JPWO2007055261A1 - UGT1A1 gene polymorphism testing method - Google Patents

Abstract

The present invention is a method for simultaneously examining a plurality of polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, which comprises a single nucleotide polymorphism in addition to a gene region containing a polymorphic site for UGT1A1 * 28 ( A result of capillary electrophoresis comprising a step of amplifying at least one gene region in which SNP) is present by the same gene amplification reaction, a step of digesting the obtained amplified fragment with a restriction enzyme, and subjecting the amplified fragment to capillary electrophoresis A plurality of polymorphisms are simultaneously determined based on By the same method, gene polymorphisms other than the UGT1A1 gene can be easily and quickly examined with high accuracy.

Description

  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 UGT1A1 * 28 and others. It is related with the method of test | inspecting the single nucleotide polymorphism (SNP) which is an important polymorphism of (1) simply, quickly and accurately. The present invention provides a technique useful for, for example, a test for examining the efficacy or side effects of a drug metabolized by UGT1A1.

  UDP-glucuronosyltransferase 1A1 (hereinafter referred to as “UGT1A1”), which is an in vivo conjugate enzyme, is an enzyme involved in the metabolism of bilirubin and foreign drugs. There have been several reports on the presence of gene polymorphisms affecting the expression level and enzyme activity of this enzyme (Non-Patent Documents 1 to 4 below). Furthermore, some UGT1A1 gene polymorphisms have been reported to be related to side effects of irinotecan, which is an anticancer drug, or to neonatal jaundice, which is relatively common in Japanese (Non-Patent Documents 5 to 7 below). ).

  Up to now, more than 30 gene polymorphisms have been reported in the UGT1A1 gene. Here, six gene polymorphisms considered to be clinically important will be described. One is a polymorphism related to UGT1A1 * 28 existing in the promoter region upstream of the UGT1A1 gene. In the UGT1A1 * 28 allele, the number of repeats of the TA sequence in the promoter region TATA box is 6 times in the wild type allele (UGT1A1 * 1 allele) occupying a large number, and the repeat sequence is 7 times. Since it is an allele having a TA sequence inserted at −40 to −39 on the genome, it is also expressed as “-40_-39insTA”. (Translation start point of UGT1A1 gene is “+1”.)

  The other five gene polymorphisms are polymorphisms related to UGT1A1 * 6, UGT1A1 * 27, UGT1A1 * 29, UGT1A1 * 7, and UGT1A1 * 60. These five gene polymorphisms are all single nucleotide polymorphisms (SNPs) and exist in exons except for the polymorphism related to UGT1A1 * 60, and cause amino acid substitution in the translation product. In the UGT1A1 * 6 allele, the 211th base on exon 1 which is guanine (G) in the wild type is adenine (A), and the polymorphic site is represented 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 represented as “C686A”. In the UGT1A1 * 29 allele, the 1099th base on exon 4 which is cytosine (C) in the wild type is guanine (G), and the polymorphic site is represented as “C1099G”. 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 represented as “T1456G”.

  The UGT1A1 * 60 allele is thymine (T) in the wild type—the 3279th base is guanine (G), and the polymorphic site is denoted as “T-3279G”. (It may be expressed as “T-3263G”.) Thus, the polymorphic site of UGT1A1 * 60 allele is located further upstream than the polymorphic site of UGT1A1 * 28 existing in the promoter region. It has become.

  All of the six genetic polymorphisms are considered to be clinically important, but their presence has been confirmed in Japanese, and clinically particularly important are UGT1A1 * 28, UGT1A1 * 6, and UGT1A1 *. There are 60 polymorphs. In particular, it is said that when a patient with UGT1A1 * 28 receives irinotecan, the risk of side effects increases, but as described above, UGT1A1 * 28 has one repeat of the TA sequence in the TATA box. Since 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 the ordinary PCR-RFLP method or the like.

  As a method for detecting the polymorphism of UGT1A1 * 28, detection examples by the melting curve method, the direct sequence method, and the pyrosequence 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 device manufactured by Roche, and the time required for detecting the polymorphism is short, but the equipment cost is initially high. The direct sequencing method is a method in which a PCR product is directly sequenced by a sequencer and a polymorphism is detected. The determination is accurate, but it takes cost, time and labor. The pyrosequencing method is a method for detecting a polymorphism by observing chemiluminescence, detecting complementary strand synthesis from the luminescence, and sequencing a DNA molecule. This method is used in Biotage's equipment and requires a short equipment cost, although the required time is short.

In addition, any of the above methods is a method for detecting each polymorphism individually. Together with the polymorphism of UGT1A1 * 28, other clinically important single nucleotide polymorphisms can be easily and quickly and accurately obtained at low cost. Currently, no detection method has been established yet.
N Engl J Med 1995; 333: 1171-5 Biochim Biophys Acta 1998; 1406: 267-73 Drug Metab Dispos 2003; 31: 108-13 Biochem Biophys Res Commun 2002; 292: 492-7 Cancer Res 2000; 60: 6921-6 Pharmacogenet Genomics 2005; 15: 35-41 Pediatrics 1999; 103: 1224-7

  The present invention has been made paying attention to the above-mentioned problems, and the first object thereof is to provide a method for simultaneously testing a plurality of polymorphisms related to the UGT1A1 gene, and more specifically, UGT1A1. * To provide a simple, quick and accurate method for examining other single nucleotide polymorphisms clinically important together with the 28 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 nucleotide sequence is inserted and a deletion lacking the same. The object is to provide a method for simply and quickly and accurately inspecting a plurality of polymorphs comprising a combination of a polymorphism between types and a single nucleotide polymorphism at a low cost.

  As mentioned above, the polymorphism for UGT1A1 * 28 is only 2 bases in length and needs to be detected visually. Therefore, an attempt was made to detect a difference in length of two bases by shortening the PCR product to 100 bp or less and using high-resolution capillary electrophoresis. Furthermore, by using the same gene amplification reaction and the same electrophoresis, it was attempted to simultaneously examine two other single nucleotide polymorphisms (UGT1A1 * 6 and UGT1A1 * 60 polymorphisms) that are clinically important. Therefore, a mismatch primer was used for amplification of a gene region containing each single nucleotide polymorphism to create a restriction enzyme cleavage site that is cleaved according to the base of the polymorphic site. In addition, conditions for simultaneously amplifying three gene regions with three sets of primers were examined. As a result, it has been found that multiple polymorphisms can be simultaneously and easily tested with high accuracy by performing capillary electrophoresis after amplifying three gene regions in the same PCR reaction and digesting with a restriction enzyme. The invention has been completed.

That is, the present invention is, as industrially and medically-medically useful inventions, it is intended to embrace the following inventions A) ~K).
A) A method for simultaneously examining a plurality of polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, which includes a single nucleotide polymorphism (SNP) in addition to a gene region containing a polymorphic site for UGT1A1 * 28 And a step of amplifying at least one gene region in which the DNA is present by the same gene amplification reaction, a step of digesting the obtained amplified fragment with a restriction enzyme, and then subjecting it to capillary electrophoresis, based on the result of capillary electrophoresis To determine multiple polymorphisms simultaneously.
B) The testing method according to A), wherein a gene region in which a single nucleotide polymorphism relating to UGT1A1 * 60 and / or UGT1A1 * 6 exists is amplified as a gene region in which a single nucleotide polymorphism exists.
C) The test method according to B) above, wherein a mismatched primer is used to amplify a gene region where a single nucleotide polymorphism exists.
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 with respect to the gene region where the single nucleotide polymorphism relating to UGT1A1 * 60 exists is restricted. The test method according to C) above, wherein HincII is used as an enzyme.
E) 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 with respect to the gene region where the single nucleotide polymorphism relating to UGT1A1 * 6 exists is restricted. The test method according to C) above, wherein MspI is used as an enzyme.
F) A gene region including a polymorphic site related to UGT1A1 * 28 is amplified by a PCR method using a forward primer having a base sequence of SEQ ID NO: 3 and a reverse primer having a base sequence of SEQ ID NO: 4. The inspection method according to A) above.
G) The inspection method according to the above B), wherein three polymorphic sites relating to UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6 are simultaneously tested.
H) A method for simultaneously examining a plurality of polymorphisms related to a target gene, wherein at least one gene region containing a single nucleotide polymorphism (SNP) is included in addition to a gene region containing a polymorphism due to the presence or absence of an insertion portion. It includes a step of amplifying by the same gene amplification reaction and a step of digesting the obtained amplified fragment with a restriction enzyme and then subjecting it to capillary electrophoresis, and simultaneously judging a plurality of polymorphisms based on the result of capillary electrophoresis Method.
I) A method for predicting the reactivity of a subject to a drug or the degree of side effects thereof using the test method according to any one of A) to H).
J) A method for predicting the reactivity of a subject to an anticancer agent or the degree of its side effects using the test method according to any one of A) to H) above.
K) A genetic polymorphism test kit using the test method according to any one of A) to H) above.

  According to the present invention, after a gene region containing a polymorphic site for UGT1A1 * 28 and at least one gene region in which a single nucleotide polymorphism exists are amplified by the same gene amplification reaction, the base difference in the single nucleotide polymorphism The digestion reaction is carried out with a restriction enzyme so that DNA fragments with different lengths can be obtained depending on the conditions. Thereafter, the DNA fragments in each sample are separated and detected according to the fragment length by capillary electrophoresis. Capillary electrophoresis has high resolution, so it is possible to detect the difference in length of two bases in UGT1A1 * 28 polymorphism, and typing of single nucleotide polymorphism from the length of other DNA fragments detected at the same time It is. In this way, a plurality of clinically important polymorphisms of UGT1A1 gene can be examined and determined with high accuracy by a series of simple and rapid methods such as amplification reaction by PCR, digestion reaction by restriction enzyme, and capillary electrophoresis. Can do. Capillary 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 low-cost inspection.

  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 research results have been reported that there is a correlation between the side effect of the anticancer drug irinotecan and the UGT1A1 gene polymorphism (Non-patent Documents 5 and 6, etc.). Based on such research results, The degree of side effects when irinotecan is administered can be predicted from the test results according to the present invention. Although the present invention examines a plurality of gene polymorphisms at the same time, it is preferable to predict drug side effects and drug efficacy based on a plurality of gene polymorphisms, and an appropriate drug suitable for individual patients based on genomic information. It contributes to the realization of treatment, and can contribute to improvement of medical quality, safety of medicine and improvement of treatment efficiency.

  By testing for UGT1A1 gene polymorphism, it can be determined whether or not it has a genetic predisposition affecting serum high levels of bilirubin, that is, high serum bilirubin levels without abnormal liver function test values such as AST and ALT It is possible to explain it. The inspection method of the present invention can also be used for such inspection.

  Furthermore, according to the present invention, it is possible to provide a method for simply and quickly and accurately testing a plurality of polymorphisms related to a target gene at low cost by applying the above-described testing method to other gene polymorphism testing methods. it can.

It is a gel image which shows the result of capillary electrophoresis in the test | inspection method of UGT1A1 gene polymorphism which concerns on the Example of this invention. (A)-(c) is a graph which shows the result of the said capillary electrophoresis.

  Hereinafter, preferred embodiments of the present invention will be described. In the present specification and drawings, when bases, amino acids, etc. are represented by abbreviations, the representation is based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or common abbreviations in the field, for example, the representation of DNA bases. Is as follows.

  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.

  In addition, numbers, numerical values, and other information related to gene polymorphisms, gene sequences, etc. are used in major gene databases available on the National Center for Biotechnology Information (NCBI) website, etc. Interpretation shall be made in consideration of numbers and numerical values.

[1] UGT1A1 gene polymorphism testing method First, the UGT1A1 gene polymorphism testing method (Example) actually performed by the present inventors will be described.
[Method for testing UGT1A1 gene polymorphism of the present invention]
Using a test method of the present invention, a plurality of polymorphisms related to UGT1A1 gene were simultaneously examined and determined for 155 samples derived from Japanese. In this example, three polymorphisms related to UGT1A1 * 60, UGT1A1 * 28, and UGT1A1 * 6 were examined, and the PCR method was used to amplify a 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 as shown in Table 1 below.

  In the column of “Sequence” in Table 1, the numbers in parentheses indicate the SEQ ID NOs in the sequence listing in which the primer sequences are described. The underlined base is an artificially inserted base in order to create a restriction enzyme cleavage site. Thus, a mutation primer (mismatch primer) was prepared for one of the primers for UGT1A1 * 60 and UGT1A1 * 6, and PCR reaction was performed using this.

  The PCR reaction was performed by putting six primers shown in Table 1 in one tube. The reaction solution (20 μL) is 60 ng genomic DNA, 1.0 unit AmpliTaq DNA polymerase, 0.25 μM UGT1A1 * 60 primer, 0.2 μM UGT1A1 * 28 primer, 0.075 μM UGT1A1 * 6 primer , Each containing 0.2 mM deoxynucleotide triphosphate. PCR conditions are as follows: (1) 94 ° C for 5 minutes, (2) 94 ° C for 30 seconds, 55 ° C for 40 seconds, 72 ° C for 40 seconds as one cycle (see Table 1). Went.

As described above, three sets of primers including two mismatched primers were mixed with the reaction solution and PCR was performed. Then, the low PCR buffer and the restriction enzymes HincII and MspI were added to the obtained PCR product at 37 ° C. Digestion reaction was performed for 1 hour. The reaction time may be about 30 minutes. After digestion reaction with restriction enzymes, each reaction solution is subjected to electrophoresis using a simplified multi-sample capillary electrophoresis apparatus “HDA GT-12 ^™ (eGene)”, and the DNA fragments in each reaction solution are sized ( According to the fragment length).

  Regarding the polymorphic site of UGT1A1 * 6, when it is the wild type allele “W”, since the polymorphic site is cleaved by the restriction enzyme MspI, the fragment lengths are 203 bp and 32 bp as shown in Table 1. On the other hand, in the case of the mutant allele “M”, since the polymorphic site is not cleaved by the restriction enzyme MspI, the fragment length is 235 bp.

  Regarding the polymorphic site of UGT1A1 * 60, when it is the wild type allele “W”, the polymorphic site is not cleaved by the restriction enzyme HincII, so that the fragment length is 115 bp and 93 bp as shown in Table 1 (amplified fragment) (There is one restriction enzyme cleavage site in it). On the other hand, in the case of the mutant allele “M”, the polymorphic site is cleaved by the restriction enzyme HincII, so that the fragment length becomes 115 bp, 67 bp and 26 bp.

  When the polymorphic site of UGT1A1 * 28 is not cleaved by a restriction enzyme and the double base repeat sequence (TA repeat) is a mutant allele “M” which is one more than the wild type allele “W”, Table 1 As shown in FIG. 5, the fragment length is 85 bp, and when the wild type allele “W”, the fragment length is 83 bp.

[Judgment by the conventional method for checking the accuracy of the inspection method of the present invention]
In order to confirm the accuracy of the inspection method of the present invention, conventional methods such as PCR-RFLP method, melting curve method, direct sequence method and the like were used for inspection and determination of each polymorphism. In addition to the three polymorphisms UGT1A1 * 60, UGT1A1 * 28, and UGT1A1 * 6, the inspection target is a total of 6 polymorphisms including UGT1A1 * 7, UGT1A1 * 27, and UGT1A1 * 29, which are relatively minor polymorphisms. It is a type. About these 6 polymorphisms, the determination method by the PCR method reported so far was arranged, and each polymorphism was determined individually. (1 cycle setting of the amplification process) each polymorphism using the determined primers and PCR conditions are as described in Table 2 below.

  As in Table 1, the numbers in parentheses in the “Sequence” column indicate the SEQ ID NOs of the Sequence Listing in which the primer sequences are described. The underlined base is an artificially inserted base in order to create a restriction enzyme cleavage site. About 5 polymorphisms except UGT1A1 * 28, it 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). In addition, when the result was not clear by this determination method, the genotype of UGT1A1 * 28 was determined using the direct sequence method.

  The UGT1A1 * 6 allele was determined using a method slightly improved from the previously reported method (Cancer Res 2000; 60: 6921-6). In this determination, as described above, a mismatch primer having an MspI 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 having Cfr13I and DraI recognition sites were designed 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 BsrI according to the method already reported (Cancer Res 2000; 60: 6921-6).

  Further, when both UGT1A1 * 28 and UGT1A1 * 6 are heterogeneous, or when both UGT1A1 * 28 and UGT1A1 * 27 are heterogeneous, UGT1A1 * 6 or UGT1A1 * is used to determine the haplotype of the subject. 27, an allele specific PCR method (Allele specific PCR method) was performed 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 contains 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 150 ng genomic DNA, 2.5 units AmpliTaq DNA polymerase, 200 nM primers each, 0.2 mM deoxynucleotide triphosphates 50 μL of PCR reaction solution was used. The primers used and PCR conditions (setting of one cycle in the amplification process) are as shown in Table 3 below.

  In the reverse primer (W) for detecting the wild type allele, only the wild type allele is amplified, whereas in the reverse primer (M) for detecting the mutant type allele, only the mutant type allele is amplified. The haplotype was determined together with the determination result of UGT1A1 * 28 in each amplified fragment.

[result]
As a result of this example, the result of simultaneously determining a plurality of polymorphisms by the test method of the present invention and the result of individually determining each polymorphism by the conventional method for 155 samples derived from Japanese completely coincided. FIG. 1 is a gel image showing a part of the result of capillary electrophoresis according to the test method of the present invention, and FIGS. 2A to 2C are graphs showing a part of the result of 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 capillary electrophoresis. . At the same time, it was possible also to accurately detect the single nucleotide polymorphism relates UGT1A1 * 60 and UGT1A1 * 6.

  No subject carrying the UGT1A1 * 7 allele and the UGT1A1 * 29 allele was present in the 155 samples investigated this time. Moreover, it was confirmed from the analysis result by the allele specific PCR method that the UGT1A1 * 27 allele is included in the UGT1A1 * 28 allele.

  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 easily and quickly and accurately obtained by the test method of the present invention. It was found that inspection / judgment is possible.

[2] Modification of Inspection Method of the Present Invention 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, in the above method, three polymorphisms of UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6 are inspected at the same time, but the present invention is not limited to the examination of these three polymorphisms. Absent. 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, UGT1A1 * 29, UGT1A1 * 7, etc. In the present invention, single nucleotide polymorphisms to be tested simultaneously with UGT1A1 * 28 polymorphisms are these polymorphisms. it may be. Thus, when testing single nucleotide polymorphisms such as UGT1A1 * 27, UGT1A1 * 29, UGT1A1 * 7, etc. simultaneously with the polymorphisms of UGT1A1 * 28, for example, PCR reaction is performed using the primer sequences listed in Table 2. Thus, a gene region containing a single nucleotide polymorphism can be amplified. The genome sequence of UGT1A1 gene is registered / published in gene sequence databases such as GenBank with accession number “NG_002601” (GeneID: 54658), and the UGT1A1 cDNA sequence is registered with accession number “NM_000463”, etc. Each primer may be designed with reference to the nucleotide sequences disclosed in these databases.

  Although the method of the said Example was test | inspected 3 polymorphism simultaneously, this invention may test | inspect 2 polymorphism simultaneously. In this case, in addition to the polymorphism related to UGT1A1 * 28, other single nucleotide polymorphisms clinically important 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) For UGT1A1 * 28, the amplification fragment was set to 100 bp or less in order to detect a difference of 2 bp, and (2) each DNA fragment after digestion with restriction enzyme (3) Consideration was made so that polymorphism could be easily detected from the relative distance and positional relationship of each DNA fragment. 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 of UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6, it is preferable to use 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 it should be noted that three gene regions can be amplified under the same reaction conditions.

  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 any organ / tissue of the subject. DNA may be purified and extracted from cells (including blood, cells in amniotic fluid, and cells obtained by culturing collected tissues) according to a conventional method. In addition, as long as gene amplification by PCR method (or other gene amplification method) is possible, the DNA purification / extraction step may be omitted or simplified.

  When a genomic region in a sample prepared from a subject is used as a template and a gene region containing a polymorphic site is amplified by the PCR method, the reaction conditions in the PCR method, the composition of the reagents and reaction solution used, the equipment used, etc. Although not particularly limited to the method of the above example, it is advisable to pay attention to the reaction conditions so that the entire target gene region is amplified. Since the PCR method is a simple method and has good accuracy, the PCR method is preferably used for the gene amplification method, but other amplification methods (for example, the RCA method, etc.) other than the PCR method may be used. (See "Post-Sequence Genomic Science (1) SNP Gene Polymorphism Strategies" (Nakayama Shoten)).

  Regarding the digestion reaction using restriction enzymes, the type of buffer, reaction temperature, reaction time, etc. may be determined according to the type of restriction enzyme used. It is preferable to use two types of restriction enzymes at the same time and detect two types of SNPs by the PCR-RFLP method as in the method of the above example.

  Capillary electrophoresis separates and detects DNA at a higher resolution than ordinary gel electrophoresis by electrophoresis of DNA in the capillary, and can be performed using a commercially available apparatus. Any device may be used as long as it can detect a 2 bp difference in the polymorphism of UGT1A1 * 28.

  The gene polymorphism test kit using the test method of the present invention may be any kit including primers designed for testing a plurality of polymorphisms related to the UGT1A1 gene. (1) Preparation of sample It may contain one or more of (2) enzymes and reagents used in PCR, (2) enzymes and reagents used in PCR, and (3) enzymes and reagents used in restriction enzyme digestion reactions. When testing three polymorphisms of 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, but the length of each primer May be longer or shorter by about 1 to 5 bases. Moreover, when testing a plurality of polymorphisms in other combinations, a kit configuration including one or a plurality of sets of primers selected from the primer group listed in Table 2 may be used. 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 present in the UGT1A1 gene or in the vicinity thereof on the genome. It means polymorphism, and it is not limited to the examination of polymorphism existing in exon region or intron region of UGT1A1 genomic gene, but it examines polymorphism existing in transcription regulatory region such as promoter region, control region, etc. Also good. In the present specification, “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.

  By conducting a polymorphism test of the UGT1A1 gene according to the present invention, it is possible to predict the efficacy or side effect of a drug metabolized by UGT1A1. For example, UGT1A1 * 28 and UGT1A1 * 60 alone and UGT1A1 * 6 together with UGT1A1 * 28 have been reported to increase the risk of irinotecan side effects. Therefore, it is possible to predict patients who are likely to cause serious side effects of irinotecan.

[3] Method for testing gene polymorphisms other than UGT1A1 gene The test method of the present invention can also be applied to testing for gene polymorphisms other than UGT1A1 gene, and a plurality of polymorphisms related to the target gene can be obtained at low cost. A simple and quick inspection method can be provided.

  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 part (an insertion type in which a certain base sequence of 2 or more bases is inserted and a lack of the base sequence). The present invention can also be applied to a case where a plurality of polymorphisms composed of a combination of a single nucleotide polymorphism (SNP) and a single nucleotide polymorphism (SNP) are simultaneously examined. Examples of such genes having a plurality of polymorphisms include the following. (In Table 4 below, molecular names encoded by each gene are shown.)

  Each molecule in the table includes 1: serotonin transporter, 2: monoamine oxidase A, 3: androgen receptor, 4: tyrosine hydroxylase, 5: serotonin receptor 5-HT2C, 6: aromatic amino acid decarboxylase (AADC) , 7: dopamine D2 receptor DRD2, 8: serotonin receptor 5-HTR3B, 9: serotonin receptor 5-HTR6, 10: GABA type A receptor α5 subunit (GABRA5), 11: dopamine transporter (SLC6A3 ( DAT)), 12: Dopamine D4 receptor DRD4, 13: Dopamine D5 receptor DRD5, 14: Phosphatidylinositol-4-phosphate-5-kinase type 2 α, 15: Adenylate cyclase 9 (ADCY9), 16: GABA receptor β3 Subunit (GABRB3), 17: Adrenergic receptor alpha 2B (ADRA2B), 18: Adrenergic receptor alpha 2C (ADRA2C), 19: Angiotensin converting enzyme, 20: Adenylate cyclase 7 (ADCY7), 21: Leptin receptor, 22: CYP19 (Aromatase), 23: G Protein beta3 subunit (GNB3) .

In addition, each related document indicated by an abbreviation in the table is as follows.
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  About each molecule | numerator shown in Table 4, it is also possible to test | inspect a some gene polymorphism similarly to the test | inspection method of the said UGT1A1 gene polymorphism, and to utilize the test result for the responsiveness prediction with respect to a chemical | medical agent or a side effect prediction ( (See related documents above).

  The single nucleotide polymorphisms (second and third polymorphisms) shown in Table 4 can also be examined by the PCR-RFLP method using a restriction enzyme, as in the above examples, and if necessary Use a mismatch primer to create a restriction enzyme cleavage site.

  Since the inspection method of the present invention detects a difference of 2 bp in the polymorphic site with high accuracy, the polymorphism due to the presence or absence of the insertion portion is particularly advantageous when the difference is 10 bp or less (more preferably 5 bp or less). It can be said that it is a high inspection method.

  In addition, when using the test method of the present invention to simultaneously examine a polymorphism due to the presence or absence of an insertion site and a single nucleotide polymorphism, both polymorphisms may not be polymorphisms related to the same gene; The present invention may be applied to inspection of a plurality of polymorphisms related to.

  As described above, the present invention relates to UGT1A1 gene polymorphism testing methods and the like. As described above, the present invention can be used for simple and rapid polymorphism testing, as well as the efficacy or side effects of drugs metabolized by UGT1A1. It can be used for examination, diagnosis, etc. to check the degree.

Claims (11)

  A method for simultaneously testing a plurality of polymorphisms related to the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, wherein there is a single nucleotide polymorphism (SNP) in addition to a gene region containing a polymorphic site for UGT1A1 * 28 A step of amplifying at least one gene region by the same gene amplification reaction, a step of digesting the obtained amplified fragment with a restriction enzyme, and then subjecting the amplified fragment to capillary electrophoresis. A method for simultaneously determining polymorphisms.   The test method according to claim 1, wherein a gene region in which a single nucleotide polymorphism relating to UGT1A1 * 60 and / or UGT1A1 * 6 exists is amplified as a gene region in which a single nucleotide polymorphism exists.   The test method according to claim 2, wherein a gene region in which a single nucleotide polymorphism exists is amplified using a mismatch primer.   The gene region where a single nucleotide polymorphism related to UGT1A1 * 60 exists is amplified by a PCR method 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, and is used as a restriction enzyme. The inspection method according to claim 3, wherein HincII is used.   The gene region where a single nucleotide polymorphism related to UGT1A1 * 6 exists is amplified 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, and is used as a restriction enzyme. The inspection method according to claim 3, wherein MspI is used.   A gene region containing a polymorphic site related to UGT1A1 * 28 is amplified by a PCR method using a forward primer having a base sequence of SEQ ID NO: 3 and a reverse primer having a base sequence of SEQ ID NO: 4. Item 1. The inspection method according to Item 1.   3. The inspection method according to claim 2, wherein three polymorphic sites related to UGT1A1 * 28, UGT1A1 * 60 and UGT1A1 * 6 are simultaneously tested.   A method for simultaneously examining a plurality of polymorphisms related to a target gene, wherein at least one gene region containing a single nucleotide polymorphism (SNP) is identical to a gene region containing a polymorphism due to the presence or absence of an insertion portion. A method of simultaneously judging a plurality of polymorphisms based on a result of capillary electrophoresis, comprising a step of amplifying by a gene amplification reaction, and a step of digesting the obtained amplified fragment with a restriction enzyme and then subjecting it to capillary electrophoresis.   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 claim 1.   A method for predicting the reactivity of a subject to an anticancer agent or the degree of its side effects, using the test method according to claim 1. A genetic polymorphism test kit using the test method according to claim 1.