JP2011250726A - Method for determining potential risk of side effect of irinotecan, and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out determination more precisely when determining the potential risk of the side effects of irinotecan by detecting the genetic polymorphism of UDP-glucuronosyltransferase.SOLUTION: A method for determining the potential risk of the side effects of the irinotecan includes determining that to which type in a normal type, a hetero type, and a variant type the genetic polymorphism of the UDP-glucuronosyltransferase in a subject belongs, by a hybridization method of a pair of nucleic acid probes for determining whether the following genetic polymorphisms: UGT1A1*28; UGT1A1*6; UGT1A1*27; and UGT1A1*60 in the UDP-glucuronosyltransferase gene are the wild type or the variant type, with an amplified nucleic acid obtained by an amplification reaction using a genomic DNA derived from a biological sample of the subject as a template.

Description

  The present invention relates to a method for determining the risk of occurrence of side effects of irinotecan and a kit therefor.

  Irinotecan (CPT-11) is an anticancer agent synthesized from camptothecin, which is an antitumor alkaloid derived from Kanlenboku, and is known to be useful for treating cancers such as lung cancer and metastatic colorectal cancer. Irinotecan exhibits an excellent anticancer effect by inhibiting the enzyme topoisomerase that promotes DNA replication, but as a side effect, it has also been reported to have great toxicity such as leucopenia and diarrhea.

  Glucuronidase (UDP-glucuronosyltransferase: UGT) is an enzyme that catalyzes the reaction of adding glucuronic acid to drugs, foreign substances or endogenous substances such as bilirubin, steroid hormones, bile acids, etc. It is known that genetic polymorphism exists in UGT1A1, which is one of the above.

  The UGT1A1 gene polymorphism has been reported to be involved in the expression of side effects of irinotecan (CPT-11) as an anticancer agent. That is, it has been reported that a person having a UGT1A1 gene polymorphism that causes a decrease in UGT activity has an increased risk of serious side effects such as leukopenia and severe diarrhea. UGT1A1 * 28, one of the UGT1A1 gene polymorphisms, has 7 repeats compared to 6 repeats in the wild type (UGT1A1 * 1), which has a large number of TA sequences in the promoter region TATA box. This gene polymorphism is reduced, and the expression level of the gene decreases due to this TA2 base insertion, resulting in a decrease in UGT activity.

  The UGT1A1 gene has at least nine isoforms of UGT1A1, UGT1A3 to UGT1A10. As for each isoform, various gene polymorphisms are known as in the case of the UGT1A1 gene described above. Some of these gene polymorphisms affect the enzyme activity of UGT and the expression level of genes, and some have been reported to be involved in the expression of irinotecan side effects.

  On the other hand, for the purpose of diagnosing the side effects of irinotecan, the UGT1A1 gene polymorphism diagnostic kit (TWT, USA) based on the Invader method has been put into practical use as a diagnostic agent. However, these methods have a problem that the discrimination accuracy is low and the analysis cost is high. Patent Document 1 discloses a method for efficiently determining the UGT1A1 * 28 polymorphism by a hybridization method using a nucleic acid probe corresponding to the UGT1A1 * 28 polymorphism in the UGT1A1 gene.

JP 2008-072913 A

  An object of the present invention is to provide a means capable of determining with higher accuracy when determining the risk of side effects caused by irinotecan by analyzing a polymorphism of a glucuronidase gene.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the risk of side effects caused by irinotecan is further increased by combining specific polymorphisms among various polymorphisms of the glucuronidase gene. The inventors have found that the determination can be made with high accuracy, and have completed the present invention. The present invention includes the following.

(1) The following gene polymorphisms in the glucuronidase gene
UGT1A1 * 28
UGT1A1 * 6
UGT1A1 * 27
UGT1A1 * 60
And a pair of nucleic acid probes for determining these wild types and an amplified nucleic acid obtained by an amplification reaction using a genomic DNA derived from the subject's biological sample as a template, A method for determining whether a genetic polymorphism is a normal type, a hetero type, or a mutant type, and determining a risk of occurrence of a side effect due to irinotecan based on the determination result.

(2) The following gene polymorphisms in the glucuronidase gene
UGT1A7 * 12 (-57)
UGT1A7 * 2
UGT1A9 * 1b
And a pair of nucleic acid probes corresponding to these wild types, and further determining whether each of these gene polymorphisms is normal, heterozygous or mutant, and based on the determination result, the side effects of irinotecan The method according to (1), wherein the risk of occurrence is determined.

  (3) The hybridization between the nucleic acid probe corresponding to the mutant type and the amplified nucleic acid and the hybridization between the nucleic acid probe corresponding to the wild type and the amplified nucleic acid are measured by the intensity value of the label incorporated in the amplified nucleic acid. The method according to (1) or (2).

  (4) The intensity value derived from the amplified nucleic acid hybridized to the nucleic acid probe corresponding to the mutant type corresponds to the intensity value derived from the amplified nucleic acid hybridized to the nucleic acid probe corresponding to the mutant type and the wild type. Dividing by the average value of the intensity values derived from the amplified nucleic acid hybridized to the nucleic acid probe to calculate a judgment value, and comparing the judgment value with a predetermined threshold A and threshold B (threshold A> threshold B), When the judgment value exceeds a threshold A, it is judged as a mutant type, when the judgment value is below a threshold A and exceeds a threshold B, it is judged as a hetero type, and when the judgment value is below a threshold B, it is judged as a wild type ( 3) The method as described.

(5) The concentration of the pair of primers for amplifying the region containing UGT1A1 * 28 is 0.06 to 1.5 μmol / l,
The concentration of a pair of primers for amplifying the region containing UGT1A1 * 6 is 0.06 to 1.5 μmol / l,
The concentration of a pair of primers for amplifying the region containing UGT1A1 * 27 is 0.01 to 0.35 μmol / l,
The method according to (1), wherein the concentration of the pair of primers for amplifying the region containing UGT1A1 * 60 is 0.01 to 0.35 μmol / l, respectively.

(6) The concentration of the pair of primers for amplifying the region containing UGT1A7 * 12 (-57) is 0.08 to 2.00 μmol / l,
The concentration of a pair of primers for amplifying the region containing UGT1A7 * 2 is 0.08 to 2.00 μmol / l,
The method according to (2), wherein the concentration of the pair of primers for amplifying the region containing UGT1A9 * 1b is 0.12 to 3.00 μmol / l, respectively.

  (7) The method according to (1) or (2), wherein the concentration of genomic DNA serving as a template in the amplification reaction is 50 to 500 pg / μl.

  (8) The method according to (1) or (2), wherein the temperature of the hybridization solution is 52 to 56 ° C.

  (9) The method according to (3), wherein the label is a fluorescent substance.

  (10) The fluorescent substance is bound to one type of base of the substrate in the amplification reaction, and the amount of the base to which the fluorescent substance is bound is larger than that of the other three types of bases. The method according to (9), wherein the ratio is 1/25 to 2/5.

(11) The following gene polymorphisms in the glucuronidase gene
UGT1A1 * 28
UGT1A1 * 6
UGT1A1 * 27
UGT1A1 * 60
And a kit for discriminating the risk of side effects caused by irinotecan, comprising a pair of nucleic acid probes for judging these wild types.

(12) The following gene polymorphisms in the glucuronidase gene
UGT1A7 * 12 (-57)
UGT1A7 * 2
UGT1A9 * 1b
And a kit according to (11), further comprising a pair of nucleic acid probes corresponding to these wild types.

  (13) The kit according to (11) or (12), comprising a DNA chip having the pair of nucleic acid probes fixed on a substrate.

  The kit for determining the risk of occurrence of side effects due to irinotecan according to the present invention may include a primer that amplifies the DNA to be detected contained in the specimen. That is, the kit according to the present invention may include a primer that specifically amplifies the DNA to be detected contained in the specimen, and the nucleic acid probe that specifically hybridizes with the amplified DNA. The kit according to the present invention includes various reagents necessary for amplifying DNA and / or various reagents necessary for specifically hybridizing the amplified DNA and the nucleic acid probe. Also good.

  According to the present invention, a predetermined polymorphism among polymorphisms of glucuronidase gene can be identified with high accuracy, and the risk of occurrence of side effects due to irinotecan can be determined with higher accuracy.

It is a schematic diagram which shows the position of the nucleic acid probe mounted in the DNA chip.

  The present invention is a method for determining with high accuracy the risk of side effects caused by irinotecan by specifying a predetermined gene polymorphism in a glucuronidase gene. Here, the gene polymorphism means both a polymorphism present in a protein-coding region and a polymorphism present in a gene expression control region.

  Irinotecan (CPT-11), 1,4'-bipiperidine-1'-carboxylic acid (S) -4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-1H -Pyrano [3 ', 4': 6,7] Indolizino [1,2-b] quinolin-9-yl ester (CAS NO: 97682-44-5) is derived from camptothecin, an antineoplastic alkaloid derived from canrenboku. It is a synthesized compound. In the present invention, irinotecan also includes salts thereof and solvates thereof, particularly hydrates (for example, CAS NO: 136572-09-3). As a salt of irinotecan, an acid addition salt in which a pharmaceutically acceptable acid is allowed to act is preferably used as an anticancer agent. Examples of such acid addition salts include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid; oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, acetic acid And salts with organic acids such as p-toluenesulfonic acid and methanesulfonic acid, and particularly hydrochloride (irinotecan hydrochloride; CAS NO: 136572-09-3) is preferably used.

  Irinotecan is converted to the active metabolite SN-38 by carboxylesterase after in vivo administration. SN-38 is detoxified in the liver by a conjugation reaction with glucuronidase (UDP-glucuronyltransferase; UGT) and then excreted in the intestine. And the relative balance of UGT activity related to SN-38 detoxification affects the amount of SN-38 in the body, and as a result, the amount of SN-38 exposure received by the body causes individual differences in side effects. It is believed that It has been clarified that the glucuronidation reaction, which is a detoxification process of SN-38, is mainly catalyzed by UGT1A1, a molecular species of UGT. When a patient with a UGT1A1 polymorphism that reduces UGT1A1 expression or enzyme activity receives irinotecan, SN-38 detoxification is delayed after administration due to decreased UGT activity, causing severe side effects Is more likely to be caused.

  UGT1A1 * 28, one of the UGT1A1 gene polymorphisms, repeats the TA sequence in the TATA box in the promoter region 6 times in the wild type (UGT1A1 * 1), which occupies a large number of 7 times. This gene polymorphism is reduced, and the expression level of the gene decreases due to this TA2 base insertion, resulting in a decrease in UGT activity.

  In addition, UGT1A1 * 60, one of the UGT1A1 gene polymorphisms, is thymine (T) in the wild type (UGT1A1 * 1), where the -3279th promoter region occupies the majority. The gene polymorphism is, and the difference in this single base decreases the expression level of the gene, resulting in a decrease in UGT activity.

  Furthermore, UGT1A1 * 6, one of the UGT1A1 gene polymorphisms, is guanine (G) in the wild type (UGT1A1 * 1), where the 211th in exon 1 is the majority, and adenine (A ) Is a genetic polymorphism. Due to this single base difference, the 71st amino acid in the UGT1A1 protein is mutated from glycine to arginine, and the mutant UGT1A1 enzyme has a reduced enzyme activity compared to the wild type.

  Furthermore, UGT1A1 * 27, one of the UGT1A1 gene polymorphisms, is cytosine (C) in the wild type (UGT1A1 * 1), where the 686th in exon 1 occupies the majority, whereas adenine ( It is a genetic polymorphism in A). Due to this one base difference, the 229th amino acid in the UGT1A1 protein is mutated from proline to glutamine, and the enzyme activity of the mutant UGT1A1 enzyme is reduced compared to the wild type.

  The base sequence (wild type) of the region containing these 4 types of gene polymorphisms UGT1A1 * 28, UGT1A1 * 60, UGT1A1 * 6 and UGT1A1 * 27 is shown in SEQ ID NO: 1. In the base sequence shown in SEQ ID NO: 1, UGT1A1 * 28 is a polymorphism in which the number of repetitions of the TA sequence located at positions 5390 to 5401 is 6 times 7 times. In the base sequence shown in SEQ ID NO: 1, UGT1A1 * 60 is a polymorphism in which the 2164th T is G. Furthermore, in the base sequence shown in SEQ ID NO: 1, UGT1A1 * 6 is a polymorphism in which the 5653rd G is A. Furthermore, in the base sequence shown in SEQ ID NO: 1, UGT1A1 * 27 is a polymorphism in which C at position 6128 is A.

  On the other hand, UGT1A7, a molecular species of UGT, is also known to be involved in the release of xenobiotics in the same way as UGT1A1, and is inactivated by SN-38 (SN-38G production activity). It has been pointed out to be involved.

  UGT1A7 * 2, one of the polymorphisms of UGT1A7, is thymine, cytosine, and guanine in the wild type (UGT1A7 * 1), where the 387th, 391st, and 392th contained in exon 1, respectively. On the other hand, the gene polymorphisms are guanine, alanine and alanine, respectively. Due to this three base difference, the 129th amino acid in UGT1A7 protein is mutated from asparagine to lysine, and the 131st amino acid is mutated from arginine to lysine. This mutant UGT1A7 enzyme has an increased enzyme activity compared to the wild type. However, in addition to the UGT1A7 * 2 allele, for example, UGT1A7 * 3 having the UGT1A7 (622T> C) allele has a reduced enzyme activity compared to the wild type.

  In addition, UGT1A7 * 12, one of the UGT1A7 gene polymorphisms, is the -57th promoter region, the 622nd and 760th coding regions, and the wild type (UGT1A7 * 1), which occupies the majority, thymine, thymine and In contrast to cytosine, polymorphisms are guanine, cytosine and thymine, respectively. The expression level of the gene decreases due to the difference of 1 base in the promoter region, and the 208th amino acid in UGT1A7 protein is mutated from tryptophan to arginine due to the difference of 2 bases in the coding region, and the 254th amino acid is changed from arginine. It will be mutated to a stop codon. This mutant type of UGT1A7 enzyme has a reduced expression level and enzyme activity as compared to the wild type. In the following description, among UGT1A7 * 12, the polymorphism located at the −57th position in the promoter region is referred to as UGT1A7 * 12 (−57).

  The nucleotide sequence (wild type) of the region containing these two gene polymorphisms UGT1A7 * 2 and UGT1A7 * 12 (-57) is shown in SEQ ID NO: 2. In the base sequence shown in SEQ ID NO: 2, UGT1A7 * 2 is a polymorphism in which 2938th T is G, 2942th C is A, and 2943th G is A. In the base sequence shown in SEQ ID NO: 2, UGT1A7 * 12 (-57) is a polymorphism in which the 2495th T is G.

  On the other hand, as well as UGT1A1 and UGT1A7, UGT1A9, which is a molecular species of UGT, is known to be involved in xenobiotic elimination, and inactivates SN-38 (SN-38G production activity). ) Has been pointed out.

UGT1A9 * 1b, one of the polymorphisms of UGT1A9, starts at -118th in the promoter region (dT), but the wild type is 9 times ((dT) ₉ ), whereas it is 10 times. It is a gene polymorphism ((dT) ₁₀ ), and the expression level of the gene increases due to the difference of 1 base insertion of thymine, and as a result, the UGT activity increases.

  The nucleotide sequence (wild type) of the region containing the gene polymorphism of UGT1A9 * 1b is shown in SEQ ID NO: 3. In the base sequence shown in SEQ ID NO: 3, UGT1A9 * 1b is a polymorphism in which 9 Ts located in the 20th to 2025th positions become 10 Ts.

  In the present invention, the side effect caused by irinotecan refers to a side effect that occurs in a patient administered irinotecan, and is not particularly limited as long as the risk of occurrence increases in a subject having a mutation in the glucuronidase gene. Preferably, UGT1A1 gene polymorphism, especially UGT1A1 * 28, UGT1A1 * 60, UGT1A1 * 6 and UGT1A1 * 27 polymorphisms, preferably UGT1A1 gene polymorphism, UGT1A7 gene polymorphism, especially UGT1A7 * 2 and UGT1A7 * 12 (-57) polymorphism, more preferably UGT1A9 gene polymorphism in addition to UGT1A9 gene polymorphism, especially one of UGT1A9 * 1b polymorphism This refers to side effects that increase the risk of occurrence. In other words, the side effect caused by irinotecan refers to a side effect that has a higher risk of occurrence than a decrease in the expression level of the glucuronidation enzyme gene and / or a decrease in the activity of the glucuronidation enzyme. More specifically, side effects caused by irinotecan include bone marrow toxicity such as leucopenia, diarrhea, vomiting, general malaise, loss of appetite, and hair loss.

  Glucuronidase (UDP-glucuronyltransferase; UGT) is a membrane enzyme that is localized mainly in the liver endoplasmic reticulum and is fat-soluble as a foreign substance (drugs, environmental pollutants, food additives, etc.) inside and outside the body. This refers to a protein having an activity of catalyzing glucuronidation that transfers glucuronic acid to a compound. The amino acid sequence of UGT1A1, which is a molecular species of glucuronidase (UGT), and the base sequence of the enzyme gene are registered as Accession No: NM — 000463 in public databases (GenBank, EMBL, DDBJ). The wild-type base sequence of the promoter region of the glucuronidase gene, wherein the TA sequence in the TATA box is repeated 6 times, is Accession No: AY533179 in the published databases (GenBank, EMBL, DDBJ). It is registered as. The base sequence of the promoter region of the glucuronidase gene, which is a mutant base sequence in which the TA sequence repeats 7 times in the TATA box, is Accession No: AY533180 in public databases (GenBank, EMBL, DDBJ). It is registered as. Each sequence can be obtained from, for example, http://www.ncbi.nlm.nih.gov/.

  In addition, the amino acid sequences of UGT1A7 and UGT1A9, which are one molecular species of glucuronidase (UGT), and the base sequence of the enzyme gene can also be specified in published databases (GenBank, EMBL, DDBJ).

  The method for determining the risk of side effects caused by irinotecan according to the present invention is based on the results of measuring the four gene polymorphisms UGT1A1 * 28, UGT1A1 * 60, UGT1A1 * 6 and UGT1A1 * 27 described above. Can be determined. In particular, in addition to these four gene polymorphisms, three polymorphisms, UGT1A7 * 2, UGT1A7 * 12 and UGT1A9 * 1b, were measured, and side effects caused by irinotecan based on the measurement results of a total of seven gene polymorphisms. It is more preferable to determine the risk of occurrence.

  In measuring these polymorphisms, for example, a method using a pair of nucleic acid probes corresponding to the wild type and the mutant type for each polymorphism can be used. The nucleic acid probe can be designed as a base sequence containing the above-mentioned mutation site. That is, as the pair of nucleic acid probes, a wild-type detection nucleic acid probe in which the above-mentioned mutation site is a wild type and a mutation-type nucleic acid probe in which the mutation site is a mutant type can be designed. The length of the nucleic acid probe is not particularly limited, but may be, for example, 15 to 40 bases, preferably 19 to 35 bases, and more preferably 25 to 35 bases. In the present invention, the nucleic acid includes DNA and RNA, and the DNA includes single-stranded DNA and double-stranded DNA. The nucleic acid probe is preferably DNA.

  Examples of the nucleic acid probes corresponding to the above-mentioned seven types of gene polymorphic variants and wild-type include oligonucleotides having the base sequences shown in Table 1 below. The probe sequences of Nos. 1-14 in Table 1 below are shown in SEQ ID NOs: 4-17.

These nucleic acid probes can be designed based on the nucleotide sequences of UGT1A1, UGT1A7, and UGT1A9 genes shown in SEQ ID NOs: 1 to 3, the sites of gene polymorphisms, and the bases after mutation. However, the nucleic acid probe corresponding to the mutant form of UGT1A9 * 1b was designed to include (dT) ₁₁ unlike the above-mentioned base sequence of (dT) ₁₀ . This is because a nucleic acid probe containing (dT) ₁₀ may erroneously detect the wild type of (dT) ₉ . By using a nucleic acid probe containing (dT) ₁₁ , hybridization with the wild type of (dT) ₉ can be prevented, and the mutant form of (dT) ₁₀ can be detected with high accuracy.

  The nucleic acid probe designed in this way can be obtained by, for example, chemically synthesizing with a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.

  The method of using a pair of nucleic acid probes corresponding to a wild type and a mutant type for a polymorphism is specifically a genomic DNA derived from a biological sample of a subject (usually a human subject) as a template. Examples include a method of amplifying a nucleic acid fragment containing a polymorphic site to be measured by an amplification reaction and detecting hybridization between the obtained nucleic acid fragment and a pair of these nucleic acid probes. That is, the ratio of the nucleic acid fragment hybridized to the nucleic acid probe that detects the wild type and the nucleic acid fragment hybridized to the nucleic acid probe that detects the mutant type is measured, and the subject homogenizes the wild type for the gene polymorphism. Whether it has a wild type and a mutant type in a heterogeneous state or a mutant type in a homozygous state. In other words, the genotype of the subject can be determined for the gene polymorphism by measuring the hybridization of the nucleic acid fragment to a pair of nucleic acid probes.

  Here, the biological sample collected from the subject is not particularly limited as long as it contains genomic DNA. For example, blood and blood-related samples derived therefrom (blood, serum, plasma, etc.), body fluids such as lymph, sweat, tears, saliva, urine, feces, ascites and cerebrospinal fluid, and crushed cells, tissues or organs and Examples include an extract. In the present invention, a blood-related sample is preferably used.

  The extraction means for extracting genomic DNA from a biological sample collected from a subject is not particularly limited, but is preferably a means capable of directly separating, purifying and recovering DNA components from the biological sample. A nucleic acid amplification reaction is performed using the obtained genomic DNA as a template to amplify a region containing the gene polymorphism to be measured. As the nucleic acid amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), or the like can be applied.

  When amplifying the detection target region, it is desirable to add a label so that the amplified region can be identified. At this time, the method for labeling the amplified nucleic acid is not particularly limited. For example, a method in which a primer used in the nucleic acid amplification reaction is labeled in advance may be used, or a labeled nucleotide may be used as a substrate in the nucleic acid amplification reaction. You may use the method used as. The labeling substance is not particularly limited, and radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

  In addition, this reaction system includes, for example, a buffer necessary for nucleic acid amplification / labeling, a heat-resistant DNA polymerase, a detection region specific primer, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate added with a fluorescent label or the like). ), A reaction system containing nucleotide triphosphate, magnesium chloride, and the like. For example, to incorporate a fluorescent substance as a label into the nucleic acid molecule to be amplified, the fluorescent dye is bound to one of the substrates (four kinds of bases) in the amplification reaction, and the abundance of the base to which the fluorescent substance is bound Is preferably 1/25 to 2/5 as compared with the most common among the other three types of bases.

  Specifically as a primer used for nucleic acid amplification reaction, a pair of oligonucleotide shown in following Table 2 can be used.

  Primers for amplifying UGT1A1 * 6 such as UGT1A1 * 6-S and UGT1A1 * 6-AS shown in Table 2 are preferably 0.06 to 1.5 μmol / l in the reaction solution, and 0.30 μmol / l. More preferably. The primers for amplifying UGT1A1 * 27 such as UGT1A1 * 27-S and UGT1A1 * 27-AS shown in Table 2 are preferably 0.01 to 0.35 μmol / l in the reaction solution, and preferably 0.07 μmol / l. More preferably, l. Furthermore, the primers for amplifying UGT1A1 * 28 such as UGT1A1 * 28-S and UGT1A1 * 28-AS shown in Table 2 preferably have a concentration in the reaction solution of 0.06 to 1.5 μmol / l, preferably 0.30 μmol / l. More preferably, l. Furthermore, the primers for amplifying UGT1A1 * 60 such as UGT1A1 * 60-S and UGT1A1 * 60-AS shown in Table 2 preferably have a concentration in the reaction solution of 0.01 to 0.35 μmol / l, preferably 0.07 μmol. More preferably / l. Furthermore, primers that amplify UGT1A7 * 12 (-57) such as UGT1A7 * 12 (-57) -S and UGT1A7 * 12 (-57) -AS shown in Table 2 each have a concentration in the reaction solution of 0.08 to It is preferably 2.00 μmol / l, more preferably 0.40 μmol / l. Furthermore, the primers for amplifying UGT1A7 * 2 such as UGT1A7 * 2-S and UGT1A7 * 2-AS shown in Table 2 preferably have a concentration in the reaction solution of 0.08 to 2.00 μmol / l, respectively, and 0.40 μmol. More preferably / l. Furthermore, the primers for amplifying UGT1A9 * 1b such as UGT1A9 * 1b-S and UGT1A9 * 1b-AS shown in Table 2 preferably have a concentration in the reaction solution of 0.12 to 3.00 μmol / l, respectively, 0.60 μmol. More preferably / l.

  When the amplified nucleic acid fragment has a label, hybridization of the nucleic acid fragment to a pair of nucleic acid probes can be measured by detecting the label. For example, when a fluorescent dye is used as a label, the nucleic acid fragment hybridized with the nucleic acid probe can be measured by measuring the fluorescence intensity derived from the fluorescent dye. Specifically, as described above, to calculate the ratio of a nucleic acid fragment hybridized to a nucleic acid probe that detects a wild type and a nucleic acid fragment that hybridizes to a nucleic acid probe that detects a mutant type, the wild type is detected. It can be calculated from the output value when the label in the nucleic acid probe to be detected is detected and the output value when the label in the nucleic acid probe to detect the mutant type is detected.

  More specifically, the intensity value derived from the amplified nucleic acid hybridized to the nucleic acid probe corresponding to the mutant type, the intensity value derived from the amplified nucleic acid hybridized to the nucleic acid probe corresponding to the mutant type, and the nucleic acid corresponding to the wild type The judgment value can be calculated by dividing by the average value of intensity values derived from the amplified nucleic acid hybridized to the probe. This determination value approximates a value obtained by normalizing the abundance of the mutant type contained in the amplified nucleic acid. Therefore, according to the height of the determination value, it can be determined whether the genetic polymorphism in the subject has a mutant type homozygous, a mutant type and a wild type heterozygous, or a wild type homozygous.

  When using the determination value, in order to determine whether the genetic polymorphism in the subject has a mutant type homozygote, a mutant type and a wild type heterozygous, or a wild type homozygous, a two-stage It is preferable to set threshold values (threshold value A and threshold value B). Here, it is assumed that the threshold A and the threshold B have a relationship of (threshold A> threshold B). That is, when the determination value calculated as described above exceeds the threshold value A, it is determined that the mutant type is homozygous. When the determination value is equal to or less than the threshold value A and exceeds the threshold value B, the mutant type and the wild type heterotype are determined. If it is determined that there is a threshold value B or less, it is determined that the wild type is homozygous.

  These threshold A and threshold B are set for each of the seven types of genetic polymorphisms described above. The method for setting the threshold A and the threshold B is not particularly limited, but the determination value is calculated as described above using a sample whose genotype has been determined in advance, and the mutation type, the hetero type, and the wild type are calculated. A method of calculating the probability density as a normal distribution can be given. At this time, an intersection point where the probability density overlaps each other (at a position where the magnitude of the probability density is switched and between the respective maximum values) is obtained, and an average value of each of the mutant type, the hetero type and the wild type is obtained. The threshold values of the mutant type and the hetero type can be calculated as an average value of (average value of mutant type and average value of hetero type) and an average value of intersection points. Similarly, the threshold values of the hetero type and the wild type can be calculated as an average value of (average value of hetero type and average value of wild type) and an average value of intersections.

  As described above, when the genotype is determined for the above-described genetic polymorphism in the subject and all of the determined genotypes are irinotecan side effect risk genotypes, the risk of occurrence of side effects due to irinotecan is high. Judged and diagnosed. Specifically, in order to determine the risk of side effects caused by irinotecan, the above four polymorphisms of UGT1A1 * 28, UGT1A1 * 60, UGT1A1 * 6 and UGT1A1 * 27 are measured, and these gene polymorphisms are measured. When all of these are irinotecan side effect risk genotypes, it can be determined and diagnosed that the risk of occurrence is high. In particular, in addition to these four gene polymorphisms, three gene polymorphisms of UGT1A7 * 2, UGT1A7 * 12 and UGT1A9 * 1b are measured, and all of these seven gene polymorphisms are irinotecan side effect risk genotypes. If it is, it can be determined and diagnosed that the risk of occurrence is high. In particular, by using the measurement results of a total of seven types of gene polymorphisms described above, the risk of occurrence of side effects due to irinotecan can be determined and diagnosed with higher accuracy.

  On the other hand, the kit for determining the risk of occurrence of side effects due to irinotecan according to the present invention includes the above-described nucleic acid probe. That is, as long as the kit according to the present invention includes the above-described nucleic acid probe, other configurations are not particularly limited. As an example, the nucleic acid probe described above in the kit according to the present invention can be immobilized on a carrier having a predetermined shape. Here, examples of the carrier include a planar substrate and a bead-shaped spherical carrier.

  Moreover, the kit which concerns on this invention may be the structure containing the primer used for the amplification reaction mentioned above in addition to the nucleic acid probe mentioned above. As a primer, the oligonucleotide which consists of a base sequence shown to sequence number 18-31 can be mentioned, for example. The primer can be provided as a solution so as to have a predetermined concentration. In this case, a plurality of primers may be dissolved in a single solution, but a plurality of primers may be provided as individual solutions.

  Furthermore, the kit according to the present invention may include various reagents necessary for the amplification reaction described above (enzyme, dNTP (including a substrate having a fluorescent label), a solution having a predetermined salt concentration, etc.) A configuration including various reagents necessary for specifically hybridizing the amplified DNA and the nucleic acid probe may be used.

  On the other hand, for the hybridization reaction between the amplified nucleic acid and the nucleic acid probe described above, it is preferable to apply a method in which the amplified nucleic acid acts on the microarray using a microarray in which the nucleic acid probe is immobilized on a carrier. In that case, the nucleic acid probes of the present invention may be immobilized on the same carrier or may be immobilized on different carriers.

  As the material for the carrier in the microarray, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as carbon typified by graphite and carbon fiber; single crystal silicon, amorphous silicon, carbonization Silicon materials represented by silicon, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, forsterite, photosensitive Inorganic materials such as porous glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and polysulfone And organic materials. The shape of the carrier is not particularly limited, but is preferably a flat plate shape.

  In the present invention, a carrier having a carbon layer and a chemical modification group on the surface is preferably used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of the substrate made of the carbon layer. As the material for the substrate, those known in the art can be used, and there are no particular restrictions, and the same materials as those described above can be used.

  The present invention is suitably used for a carrier having a fine flat plate structure. The shape is not limited to a rectangle, a square, or a circle, but a shape of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is usually used. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. In particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slightly different orientation of the crystal axis in some parts (sometimes called a mosaic crystal), or includes atomic scale disturbances (lattice defects) Are also included.

  In the present invention, the carbon layer formed on the substrate is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene) , Carbon nanotubes), a mixture thereof, or a laminate of them is preferably used. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an incomplete diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer has excellent chemical stability, can withstand subsequent reactions in the introduction of chemical modification groups and binding to the analyte, and the binding is flexible because of the electrostatic binding to the analyte. It is advantageous in that it has the property of being transparent, it is transparent to the detection system UV because there is no UV absorption, and it can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

  In the present invention, the carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionization deposition method, arc Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the high-frequency plasma CVD method, a raw gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency, and a DLC (diamond-like carbon) layer is synthesized on the substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of the remaining methane gas.

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

  In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

  When the carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually a monomolecular layer to about 100 μm. If it is too thin, the surface of the base substrate may be locally exposed, and conversely thick. In this case, the productivity is deteriorated, so that the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

  By introducing a chemical modification group into the surface of the substrate on which the carbon layer is formed, the nucleic acid probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

  An amino group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas, and further irradiated with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

  The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. As a compound used for introducing a carboxyl group, for example, the formula: X-R1-COOH (wherein X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms). The indicated halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R2-COOH ( In the formula, R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms.) For example, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; Polyvalent carboxylic acids such as acrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; Formula: R3-CO-R4-COOH (wherein R3 is a hydrogen atom or a divalent carbon of 1 to 12 carbon atoms) A hydrogen group, R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms.) Keto acid or aldehyde acid shown: Formula: X-OC-R5-COOH (wherein X represents a halogen atom, R5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms). Examples include monohalides of dicarboxylic acids such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

  Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

  Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

  Introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

  The active ester group means an ester group having an electron-withdrawing group having high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having high reaction activity. The ester group has an electron-withdrawing group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. More specifically, examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group and the like. In particular, an N-hydroxysuccinimide group is preferably used.

  The introduction of the active ester group is performed, for example, by converting the carboxyl group introduced as described above into a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N- It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

  The nucleic acid probe of the present invention is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted on a carrier by a spotter device or the like. Thus, a microarray in which a nucleic acid probe is immobilized on a carrier can be produced. Alternatively, the spotting solution may be spotted manually with a micropipette.

  After spotting, incubation is preferably performed in order to advance the reaction of the nucleic acid probe binding to the carrier. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed under a high humidity atmosphere, for example, under conditions of a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing using a washing solution (for example, 50 mM TBS / 0.05% Tween 20) in order to remove nucleic acids not bound to the carrier.

  Furthermore, the nucleic acid probe may be immobilized as a plurality of spots in which the amount of immobilization is changed stepwise. For example, for each nucleic acid probe, a plurality of spots such as a spot with a DNA amount of 1 ng, a spot with 100 pg, and a spot with 10 pg may be formed. Thereby, the detection target area can be semi-quantified.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, a pair of genes for distinguishing genotypes of seven types of gene polymorphisms consisting of UGT1A1 * 28, UGT1A1 * 60, UGT1A1 * 6, UGT1A1 * 27, UGT1A7 * 2, UGT1A7 * 12 and UGT1A9 * 1b A DNA chip having a nucleic acid probe was prepared. Table 3 shows a list of nucleic acid probes mounted on the DNA chip. The position of the nucleic acid probe mounted on the DNA chip is schematically shown in FIG.

  In this example, a nucleic acid fragment containing the above gene polymorphism was amplified by the PCR method on the DNA chip produced as described above, and the obtained nucleic acid fragment was allowed to act on the DNA chip to obtain a nucleic acid fragment and a nucleic acid probe. Hybridization was detected. Specifically, in the PCR method, a reaction solution having the composition shown in Table 4 was prepared, and a thermal cycler (manufactured by Eppendorf, model: MasterCycler ep gradient pro S) was used.

  For dNTP, a mixed solution of 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dTTP, 0.4 mM dCTP and 0.1 mM Cy5-dCTP was used. The primer set used was a mixture of oligonucleotides shown in Table 5.

At this time, the concentrations of UGT1A1 * 6-S and UGT1A1 * 6-AS were each 0.30 μmol / l. The concentrations of UGT1A1 * 27-S and UGT1A1 * 27-AS were 0.07 μmol / l, respectively, and the concentrations of UGT1A1 * 28-S and UGT1A1 * 28-AS were each 0.30 μmol / l. The concentrations of UGT1A1 * 60-S and UGT1A1 * 60-AS were 0.07 μmol / l, respectively. The concentrations of UGT1A7 * 12 (-57) -S and UGT1A7 * 12 (-57) -AS were 0.40 μmol / l, respectively. The concentrations of UGT1A7 * 2-S and UGT1A7 * 2-AS were 0.40 μmol / l, respectively. The concentrations of UGT1A9 * 1b-S and UGT1A9 * 1b-AS were 0.60 μmol / l, respectively.
Moreover, the temperature program shown in Table 6 was set to the thermal cycler.

  When hybridizing the amplified fragment obtained as described above to a DNA chip, a wet box was first placed in a chamber set at 54 ° C. and sufficiently heated. In a new sample tube, 1.5 μL of hybridization buffer (composition: 3 × SSC / 0.3 × SDS) and 3 μL of PCR product were added and mixed. 3 μL of the mixture was dropped onto the DNA chip, covered, placed in a preheated wet box, and each wet box was placed in a champ. Then, it left still at 54 degreeC for 1 hour.

  Thereafter, washing was performed. For cleaning, cleaning liquid A (10 × SSC / 1% SDS solution), cleaning liquid B (20 × SSC) and cleaning liquid C (5 × SSC) were prepared. And the solution which diluted the washing | cleaning liquid A 10 times was prepared. This diluted solution was placed in a staining bottle and heated to 48 ° C. in an incubator. Further, a solution in which the cleaning solution B was diluted 10 times was prepared. Furthermore, a solution in which the washing solution C (5 × SSC) was diluted 10 times was prepared.

  Immediately after the completion of hybridization, the cover of the DNA chip chip was removed and placed in a washing solution A diluted solution heated to 48 ° C. In a state where the DNA chip was immersed in a washing solution A dilution at 48 ° C., it was shaken 10 times and allowed to stand for 5 minutes. Thereafter, the DNA chip was immersed in a washing solution A diluted at room temperature, shaken 10 times, and allowed to stand for 5 minutes. Next, the DNA chip was immersed in the washing solution B diluted solution, shaken 10 times, and allowed to stand for 3 minutes. Next, the DNA chip was immersed in a washing solution A dilution at 48 ° C. and shaken 10 times. Thereafter, the DNA chip was immersed in a washing solution A diluted solution at room temperature and shaken 10 times. Next, the DNA chip was immersed in the washing solution B diluted solution and shaken 10 times. Next, it was immersed in a DNA chip washing solution C diluted solution and shaken 10 times. Thereafter, the DNA chip was immersed in the washing solution B dilution, and the reaction site was covered with a cover glass.

  BIOSHOT (manufactured by Toyo Kohan Co., Ltd.) was used for detection of hybridization. At this time, the spot diameter was set to 16 pixels (about 200 μm), and the exposure time was set to 20 seconds. Further, the median value was obtained from the numerical values of the pixels within each spot diameter. For the same probe spot, the average of the median values was obtained and used as the output value of each spot.

  Using the output value from which hybridization has been detected as described above, the genotype relating to the above seven types of gene polymorphisms in the specimen can be determined as follows. When determining the genotype, the following determination values were calculated.

Judgment value = [Variant] / {Average [Wild]; [Variant]}

  In the above formula, [Variant] is the output value from the spot of the nucleic acid probe corresponding to the mutant type, and {Average [Wild]; [Variant]} is the output value from the spot of the nucleic acid probe corresponding to the mutant type. It is the average value of the output values from the spot of the nucleic acid probe corresponding to the mold. The genotype of the specimen was determined by comparing the calculated determination value with the threshold A and threshold B determined for each polymorphism. That is, when judgment value <threshold B, wild type homozygous, when threshold B <judgment value <threshold A, wild type and mutant heterozygous, and when threshold A> judgment value Was identified as a homozygous variant. The thresholds A and B defined for each gene polymorphism are summarized in Table 7.

[Example 2]
In this example, the standard nucleic acid (sample A) that becomes wild type for all gene polymorphisms, the standard nucleic acid that becomes heterogeneous for all gene polymorphisms (sample B), and the standard nucleic acid that becomes mutated for all gene polymorphisms Using (Sample C), the hybridization temperature in Example 1 was changed, and the relationship between the hybridization temperature and detection sensitivity was examined.

  In this example, genotypes were discriminated with respect to seven types of gene polymorphisms in the same manner as in Example 1 except that the hybridization temperature was 50 ° C, 52 ° C, 54 ° C, 56 ° C and 58 ° C. Table 8 shows the results of calculating the judgment values for samples A to C. In addition, Table 9 shows the result of determining the genotype by comparing the determination values shown in Table 8 with the thresholds A and B shown in Table 7.

  As can be seen from Table 9, when the hybridization temperature is 58 ° C., a plurality of gene polymorphisms are misidentified. Also, in Table 8, when the temperature is 50 ° C, the 1 * 6 judgment value of sample A is extremely high compared to the judgment values of 0.569 and 52 ° C or higher, and is close to the threshold value B. There is sex. Therefore, from this example, it was revealed that it is preferable to perform hybridization in the range of 52 to 56 ° C. in the DNA chip prepared in Example 1 and the genotyping protocol.

Example 3
In this example, four types of samples (samples D to G) with all known genetic polymorphisms were used, and the sample amount (template DNA) used in PCR in Example 1 was changed to detect the sample amount and detection. The relationship with sensitivity was examined.

  In this example, genotypes were discriminated with respect to seven types of gene polymorphisms in the same manner as in Example 1 except that the sample amount was 0.5 pg / μl, 5 pg / μl, 50 pg / μl and 500 pg / μl. . Table 10 shows the results of calculating the judgment values for samples D to G. In addition, Table 11 shows the results of determining the genotype by comparing the determination values shown in Table 10 with the thresholds A and B shown in Table 7.

  As can be seen from Table 11, when the amount of specimen is less than 50 pg / μl, it is understood that a plurality of gene polymorphisms are misjudged. Therefore, from this example, it was revealed that it is preferable to perform PCR at 50 pg / μl or more in the DNA chip prepared in Example 1 and the genotyping protocol.

Example 4
In this example, it was demonstrated that the risk of occurrence of side effects can be predicted with high accuracy using a genomic DNA sample derived from a patient actually administered irinotecan. In addition, about each patient, the presence or absence of the side effect was specified. In this example, as a comparison, the genotypes of UGT1A1 * 28 and UGT1A1 * 6 were independently determined, and it was examined whether the occurrence of side effects could be predicted from the determination results.

  Wild-type homozygotes that do not have UGT1A1 * 28 are expected to have no side effects. There were 52 patients who were wild-type homozygous without UGT1A1 * 28, of which 27 patients had no side effects. That is, only 51.9% of cases were correctly diagnosed as having no side effects based on the test result that it was a wild type homozygote not having UGT1A1 * 28. If UGT1A1 * 28 is a mutant homozygote, it is predicted that there will be side effects. There were 16 patients whose UGT1A1 * 28 was a mutant homozygote, of which 8 patients had side effects. That is, from the test result that UGT1A1 * 28 is a mutant homozygous, only 50% was correctly diagnosed as having side effects.

  In addition, wild-type homozygous that does not have UGT1A1 * 6 is expected to have no side effects. There were 45 patients who were wild-type homozygous without UGT1A1 * 6, of which 27 patients had no side effects. That is, from the test result that it was a wild-type homozygote not having UGT1A1 * 6, only 60% was correctly diagnosed as having no side effect. In addition, if UGT1A1 * 6 is a mutant homozygote, it is predicted that there will be side effects. There were 23 patients whose UGT1A1 * 6 was a mutant homozygote, of which 15 patients had side effects. That is, from the test result that UGT1A1 * 6 is a mutant homozygote, the ratio of correctly diagnosed as having side effects was only 65.2%.

  As described above, it was found that it is difficult to sufficiently determine the risk of occurrence of side effects of irinotecan from the genotype of UGT1A1 * 28 or UGT1A1 * 6.

  Therefore, in this example, in addition to the genotypes of UGT1A1 * 28 and UGT1A1 * 6, the genotypes of UGT1A9 * 1b, UGT1A7 * 12 (-57) and UGT1A1 * 60 were considered together. That is, a group of patients who are wild-type homozygous not having both UGT1A1 * 28 and UGT1A9 * 1b, a group of patients whose UGT1A1 * 28 is heterozygous and does not have a genotype of UGT1A7 * 12 (-57), The total number of patient groups that were wild-type homozygotes that did not have UGT1A1 * 6 and UGT1A1 * 60 was 29 cases, of which 22 cases had no side effects. In other words, the percentage of correctly diagnosed as having no side effects was 75.8% by considering these UGT1A9 * 1b, UGT1A7 * 12 (-57), and UGT1A1 * 60 genotypes.

  In addition, the group of patients with wild-type homology without UGT1A1 * 28 and UGT1A9 * 1b mutant homozygous, and patient groups with UGT1A1 * 28 heterozygous and UGT1A7 * 12 (-57) genotype heterogeneous 16 cases, of which 12 cases had side effects. In other words, by considering these UGT1A9 * 1b and UGT1A7 * 12 (-57) genotypes, the rate of correctly diagnosed as having side effects was 75%.

  As described above, according to this example, in addition to UGT1A1 * 28 and UGT1A1 * 6 gene polymorphisms, UGT1A9 * 1b, UGT1A7 * 12 (-57), and UGT1A1 * 60 genotypes should be considered. Thus, it became clear that the risk of side effects of irinotecan can be determined with very high accuracy.

Claims (13)

The following gene polymorphisms in the glucuronidase gene
UGT1A1 * 28
UGT1A1 * 6
UGT1A1 * 27
UGT1A1 * 60
And a pair of nucleic acid probes for determining these wild types and an amplified nucleic acid obtained by an amplification reaction using a genomic DNA derived from the subject's biological sample as a template, A method for determining whether a genetic polymorphism is a normal type, a hetero type, or a mutant type, and determining a risk of occurrence of a side effect due to irinotecan based on the determination result. The following gene polymorphisms in the glucuronidase gene
UGT1A7 * 12 (-57)
UGT1A7 * 2
UGT1A9 * 1b
And a pair of nucleic acid probes corresponding to these wild types, and further determining whether each of these gene polymorphisms is normal, heterozygous or mutant, and based on the determination result, the side effects of irinotecan The method according to claim 1, wherein the occurrence risk is determined.   The hybridization between the nucleic acid probe corresponding to the mutant type and the amplified nucleic acid and the hybridization between the nucleic acid probe corresponding to the wild type and the amplified nucleic acid are measured by the intensity value of the label incorporated in the amplified nucleic acid. The method according to 1 or 2.   The intensity value derived from the amplified nucleic acid hybridized to the nucleic acid probe corresponding to the mutant type is used as the intensity value derived from the amplified nucleic acid hybridized to the nucleic acid probe corresponding to the mutant type and the nucleic acid probe corresponding to the wild type. A judgment value is calculated by dividing by the average value of intensity values derived from the hybridized amplified nucleic acid, and the judgment value is compared with a predetermined threshold A and threshold B (threshold A> threshold B). 4 is determined to be a mutant type when the value exceeds a threshold A, a hetero type when the determination value is equal to or less than the threshold A and exceeds a threshold B, and a wild type when the determination value is equal to or less than the threshold B. the method of. The concentration of a pair of primers for amplifying the region containing UGT1A1 * 28 is 0.06 to 1.5 μmol / l,
The concentration of a pair of primers for amplifying the region containing UGT1A1 * 6 is 0.06 to 1.5 μmol / l,
The concentration of a pair of primers for amplifying the region containing UGT1A1 * 27 is 0.01 to 0.35 μmol / l,
The method according to claim 1, wherein the concentration of the pair of primers for amplifying the region containing UGT1A1 * 60 is 0.01 to 0.35 µmol / l, respectively. The concentration of a pair of primers for amplifying the region containing UGT1A7 * 12 (-57) is 0.08 to 2.00 μmol / l,
The concentration of a pair of primers for amplifying the region containing UGT1A7 * 2 is 0.08 to 2.00 μmol / l, respectively.
The method according to claim 2, wherein the concentration of the pair of primers for amplifying the region containing UGT1A9 * 1b is 0.12 to 3.00 µmol / l, respectively.   The method according to claim 1 or 2, wherein the concentration of genomic DNA serving as a template in the amplification reaction is 50 to 500 pg / µl.   The method according to claim 1 or 2, wherein the temperature of the hybridization solution is 52 to 56 ° C.   The method according to claim 3, wherein the label is a fluorescent substance. The fluorescent substance is bound to one type of base in the substrate in the amplification reaction,
10. The method according to claim 9, wherein the abundance of the base to which the fluorescent substance is bound is 1/25 to 2/5 as compared with the most abundant of the other three types of bases. The following gene polymorphisms in the glucuronidase gene
UGT1A1 * 28
UGT1A1 * 6
UGT1A1 * 27
UGT1A1 * 60
And a kit for discriminating the risk of side effects caused by irinotecan, comprising a pair of nucleic acid probes for judging these wild types. The following gene polymorphisms in the glucuronidase gene
UGT1A7 * 12 (-57)
UGT1A7 * 2
UGT1A9 * 1b
The kit according to claim 11, further comprising a pair of nucleic acid probes corresponding to these wild types.   The kit according to claim 11 or 12, comprising a DNA chip having the pair of nucleic acid probes immobilized on a substrate.

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