditions are (1) 94 ° C for 5 minutes, and (2) 94 ° C for 30 seconds, 55 ° C for 40 seconds, and 72 ° C for 40 seconds. The reaction was performed.
[0025] 上記のように、 2つのミスマッチプライマーを含む 3組のプライマーを反応液に混合 して PCRを行った後、得られた PCR産物に、 Lowバッファーおよび制限酵素の Hinc IIと Msplを加え、 37°Cで 1時間、消化反応を行った。なお、反応時間は 30分程度で もよい。制限酵素による消化反応後、簡易型多検体用キヤビラリ一電気泳動装置「H DA GT-12™(eGene社)」を用いて各反応液を電気泳動に供し、各反応液中の DNA 断片をそのサイズ (断片長）にしたがって分離し検出した。  [0025] As described above, 3 pairs of primers including two mismatched primers were mixed into the reaction mixture and PCR was performed. Then, the low PCR buffer and the restriction enzymes Hinc II and Mspl were added to the resulting PCR product. The digestion reaction was performed at 37 ° C for 1 hour. The reaction time may be about 30 minutes. After digestion with restriction enzymes, each reaction solution is subjected to electrophoresis using a simple multi-sample capillary electrophoresis device “H DA GT-12 ™ (eGene)”, and the DNA fragments in each reaction solution are subjected to electrophoresis. Separated and detected according to size (fragment length).
[0026] UGT1A1 * 6の多型部位に関し、野生型アレル「W」であるときは、多型部位は制 限酵素 Msplにより切断されるので、表 1に示すように、断片長は 203bpと 32bpにな る。他方、変異型アレル「M」であるときは、多型部位は制限酵素 Msplにより切断さ れないので、断片長は 235bpになる。  [0026] Regarding the polymorphic site of UGT1A1 * 6, when it is the wild-type allele “W”, the polymorphic site is cleaved by the restriction enzyme Mspl. Therefore, as shown in Table 1, the fragment lengths are 203 bp and 32 bp. become. On the other hand, in the case of the mutant allele “M”, the polymorphic site is not cleaved by the restriction enzyme Mspl, so the fragment length is 235 bp.
[0027] UGT1A1 * 60の多型部位に関し、野生型アレル「W」であるときは、多型部位は 制限酵素 Hindiにより切断されないので、表 1に示すように、断片長は 115bpと 93b Pになる (増幅断片中に制限酵素切断部位が 1箇所存在するため)。他方、変異型ァ レル「M」であるときは、多型部位は制限酵素 Hindiにより切断されるので、断片長は 115bpと 67bpと 26bpになる。  [0027] Regarding the polymorphic site of UGT1A1 * 60, when the wild-type allele “W” is used, the polymorphic site is not cleaved by the restriction enzyme Hindi. Therefore, as shown in Table 1, the fragment length is 115 bp and 93 bp. (Because there is one restriction enzyme cleavage site in the amplified fragment). On the other hand, in the case of the mutant allele “M”, the polymorphic site is cleaved by the restriction enzyme Hindi, so that the fragment length becomes 115 bp, 67 bp and 26 bp.
[0028] UGT1A1 * 28の多型部位は、制限酵素によっては切断されず、 2塩基繰り返し配 列 (TAリピート）が野生型アレル「W」よりも 1つ多い変異型アレル「M」であるときは、 表 1に示すように、断片長は 85bpになり、野生型アレル「W」であるときは、断片長は 83bpになる。 [0028] The polymorphic site of UGT1A1 * 28 is not cleaved by a restriction enzyme, and the double nucleotide repeat sequence (TA repeat) is a mutant allele “M” that is one more than the wild type allele “W” As shown in Table 1, the fragment length is 85 bp, and when the wild type allele “W”, the fragment length is It becomes 83bp.
[0029] [本発明の検査法の精度確認のための従来法による判定]  [Decision by Conventional Method for Confirming Accuracy of Inspection Method of Present Invention]
本発明の検査法の精度を確認するため、 PCR-RFLP法、メルティングカーブ法、 ダイレクトシークェンス法などの従来法を用いて、各多型の検査 ·判定をあわせて行 つた。検査対象は、 UGT1A1 * 60、 UGT1A1 * 28、及び UGT1A1 * 6の 3多型 のほ力に、比較的マイナーな多型である UGT1A1 * 7、 UGT1A1 * 27、及び UG T1A1 * 29をカ卩えた合計 6多型である。これらの 6多型について、これまでに報告さ れて 、る PCR法による判定方法をアレンジして、それぞれの多型に対し個別に判定 を行った。各多型の判定に使用したプライマーと PCR条件 (増幅プロセスにおける 1 サイクルの設定）は、下記表 2に記載のとおりである。  In order to confirm the accuracy of the inspection method of the present invention, conventional methods such as the PCR-RFLP method, the melting curve method, and the direct sequence method were used to inspect and determine each polymorphism. The test targets were UGT1A1 * 60, UGT1A1 * 28, and UGT1A1 * 6, and the three minor polymorphs UGT1A1 * 7, UGT1A1 * 27, and UG T1A1 * 29. There are 6 polymorphisms in total. For these 6 polymorphisms, the methods of determination by the PCR method previously reported were arranged, and each polymorphism was individually determined. The primers and PCR conditions (1 cycle setting in the amplification process) used to determine each polymorphism are shown in Table 2 below.
[0030] [表 2] 'ライマ—向き 配列 PCR条件 制限酵素 断片長（bp) [0030] [Table 2] 'Lima orientation Sequence PCR conditions Restriction enzyme Fragment length (bp)
UGT1A1*6 Forward 5'-CTAGCACCTGACGCCTCGTTGTACATCA 30sec UGT1A1 * 6 Forward 5'-CTAGCACCTGACGCCTCGTTGTACATCA 30sec
W 203+32 GAG C-3' (5) 60 40sec Mspl  W 203 + 32 GAG C-3 '(5) 60 40sec Mspl
M 235 Reverse 5 ' -CCATGAGCTCCTTGTTGTGC-3 ' (6) 72X 40sec  M 235 Reverse 5 '-CCATGAGCTCCTTGTTGTGC-3' (6) 72X 40sec
UGT1A1 *7 Forward 5 ' - AGCATAAAGAG AGG ArrGTT-3 ' (7) Imin  UGT1A1 * 7 Forward 5 '-AGCATAAAGAG AGG ArrGTT-3' (7) Imin
W 365+214 Reverse 5 ' -CACCAGAGGGGGCACG ATAC-3 ' (8) 60 lmin Bsrl  W 365 + 214 Reverse 5 '-CACCAGAGGGGGCACG ATAC-3' (8) 60 lmin Bsrl
M 579 x Imin  M 579 x Imin
UGT1A1*27 Forward 5 ' - AGTACCTGTCTCTGCCC AC-3 ' (9) 30sec  UGT1A1 * 27 Forward 5 '-AGTACCTGTCTCTGCCC AC-3' (9) 30sec
W 331+68 Reverse 5 ' -GTCCCACTCC A ATAC ACAC-3 ' (10) 40 sec Bsrl  W 331 + 68 Reverse 5 '-GTCCCACTCC A ATAC ACAC-3' (10) 40 sec Bsrl
M 199+32+68 40 sec  M 199 + 32 + 68 40 sec
UGT1A1*28 Forward 5'-AAGTGAACTCCCTGCTACCTT -3' (11) 95°C Osec メルティング力一ブ法  UGT1A1 * 28 Forward 5'-AAGTGAACTCCCTGCTACCTT -3 '(11) 95 ° C Osec Melting force method
Reverse 5'CCACTGGGATCAACAGTATCT -3' (12) 55°C 7 sec または  Reverse 5'CCACTGGGATCAACAGTATCT -3 '(12) 55 ° C 7 sec or
72°C 12sec ダイレクトシ一ク 法 72 ° C 12sec direct seek method
UGT1A1 *29 Forward 5'-TCCTCCCTATTTTGCATCTCAGGTCACC 9A 30sec UGT1A1 * 29 Forward 5'-TCCTCCCTATTTTGCATCTCAGGTCACC 9A 30sec
W 252+33 CGATGGCC-3' (13) 60 40sec QH3I  W 252 + 33 CGATGGCC-3 '(13) 60 40sec QH3I
M 28 M 28
Reverse 5'-TGAATGCCATGACCAAA-3' (14) 72で 40 sec Reverse 5'-TGAATGCCATGACCAAA-3 '(14) 40 sec at 72
UGT1A1 *60 Forward 5'- TTAACCAAAGAACATTCTAACGG-3 ' (1) 94°C 30sec  UGT1A1 * 60 Forward 5'- TTAACCAAAGAACATTCTAACGG-3 '(1) 94 ° C 30sec
W 183+25 Reverse 5 ' -TTGATGTTCTCAAATTGCI TGT1TA-3 ' 60 lmin Oral  W 183 + 25 Reverse 5 '-TTGATGTTCTCAAATTGCI TGT1TA-3' 60 lmin Oral
M 208 M 208
(15) 72"C lmin 表 1と同様に、「配列」の欄のカツコ内の数字は、同プライマー配列が記載される配 列表の配列番号を示す。また、下線部の塩基は、制限酵素切断部位^ iij出するため(15) 72 "C lmin As in Table 1, the numbers in brackets in the" Sequence "column indicate the SEQ ID NOs of the Sequence Listing in which the primer sequences are described. In addition, underlined bases are used to generate restriction enzyme cleavage sites ^ iij
、人為的に異なる塩基を挿入した箇所である。 UGT1A1 * 28を除く 5つの多型につ いては、 PCR-RFLP法により判定した。 UGT1A1 * 28は、以前報告された方法（H epatology 2000;32:792-5)に従って、ロシュ社のライトサイクラ を用いたメルティング カーブ法により判定した。また、この判定法で結果がはっきりしない場合には、ダイレ タトシークェンス法を用いて UGT1A1 * 28の遺伝子型を決定した。 This is the place where an artificially different base was inserted. Five polymorphisms except UGT1A1 * 28 were determined by PCR-RFLP method. UGT1A1 * 28 was determined by a melting curve method using a Roche light cycler according to a previously reported method (Hepatology 2000; 32: 792-5). If the result is not clear by this method, The genotype of UGT1A1 * 28 was determined using the Tato Sequence method.
[0032] UGT1A1 * 6アレルにつ!、ては、既に報告されて!、る方法（Cancer Res 2000;60:6 921-6)を若干改良した方法を用いて判定した。この判定においては、上記のように、 野生型アレル「W」であるときのみ、 Mspl認識部位を持つようなミスマッチプライマー を設計した（表 2参照）。 UGT1A1 * 29アレル、及び UGT1A1 * 60アレルにつ!ヽ ても同様に、野生型アレル「W」であるときのみ、それぞれ Cfrl3I、 Dral認識部位を 持つようなミスマッチプライマーを設計した。 UGT1A1 * 7アレル、及び UGT1A1 * 27アレルについては、既に報告されている方法（Cancer Res 2000;60:6921-6)にし たがって、制限酵素 Bsrlを用いた PCR-RFLP法により判定した。 [0032] The UGT1A1 * 6 allele was determined using a method slightly improved from the previously reported method (Cancer Res 2000; 60: 6 921-6). In this determination, as described above, a mismatch primer having an Mspl recognition site was designed only when the wild type allele was “W” (see Table 2). Similarly, for the UGT1A1 * 29 allele and the UGT1A1 * 60 allele, mismatch primers were designed to have Cfrl3I and Dral recognition sites only when the wild type allele was “W”. The UGT1A1 * 7 allele and the UGT1A1 * 27 allele were determined by the PCR-RFLP method using the restriction enzyme Bsrl according to the method already reported (Cancer Res 2000; 60: 6921-6).
[0033] さらに、 UGT1A1 * 28と UGT1A1 * 6の両方がヘテロの場合、あるいは、 UGT1 A1 * 28と UGT1A1 * 27の両方がヘテロの場合に、被検者のハプロタイプを決定 するために、 UGT1A1 * 6または UGT1A1 * 27に対してアレルスぺシフィック PCR 法 (Allele specific PCR法）を次のように行った。 UGT1A1 * 28アレルについては、 各々の PCR産物を精製し、ダイレクトシークェンスを行って UGT1A1 * 28の多型を 確認した。 [0033] Furthermore, in order to determine the haplotype of a subject when both UGT1A1 * 28 and UGT1A1 * 6 are heterogeneous, or when both UGT1A1 * 28 and UGT1A1 * 27 are heterogeneous, UGT1A1 * Allele specific PCR method (Allele specific PCR method) was performed on 6 or UGT1A1 * 27 as follows. For the UGT1A1 * 28 allele, each PCR product was purified and subjected to direct sequencing to confirm the polymorphism of UGT1A1 * 28.
[0034] PCRは、 10mMのトリス塩酸（pH8. 3)、 1. 5mMの MgCl 、 150ngのゲノム DNA  [0034] PCR was performed using 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2, 150 ng genomic DNA.
2  2
、 2. 5ユニットの AmpliTaq DNAポリメラーゼ、各 200nMプライマー、各 0. 2mMのデ ォキシヌクレオチド三リン酸を含む 50 Lの PCR反応液で行った。使用したプライマ 一と PCR条件 (増幅プロセスにおける 1サイクルの設定）は、下記表 3に記載のとおり である。  The reaction was performed in a 50 L PCR reaction solution containing 2.5 units of AmpliTaq DNA polymerase, 200 nM of each primer, and 0.2 mM of dioxynucleotide triphosphate. The primers used and the PCR conditions (one cycle setting in the amplification process) are shown in Table 3 below.
[0035] [表 3] "向き 配列 PCR条件 制限酵素 断片長 （bp)[0035] [Table 3] Orientation Sequence PCR Conditions Restriction Enzyme Fragment Length (bp)
UGT1A1*28 Forward 5'-AAGTGAACTCCCTGCTACCTT-3' (11) 94V 40 sec UGT1A1 * 28 Forward 5'-AAGTGAACTCCCTGCTACCTT-3 '(11) 94V 40 sec
And Reverse(W) 5'-TTCAAGGTGTAAAATGCTCC-3' (16) 56V 40 sec ― 377  And Reverse (W) 5'-TTCAAGGTGTAAAATGCTCC-3 '(16) 56V 40 sec ― 377
UGT1A1*6 Reverse(M) 5'-TTCAAGGTGTAAAATGCTCT-3' (17) 72°C 40 sec  UGT1A1 * 6 Reverse (M) 5'-TTCAAGGTGTAAAATGCTCT-3 '(17) 72 ° C 40 sec
UGT1A1*28 Forward 5'-AAGTGAACTCCCTGCTACCTT-3' (11) 94°C lmia  UGT1A1 * 28 Forward 5'-AAGTGAACTCCCTGCTACCTT-3 '(11) 94 ° C lmia
And Re verse (W) 5'-CTGAGGCAAGGGTTGCATACG- 3' (18) 63で lmin ― 853  And Re verse (W) 5'-CTGAGGCAAGGGTTGCATACG-3 '(18) 63 lmin ― 853
UGT1A1*27 Reverse(M) 5'-CTGAGGCAAGGGTTGCATACT-3' (19) 72°C Imin 野生型アレル検出用のリバースプライマー (W)では野生型アレルのみ増幅される 一方、変異型アレル検出用のリバースプライマー（M)では変異型アレルのみ増幅さ れる。各増幅断片における UGTIAI * 28の判定結果とあわせてハプロタイプを判 し 7こ。 UGT1A1 * 27 Reverse (M) 5'-CTGAGGCAAGGGTTGCATACT-3 '(19) 72 ° C Imin Only the wild type allele is amplified with the reverse primer (W) for detecting the wild type allele, while the reverse primer for detecting the mutant type allele In (M), only the mutant allele is amplified. It is. Judge the haplotype together with the UGTIAI * 28 judgment result for each amplified fragment.
[0037] [結果] [0037] [Result]
本実施例の結果、日本人由来の 155サンプルについて、本発明の検査法により複 数の多型を同時に判定した結果と、従来法により個別に各多型を判定した結果とが 完全に一致した。図 1は、本発明の検査法によるキヤピラリー電気泳動の結果の一部 を示すゲルイメージであり、図 2 (a)〜（c)は、当該キヤピラリー電気泳動の結果の一 部を示すグラフである。各図において、野生型アレルは「* 1」として示される。これら の図に示すように、 UGT1A1 * 28に関する多型部位の 2塩基の差異を、キヤビラリ 一電気泳動を用いることによって増幅断片を 85bpと 83bpとに分け、視覚的に検出 することが可能となった。同時に、 UGT1A1 * 60及び UGT1A1 * 6に関する一塩 基多型についても精度良く検出することができた。  As a result of this example, for 155 samples derived from Japanese, the results of simultaneous determination of multiple polymorphisms by the test method of the present invention completely matched the results of individual determination of each polymorphism by the conventional method. . Fig. 1 is a gel image showing a part of the result of capillary electrophoresis by the test method of the present invention, and Fig. 2 (a) to (c) are graphs showing a part of the result of the capillary electrophoresis. . In each figure, the wild type allele is indicated as “* 1”. As shown in these figures, the difference between the two bases of the polymorphic site of UGT1A1 * 28 can be visually detected by dividing the amplified fragment into 85 bp and 83 bp by using single electrophoresis. It was. At the same time, the monobasic polymorphism related to UGT1A1 * 60 and UGT1A1 * 6 could be detected with high accuracy.
[0038] UGT1A1 * 7アレルと UGT1A1 * 29アレルを保有する被検者は、今回調査した 155サンプル中には存在しなかった。また、アレルスぺシフィック PCR法による分析 結果から、 UGT1A1 * 27アレルは UGT1A1 * 28アレルに含まれることが確認され た。 [0038] Subjects with the UGT1A1 * 7 allele and the UGT1A1 * 29 allele were not present in the 155 samples investigated. In addition, from the results of analysis by the allele specific PCR method, it was confirmed that the UGT1A1 * 27 allele was included in the UGT1A1 * 28 allele.
[0039] 以上のように、本実施例の結果から、 UGT1A1遺伝子に関連する臨床的に重要 な 3つの多型 UGT1A1 * 28、 UGT1A1 * 60及び UGT1A1 * 6を、本発明の検 查法によって簡易迅速かつ精度良く検査 ·判定できることがわ力つた。  [0039] As described above, from the results of this Example, three clinically important polymorphisms UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6 related to the UGT1A1 gene can be simplified by the test method of the present invention. I was able to test and judge quickly and accurately.
[0040] [2]本発明の検査法の変更態様  [0040] [2] Modification of inspection method of the present invention
勿論、本発明の検査法は、上記方法に限定されるものではなぐ本発明の範囲内 で種々の変更が可能である。例えば、上記方法は、 UGT1A1 * 28、 UGT1A1 * 6 0及び UGT1A1 * 6の 3箇所の多型を同時に検査するものであった力 本発明はこ れら 3箇所の多型の検査に制限されるものではない。これら 3箇所の多型は、日本人 に観察される臨床的に重要な多型であるので、これら多型の検査に本発明の検査法 を適用することは好ましいが、臨床的に重要な UGT1A1遺伝子多型として、他にも UGT1A1 * 27、 UGTIAI * 29、 UGTIAI * 7などが知られており、本発明にお いて、 UGT1A1 * 28の多型と同時に検査する一塩基多型はこれらの多型であって もよ ヽ。このように、 UGT1A1 * 27、 UGT1A1 * 29、 UGT1A1 * 7などの一塩基 多型を UGT1A1 * 28の多型と同時に検査する場合は、例えば表 2に掲げるプライ マー配列を使用して PCR反応を行うことで、一塩基多型を含む遺伝子領域を増幅す ることができる。また、 UGT1A1遺伝子のゲノム配列は、 GenBank等の遺伝子配列 データベースにァクセッション番号「NG_002601」（GeneID : 54658)で登録'掲載され ており、 UGT1A1の cDNA配列はァクセッション番号「NM_000463」などで登録 '掲 載されているので、これらのデータベースに開示されている塩基配列などを参考に各 プライマーを設計してもよい。 Of course, the inspection method of the present invention is not limited to the above method, and various modifications can be made within the scope of the present invention. For example, the above method is a force that simultaneously inspects three polymorphisms of UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6.The present invention is limited to the inspection of these three polymorphisms. It is not a thing. Since these three polymorphisms are clinically important polymorphisms observed in Japanese, it is preferable to apply the test method of the present invention to the examination of these polymorphisms, but clinically important UGT1A1 Other gene polymorphisms are known, such as UGT1A1 * 27, UGTIAI * 29, UGTIAI * 7, etc.In the present invention, single nucleotide polymorphisms to be tested simultaneously with UGT1A1 * 28 polymorphisms are those polymorphisms. Type Moyo! Thus, when testing single nucleotide polymorphisms such as UGT1A1 * 27, UGT1A1 * 29, UGT1A1 * 7 at the same time as the polymorphism of UGT1A1 * 28, for example, PCR reaction using the primer sequences listed in Table 2 is performed. By doing so, a gene region containing a single nucleotide polymorphism can be amplified. The genomic sequence of the UGT1A1 gene is registered in the gene sequence database such as GenBank with the accession number “NG_002601” (GeneID: 54658). The UGT1A1 cDNA sequence is the accession number “NM_000463”, etc. Since registration is listed, each primer may be designed with reference to the nucleotide sequences disclosed in these databases.
[0041] 上記実施例の方法は 3箇所の多型を同時に検査するものであった力 本発明は 2 箇所の多型を同時に検査するものであってもよい。この場合は、 UGT1A1 * 28に関 する多型のほかに、臨床的に重要な他の一塩基多型を検査する。 4箇所以上の多型 を同時に検査することも可能であるが、異なる増幅断片が重なって検出困難になるこ とがないよう、プライマーの設計などに十分留意する必要がある。上記実施例の検査 法では、プライマーの設計にあたり、（1) 110丁1八1 * 28にっぃては21^の差異を検 出するため増幅断片を lOObp以下になるようにし、（2)制限酵素による消化後の各 D NA断片が互いに重なって検出困難にならないようにし、また、（3)各 DNA断片の相 対的な距離、位置関係力も多型の検出が容易になるように配慮した。具体的には、 U GT1A1 * 28の多型検出のため、当該増幅断片の前後いずれか約 30bp以内に他 の増幅断片が少なくとも 1つ (好ましくは複数）出現するようにプライマーを設計した。 したがって、 UGT1A1 * 28、 UGT1A1 * 60及び UGT1A1 * 6の 3箇所の多型を 検査する場合に、表 1に掲げる 3組のプライマーを使用することは好ましい。各プライ マーの長さは、表 1に掲げるものより多少短くても長くてもよいが、同一の反応条件で 3箇所の遺伝子領域を増幅できるよう留意する。  [0041] The method of the above-described embodiment has the power to inspect three polymorphisms at the same time. The present invention may inspect two polymorphisms at the same time. In this case, in addition to the polymorphism related to UGT1A1 * 28, other clinically important single nucleotide polymorphisms are examined. Although it is possible to simultaneously examine four or more polymorphisms, it is necessary to pay careful attention to the design of the primers so that different amplified fragments do not overlap and become difficult to detect. In the inspection method of the above example, in designing the primer, (1) in order to detect a difference of 110 ^ 1 18 1 * 28, 21 ^, the amplified fragment should be less than lOObp, and (2) Make sure that DNA fragments after digestion with restriction enzymes do not overlap each other and make it difficult to detect. (3) Consider the relative distance and positional relationship of each DNA fragment so that polymorphism can be easily detected. did. Specifically, in order to detect the polymorphism of UGT1A1 * 28, primers were designed so that at least one (preferably a plurality) of other amplified fragments appeared within about 30 bp before or after the amplified fragment. Therefore, when testing three polymorphisms UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6, it is preferable to use the three sets of primers listed in Table 1. The length of each primer may be slightly shorter or longer than those listed in Table 1, but care must be taken to amplify the three gene regions under the same reaction conditions.
[0042] 上記実施例の検査法において、遺伝子試料は各被検者の末梢血からゲノム DNA を抽出して調製したが、本発明において、検査に供する遺伝子試料は、被検者の任 意の器官 ·組織 ·細胞 (血液、羊水中の細胞、採取した組織等を培養した細胞を含む )から常法に従って DNAを精製'抽出すればよい。なお、 PCR法 (又は他の遺伝子 増幅法）による遺伝子増幅が可能な限りにおいて、 DNAの精製 ·抽出工程は省略又 は簡略ィ匕してもよい。 [0042] In the test methods of the above examples, the gene sample was prepared by extracting genomic DNA from the peripheral blood of each subject. In the present invention, the gene sample to be used for the test is the subject's arbitrary. DNA can be purified and extracted from organs / tissues / cells (including blood, cells in amniotic fluid, cells cultured from collected tissues, etc.) according to a conventional method. As long as gene amplification by PCR (or other gene amplification) is possible, DNA purification and extraction steps are omitted or May be simplified.
[0043] 被検者カゝら調製した試料中のゲノム DNAを铸型にして、多型部位を含む遺伝子領 域を PCR法によって増幅する場合、 PCR法における反応条件、使用する試薬'反応 液の組成、使用機器などは特に上記実施例の方法に制限されるものではないが、目 的の遺伝子領域がすべて増幅されるように反応条件など留意するとよい。 PCR法は 簡便な方法であり、精度も良好であるので、遺伝子増幅法には PCR法の使用が好ま しいが、 PCR法以外の他の増幅法（例えば、 RC A法など）を使用してもよい（「ポスト シークェンスのゲノム科学（1) SNP遺伝子多型の戦略」（中山書店)など参照)。  [0043] When genomic DNA in a sample prepared by a subject is used as a saddle and a gene region containing a polymorphic site is amplified by PCR, the reaction conditions in the PCR and the reagents used The composition and the equipment to be used are not particularly limited to the methods of the above examples, but it is advisable to pay attention to the reaction conditions so that the entire target gene region is amplified. The PCR method is a simple method and has good accuracy. Therefore, it is preferable to use the PCR method for the gene amplification method, but other amplification methods (eg, RCA method) other than the PCR method are used. Good (see “Post-Sequence Genomic Sciences (1) Strategies of SNP gene polymorphism” (Nakayama Shoten)).
[0044] 制限酵素による消化反応についても、使用する制限酵素の種類等に応じてバッフ ァ一の種類、反応温度、反応時間などを決定すればよい。上記実施例の方法のよう に、 2種類の制限酵素を同時に使用し、 2種の SNPを PCR-RFLP法によって検出 することは好ましい。  Regarding the digestion reaction with a restriction enzyme, the type of buffer, reaction temperature, reaction time, etc. may be determined according to the type of restriction enzyme used. As in the method of the above example, it is preferable to use two kinds of restriction enzymes at the same time and detect two kinds of SNPs by the PCR-RFLP method.
[0045] キヤピラリー電気泳動は、キヤピラリー内で DNAを電気泳動させることによって通常 のゲル電気泳動に比べて高分解能で DNAを分離 ·検出するものであり、市販の装 置を用いて行うことができる。 UGT1A1 * 28の多型における 2bpの差異を検出でき るものであれば、いずれの装置を用いてもよい。  [0045] In capillary electrophoresis, DNA is separated and detected at higher resolution than normal gel electrophoresis by electrophoresis of DNA in the capillary, and can be performed using a commercially available apparatus. . Any device that can detect a 2 bp difference in the UGT1A1 * 28 polymorphism may be used.
[0046] 本発明の検査法を用いた遺伝子多型検査キットとしては、 UGT1A1遺伝子に関連 する複数の多型を検査するために設計されたプライマーを含むものであればよぐさ らに、（1)試料の調製に用いる酵素や試薬、（2) PCR法に用いる酵素や試薬、 (3) 制限酵素による消化反応に用いる酵素や試薬、などのうち 1又は 2以上を含むもので あってもょ ヽ。 UGT1A1 * 28、 UGT1A1 * 60及び UGT1A1 * 6の 3箇所の多型 を検査する場合、検査キットに含まれるプライマーとしては、表 1に掲げる 3組のプライ マーが例示されるが、各プライマーの長さはこれより 1〜5塩基程度長くても短くてもよ い。また、他の組み合わせの複数の多型を検査する場合は、表 2に掲げるプライマー 群の中から選択された 1組又は複数組のプライマーを含んだキット構成としてもょ ヽ。 なお、本発明は、「UGT1A1遺伝子に関連する複数の多型」を同時に検査するもの であるが、 UGT1A1遺伝子に関連する多型とは、換言すれば、 UGT1A1遺伝子ま たはゲノム上その近傍に存在する多型を意味し、 UGT1A1ゲノム遺伝子のェキソン 領域あるいはイントロン領域に存在する多型の検査に制限されず、そのプロモーター 領域など転写調節領域、制御領域などに存在する多型を検査するものであってもよ い。本明細書において、「遺伝子多型」とは、このようなプロモーター領域などに存在 する多型を含み、当該遺伝子に関連する多型という広義の意味である。 [0046] As a genetic polymorphism testing kit using the testing method of the present invention, any kit including primers designed for testing a plurality of polymorphisms related to the UGT1A1 gene may be used. (1) Enzymes and reagents used in sample preparation, (2) Enzymes and reagents used in PCR, (3) Enzymes and reagents used in restriction enzyme digestion reactions, etc.ヽ. When testing three polymorphisms, UGT1A1 * 28, UGT1A1 * 60, and UGT1A1 * 6, the primer included in the test kit is exemplified by the three sets of primers listed in Table 1. The length may be 1 to 5 bases longer or shorter than this. In addition, when testing multiple polymorphisms in other combinations, the kit configuration may include one or more primers selected from the primer group listed in Table 2. In the present invention, “a plurality of polymorphisms related to the UGT1A1 gene” are simultaneously examined. In other words, the polymorphisms related to the UGT1A1 gene are, in other words, the UGT1A1 gene or its vicinity in the genome. An existing polymorphism, exon of UGT1A1 genomic gene The present invention is not limited to testing for polymorphisms existing in the region or intron region, but may be those for testing polymorphisms present in transcriptional regulatory regions such as the promoter region and control regions. In the present specification, the “gene polymorphism” includes a polymorphism existing in such a promoter region and the like, and has a broad meaning of a polymorphism related to the gene.
[0047] 本発明によって UGT1A1遺伝子の多型検査を行うことで、 UGT1A1で代謝される 薬剤の薬効または副作用の程度を予測することができる。例えば、 UGT1A1 * 28と UGT1A1 * 60は単独で、また、 UGT1A1 * 6は UGT1A1 * 28と一緒に存在する ことでイリノテカンの副作用のリスクが高まることが報告されており、これらの多型を判 定することでイリノテカンの重篤な副作用を起こす可能性の高い患者を予測すること ができる。  [0047] By conducting a polymorphism test of the UGT1A1 gene according to the present invention, the efficacy or side effect of a drug metabolized by UGT1A1 can be predicted. For example, the presence of UGT1A1 * 28 and UGT1A1 * 60 alone and UGT1A1 * 6 together with UGT1A1 * 28 has been reported to increase the risk of irinotecan side effects. By doing so, it is possible to predict patients who are likely to cause serious side effects of irinotecan.
[0048] [3]UGT1A1遺伝子以外の遺伝子多型の検査法  [0048] [3] Testing methods for polymorphisms other than UGT1A1 gene
本発明の検査法は、 UGT1A1遺伝子以外の遺伝子多型の検査にも応用可能で あり、目的遺伝子に関連する複数の多型を、低コストで簡易迅速かつ精度良く検査 する方法を提供することができる。  The test method of the present invention can also be applied to the test of gene polymorphisms other than the UGT1A1 gene, and can provide a method for quickly and accurately testing a plurality of polymorphisms related to the target gene at low cost. it can.
[0049] 即ち、本発明は、 UGT1A1遺伝子以外の遺伝子に関連する複数の多型であって 、挿入部分の有無による多型 (ある 2塩基以上の塩基配列が挿入された挿入型と当 該塩基配列を欠く欠失型との間の多型）と、一塩基多型 (SNP)との組み合わせから なる複数の多型を同時に検査する場合にも適用することができる。このような複数の 多型をもつ遺伝子としては、例えば次のようなものを挙げることができる。（下記表 4で は、各遺伝子によってコードされる分子名を示す。 )  That is, the present invention relates to a plurality of polymorphisms related to genes other than the UGT1A1 gene, which are polymorphisms depending on the presence or absence of an insertion portion (an insertion type in which a certain base sequence of 2 or more bases is inserted and the base) It can also be applied to the case where multiple polymorphisms consisting of a combination of single nucleotide polymorphism (SNP) and a polymorphism between deletion type lacking sequence) and single nucleotide polymorphism (SNP) are simultaneously examined. Examples of such genes having a plurality of polymorphisms include the following. (Table 4 below shows the names of molecules encoded by each gene.)
[0050] [表 4] [0050] [Table 4]
挿部多第型入分のの 1： « G Q Insert part 1st type: «G Q
多第多型第型第多型場所多繰 2の S 3の SNP有無よる型（り返名略称NPののに 1分（）子s：s：： σ> -H >  Multi-polymorphic polymorphic polymorphic type polymorphic location type 2 S 3 SNP type with or without SNP (repeat name abbreviation NP but one minute s: s :: σ> -H>
m  m
多型し配列）の Q G Q D Q Q a Q Q Q Q Q Q Q Q Q Q D a G Ω G D or  Q G Q D Q Q a Q Q Q Q Q Q Q Q Q Q D a G Ω G D or
m σ m σ
n  n
>  >
- // aaaoi  -// aaaoi
σ> σ>
>  >
c or c or
ra  ra
卜  卜
- >  ->
>. >.
¾  ¾
さ さ  Sasa
表中の各分子は、 1:セロトニントランスポーター、 2:モノアミンォキシダーゼ八、 3:ァ ンドロゲン受容体、 4:チロシンヒドロキシラーゼ、 5:セロトニン受容体 5-HT2C 6:芳香 族アミノ酸デカルボキシラーゼ (AADC)、 7:ドーノミン D2受容体 DRD2、 8:セロトニン 受容体 5-HTR3B、 9：セロトニン受容体 5-HTR6、 10： GABAの A型受容体 oc 5サブュニ ット (GABRA5)、 11:ドーパミントランスポーター (SLC6A3(DAT))、 12:ドーパミン D4受容 体 DRD4、 13：ドーパミン D5受容体 DRD5、 14： Phosphatidylinositoト 4- phosphate- 5- ki nase type 2 a、 15 denylate cyclase 9(ADCY9)、 16: GABA受容体 j83サブユニット (GABRB3)、 17: Adrenergic receptor alpha 2B(ADRA2B)ゝ 18: Adrenergic receptor al pha 2C(ADRA2C)、 19:アンギオテンシン変換酵素、 20 denylate cyclase 7(ADCY7) 、 21:レプチン受容体、 22:CYP19(ァロマターゼ)、 23:G Protein beta3 subunit(GNB3) 、である。 Each molecule in the table consists of 1: serotonin transporter, 2: monoamine oxidase-8, 3: androgen receptor, 4: tyrosine hydroxylase, 5: serotonin receptor 5-HT2C 6: aroma Amino acid decarboxylase (AADC), 7: donomin D2 receptor DRD2, 8: serotonin receptor 5-HTR3B, 9: serotonin receptor 5-HTR6, 10: GABA type A receptor oc 5 subunit (GABRA5) 11: Dopamine transporter (SLC6A3 (DAT)), 12: Dopamine D4 receptor DRD4, 13: Dopamine D5 receptor DRD5, 14: Phosphatidylinosito 4-phosphate-5-kinase type 2 a, 15 denylate cyclase 9 ( ADCY9), 16: GABA receptor j83 subunit (GABRB3), 17: Adrenergic receptor alpha 2B (ADRA2B) ゝ 18: Adrenergic receptor al pha 2C (ADRA2C), 19: Angiotensin converting enzyme, 20 denylate cyclase 7 (ADCY7), 21: Leptin receptor, 22: CYP19 (aromatase), 23: G Protein beta3 subunit (GNB3).
また表中、略号で示した各関連文献は、以下のとおりである。  In addition, each related document indicated by an abbreviation in the table is as follows.
Dl: Science. 1996 Nov 29;274(5292):1527-31 Dl: Science. 1996 Nov 29; 274 (5292): 1527-31
D2:Am J Med Genet B Neuropsychiatr Genet. 2004 May 15;127(1):104— 12  D2: Am J Med Genet B Neuropsychiatr Genet. 2004 May 15; 127 (1): 104—12
D3: Lancet. 1996 Mar 16;347(9003):731— 3  D3: Lancet. 1996 Mar 16; 347 (9003): 731— 3
D4:Hum Genet. 1998 Sep;103(3):273-9  D4: Hum Genet. 1998 Sep; 103 (3): 273-9
D5:Exp Gerontol. 2004;39:1603-11  D5: Exp Gerontol. 2004; 39: 1603-11
D6:Eur J Pharmacol. 2000 Dec 27;410:215- 226  D6: Eur J Pharmacol. 2000 Dec 27; 410: 215-226
D7:Diabetologia. 2000 Mar;43(3):373- 6  D7: Diabetologia. 2000 Mar; 43 (3): 373-6
D8:J Neural Transm. 2002 May;109:939— 46  D8: J Neural Transm. 2002 May; 109: 939— 46
D9:Hum Mol Genet. 2005 Jun 15;14:1691— 8  D9: Hum Mol Genet. 2005 Jun 15; 14: 1691— 8
D10:Hum. Mol. Genet.1997; 6: 577-582  D10: Hum. Mol. Genet. 1997; 6: 577-582
D11:J. Biol. Chem 1996; 271: 26013-26017  D11: J. Biol. Chem 1996; 271: 26013-26017
D 12 : Pharmacogenetics . 1997 Dec;7(6):479— 84  D 12: Pharmacogenetics. 1997 Dec; 7 (6): 479- 84
D13:J Clin Oncol. 2003 Jun l;21(ll):2147-55  D13: J Clin Oncol. 2003 Jun l; 21 (ll): 2147-55
D14:Schizophr Res. 2002 Nov 1;58(1):93- 7.  D14: Schizophr Res. 2002 Nov 1; 58 (1): 93-7.
D15: Psychiatry Clin Neurosci. 2005 Jun;59(3):345- 9  D15: Psychiatry Clin Neurosci. 2005 Jun; 59 (3): 345-9
D16:Am J Med Genet B Neuropsychiatr Genet. 2003 Apr 1;118(1):36— 40  D16: Am J Med Genet B Neuropsychiatr Genet. 2003 Apr 1; 118 (1): 36— 40
Dl 7: Neurosci Lett. 2005 Jun 10- 17;381(1- 2):108- 13  Dl 7: Neurosci Lett. 2005 Jun 10-17; 381 (1-2): 108-13
D18:J Nucl Med. 2005 May;46(5):745-51 D19 : Biol Psychiatry. 2005 May 1;57(9):999- 1003 D18: J Nucl Med. 2005 May; 46 (5): 745-51 D19: Biol Psychiatry. 2005 May 1; 57 (9): 999-1003
D20： Mol. Psychiatry 2000； 5： 64-69  D20: Mol. Psychiatry 2000; 5: 64-69
D21： Am J Med Genet B Neuropsychiatr Genet. 2004 Feb 15;125(1):38- 42 D21: Am J Med Genet B Neuropsychiatr Genet. 2004 Feb 15; 125 (1): 38- 42
D22 :Am J Med Genet B Neuropsychiatr Genet. 2003 Nov 15;123(1):50— 8 D22: Am J Med Genet B Neuropsychiatr Genet. 2003 Nov 15; 123 (1): 50— 8
D23 : Psychiatr Genet. 2005 Sep;15(3):223- 7.  D23: Psychiatr Genet. 2005 Sep; 15 (3): 223-7.
D24 :Am J Med Genet. 2002 Jan 8;114(1):84- 92  D24: Am J Med Genet. 2002 Jan 8; 114 (1): 84-92
D25 : Psychiatry Res. 2001 Nov 1;104(2):109— 17  D25: Psychiatry Res. 2001 Nov 1; 104 (2): 109-17
D26 :J Clin Endocrinol Metab. 2003 Mar;88(3): 1184-7  D26: J Clin Endocrinol Metab. 2003 Mar; 88 (3): 1184-7
D27 : Br J Clin Pharmacol. 2005 Oct;60(4):414-7  D27: Br J Clin Pharmacol. 2005 Oct; 60 (4): 414-7
D28 : Hypertens Res. 2002 Nov;25(6):843— 8  D28: Hypertens Res. 2002 Nov; 25 (6): 843— 8
D29 :Am J Med Genet. 1997 Feb 21;74(l):95-8  D29: Am J Med Genet. 1997 Feb 21; 74 (l): 95-8
D30 :J Atheroscler Thromb. 2004;l l(2):73-8  D30: J Atheroscler Thromb. 2004; l l (2): 73-8
D31 : Anticancer Res. 2003 Nov— Dec;23(6D):4941— 6  D31: Anticancer Res. 2003 Nov— Dec; 23 (6D): 4941— 6
D32 : Pharmacogenetics 2002, 12:209-220  D32: Pharmacogenetics 2002, 12: 209-220
[0053] 表 4に示した各分子について、複数の遺伝子多型を上記 UGT1A1遺伝子多型の 検査法と同様に検査し、その検査結果を薬剤に対する応答性予測や副作用予測に 利用することも可能である（上記関連文献など参照)。 [0053] For each molecule shown in Table 4, multiple gene polymorphisms can be examined in the same way as the above UGT1A1 gene polymorphism testing method, and the test results can be used for drug response prediction and side effect prediction. (See the above-mentioned related literature).
[0054] 表 4に示す一塩基多型 (第 2、第 3の多型)の検査についても、上記実施例の方法と 同様に、制限酵素を用いた PCR-RFLP法によって行うことができ、必要に応じてミス マッチプライマーを使用し、制限酵素切断部位を作り出すとよい。 [0054] The single nucleotide polymorphisms (second and third polymorphisms) shown in Table 4 can also be examined by PCR-RFLP using restriction enzymes, as in the above-described methods. If necessary, use a mismatch primer to create a restriction enzyme cleavage site.
[0055] 本発明の検査法は、多型部位における 2bpの差異を高精度に検出するため、挿入 部分の有無による多型について、その差異が 10bp以下 (より好ましくは 5bp以下）の 場合に特に優位性の高 ヽ検査法と ヽうことができる。 [0055] The test method of the present invention detects a difference of 2 bp in the polymorphic site with high accuracy, and therefore, especially when the difference is 10 bp or less (more preferably 5 bp or less) for the polymorphism due to the presence or absence of the insertion site. It can be described as a highly superior inspection method.
[0056] また、本発明の検査法を用いて、挿入部分の有無による多型と一塩基多型を同時 に検査する場合に、両多型は同一遺伝子に関連する多型でなくてもよぐ別々の遺 伝子に関連する複数の多型の検査に本発明を適用してもよい。 [0056] Further, when a polymorphism due to the presence or absence of an insertion site and a single nucleotide polymorphism are simultaneously examined using the test method of the present invention, both polymorphisms may not be polymorphisms related to the same gene. The present invention may be applied to examination of a plurality of polymorphisms related to different genes.
産業上の利用可能性  Industrial applicability
[0057] 本発明は、以上のように、 UGT1A1遺伝子多型の検査法等に関するものであり、 前述したとおり、簡易迅速な複数の多型検査に利用できるほか、 UGT1A1で代謝さ れる薬剤の薬効または副作用の程度を調べる検査、診断などに利用することができ る。 [0057] As described above, the present invention relates to a method for testing a UGT1A1 gene polymorphism and the like, As mentioned above, it can be used for multiple quick and simple polymorphism tests, and it can be used for tests, diagnoses, etc. to check the efficacy or side effects of drugs metabolized by UGT1A1.

Claims

請求の範囲 The scope of the claims
[1] UDP-グルクロノシルトランスフェラーゼ 1A1 (UGT1A1)遺伝子に関連する複数 の多型を同時に検査する方法であって、 UGT1A1 * 28に関する多型部位を含む 遺伝子領域のほか、一塩基多型（SNP)が存在する少なくとも 1つの遺伝子領域を同 一の遺伝子増幅反応によって増幅する工程と、得られた増幅断片を制限酵素によつ て消化した後、キヤピラリー電気泳動に供する工程とを含み、キヤビラリ一電気泳動の 結果に基づいて複数の多型を同時に判定する方法。  [1] A method for simultaneously testing multiple polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, which includes a single nucleotide polymorphism (SNP) in addition to the gene region containing the polymorphic site for UGT1A1 * 28. ) Is present by the same gene amplification reaction, and the amplified fragment obtained is digested with restriction enzymes and then subjected to capillary electrophoresis. A method for simultaneously judging multiple polymorphisms based on the results of electrophoresis.
[2] 一塩基多型が存在する遺伝子領域として、 UGT1A1 * 60及び Z又は UGT1A1  [2] UGT1A1 * 60 and Z or UGT1A1 are gene regions where single nucleotide polymorphisms exist
* 6に関する一塩基多型が存在する遺伝子領域を増幅する、請求項 1記載の検査 方法。  2. The test method according to claim 1, wherein a gene region in which a single nucleotide polymorphism relating to 6 exists is amplified.
[3] ミスマッチプライマーを使用して一塩基多型が存在する遺伝子領域を増幅する、請 求項 2記載の検査方法。  [3] The testing method according to claim 2, wherein a mismatched primer is used to amplify a gene region where a single nucleotide polymorphism exists.
[4] UGT1A1 * 60に関する一塩基多型が存在する遺伝子領域について、配列番号 1 の塩基配列を有するフォワードプライマー及び配列番号 2の塩基配列を有するリバ ースプライマーを用いた PCR法による増幅を行うと共に、制限酵素に Hindiを用い ることを特徴とする、請求項 3記載の検査方法。  [4] Amplification by PCR using a forward primer having the nucleotide sequence of SEQ ID NO: 1 and a reverse primer having the nucleotide sequence of SEQ ID NO: 2 for the gene region in which a single nucleotide polymorphism related to UGT1A1 * 60 exists, The test method according to claim 3, wherein Hindi is used as a restriction enzyme.
[5] UGT1A1 * 6に関する一塩基多型が存在する遺伝子領域について、配列番号 5 の塩基配列を有するフォワードプライマー及び配列番号 6の塩基配列を有するリバ ースプライマーを用いた PCR法による増幅を行うと共に、制限酵素に Msplを用いる ことを特徴とする、請求項 3記載の検査方法。  [5] Amplification by PCR using a forward primer having the nucleotide sequence of SEQ ID NO: 5 and a reverse primer having the nucleotide sequence of SEQ ID NO: 6 for the gene region where the single nucleotide polymorphism relating to UGT1A1 * 6 exists, The test method according to claim 3, wherein Mspl is used as a restriction enzyme.
[6] UGT1A1 * 28に関する多型部位を含む遺伝子領域について、配列番号 3の塩基 配列を有するフォワードプライマー及び配列番号 4の塩基配列を有するリバースプラ イマ一を用いた PCR法による増幅を行うことを特徴とする、請求項 1記載の検査方法  [6] Amplification by PCR using a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4 for the gene region containing the polymorphic site related to UGT1A1 * 28 The inspection method according to claim 1, characterized in that
[7] UGT1A1 * 28、 UGT1A1 * 60及び UGT1A1 * 6に関する 3箇所の多型部位を 同時に検査することを特徴とする、請求項 2記載の検査方法。 [7] The inspection method according to claim 2, wherein three polymorphic sites related to UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6 are simultaneously tested.
[8] 目的遺伝子に関連する複数の多型を同時に検査する方法であって、挿入部分の 有無による多型を含む遺伝子領域のほか、一塩基多型（SNP)が存在する少なくとも 1つの遺伝子領域を同一の遺伝子増幅反応によって増幅する工程と、得られた増幅 断片を制限酵素によって消化した後、キヤビラリ一電気泳動に供する工程とを含み、 キヤピラリー電気泳動の結果に基づいて複数の多型を同時に判定する方法。 [8] A method for simultaneously testing a plurality of polymorphisms related to a target gene, wherein at least a single nucleotide polymorphism (SNP) exists in addition to a gene region containing a polymorphism due to the presence or absence of an insertion site A step of amplifying one gene region by the same gene amplification reaction, and a step of digesting the obtained amplified fragment with a restriction enzyme and then subjecting it to a capillary electrophoresis. A method for simultaneously determining polymorphisms.
[9] 請求項 1〜8のいずれか 1項に記載の検査方法を用いて、被検者の薬剤に対する 反応性またはその副作用の程度を予測する方法。  [9] A method for predicting the reactivity of a subject to a drug or the degree of its side effects using the test method according to any one of claims 1 to 8.
[10] 請求項 1〜8のいずれか 1項に記載の検査方法を用いて、被検者の杭がん剤に対 する反応性またはその副作用の程度を予測する方法。 [10] A method for predicting the reactivity of a subject to a pile cancer agent or the degree of its side effects using the inspection method according to any one of claims 1 to 8.
[11] 請求項 1〜8のいずれか 1項に記載の検査方法を用いた、遺伝子多型検査キット。 [11] A genetic polymorphism test kit using the test method according to any one of claims 1 to 8.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011152272A1 (en) * 2010-06-01 2011-12-08 東洋鋼鈑株式会社 Method of determining the risk of adverse effects of irinotecan, and kit therefore
CN110438224A (en) * 2019-07-12 2019-11-12 广州迈景基因医学科技有限公司 A kind of primer, kit and detection method for UGT1A1 genetic polymorphism detection
CN111534591A (en) * 2020-04-30 2020-08-14 北京和合医学诊断技术股份有限公司 Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032042A2 (en) * 1996-03-01 1997-09-04 The University Court Of The University Of Dundee Drug trial assay system
WO2002048400A1 (en) * 2000-12-12 2002-06-20 Nagoya Industrial Science Research Institute Method of estimating risk of the expression of side effect caused by the administration of compound metabolized, either per se or as its metabolic intermediate, by ugt1a1 enzyme
WO2004108954A1 (en) * 2003-05-30 2004-12-16 University Of Chicago Methods and compositions for predicting irinotecan toxicity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032042A2 (en) * 1996-03-01 1997-09-04 The University Court Of The University Of Dundee Drug trial assay system
WO2002048400A1 (en) * 2000-12-12 2002-06-20 Nagoya Industrial Science Research Institute Method of estimating risk of the expression of side effect caused by the administration of compound metabolized, either per se or as its metabolic intermediate, by ugt1a1 enzyme
WO2004108954A1 (en) * 2003-05-30 2004-12-16 University Of Chicago Methods and compositions for predicting irinotecan toxicity

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDO Y. ET AL.: "Polymorphisms of UDP-glucuronosyltransferase gene and irinotecan toxicity: a pharmacogenetic analysis", CANCER RES., vol. 60, no. 24, 2000, pages 6921 - 6926, XP002331169 *
KANIWA N. ET AL.: "Racial variability in haplotype frequencies of UGT1A1 and glucuronidation activity of a novel single nucleotide polymorphism 686C> T (P229L) found in an African-American", DRUG METAB. DISPOS., vol. 33, no. 3, March 2005 (2005-03-01), pages 458 - 465, XP003012790 *
SUGATANI J. ET AL.: "Identification of a defect in the UGT1A1 gene promoter and its association with hyperbilirubinemia", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 292, no. 2, 2002, pages 492 - 497, XP003012789 *
TANABE T. ET AL.: "UGT1A1 Idenshi Tagata no Kanben na Kenshutsu Hoho", JAPANESE JOURNAL OF CLINICAL PHARMACOLOGY, vol. 36, no. SUPPL., 15 November 2005 (2005-11-15), pages S187 (2P-069), XP003012791 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011152272A1 (en) * 2010-06-01 2011-12-08 東洋鋼鈑株式会社 Method of determining the risk of adverse effects of irinotecan, and kit therefore
JP2011250726A (en) * 2010-06-01 2011-12-15 Toyo Kohan Co Ltd Method for determining potential risk of side effect of irinotecan, and kit therefor
CN110438224A (en) * 2019-07-12 2019-11-12 广州迈景基因医学科技有限公司 A kind of primer, kit and detection method for UGT1A1 genetic polymorphism detection
CN111534591A (en) * 2020-04-30 2020-08-14 北京和合医学诊断技术股份有限公司 Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene

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