JP5661251B2 - UGT1A1 gene polymorphism detection method - Google Patents

Description

  The present invention belongs to the technical field of pharmacogenetics or pharmacogenomics relating to the correlation between polymorphisms of UGT1A1 gene and side effects of irinotecan. Moreover, it belongs to the technical field of the detection method of single nucleotide polymorphism or genetic polymorphism, and more specifically, belongs to the technical field of TaqMan PCR method.

  In recent years, the involvement of genetic polymorphisms related to drug metabolism and kinetics has attracted attention as a factor of individual differences in drug efficacy and side effects, and tailor-made medical care (patient individualized therapy) that selects and administers appropriate drugs based on genetic diagnosis. ) Genomic pharmacology research aiming at realization has been progressing rapidly in the last few years. In many cases, the utility of genetic polymorphism diagnosis has been clarified, including haplotypes (combinations of individual gene polymorphisms on a chromosome), and tailor-made medicine is becoming a reality (Non-patent Document 20).

The camptothecin derivative irinotecan has been widely applied to lung cancer, gastrointestinal cancer, and the like, but it has sometimes been problematic to cause severe diarrhea, leukopenia, and the like (Non-patent Document 20). In addition, Iyer et al. Can improve the therapeutic index of irinotecan by glucuronidation of SN-38, an active metabolite of irinotecan, being catalyzed by the UGT1A1 enzyme, and by pharmacological manipulation of glucuronidation of SN-38. It has been shown that these findings may be particularly important for populations with reduced UGT1A1 activity, such as Gilbert's syndrome (Non-patent Document 9). In normal individuals the region of UGT1A1 promoter that is A (TA) ₆ TAA, but in Gilbert's syndrome has become A (TA) ₇ TAA (Non-Patent Document 9).

  Since the first report by Ando (Non-patent Document 2) et al. Of the UGT1A1 gene polymorphism * 28 (-40_-39insTA) and severe side effects in patients treated with irinotecan, many The importance of * 28 has been suggested by this research (Non-patent document 2, Non-patent document 10, Non-patent document 8, Non-patent document 14, Non-patent document 17). As an important polymorphism that leads to decreased expression of UGT1A1, the number of TA repeats present in the promoter region has increased from the usual 6 to 7 times (note that the TA of the last TAA cannot be counted). UGT1A1 * 28 (-40_-39insTA) (Non-patent document 20, Non-patent document 3) and UGT1A1 * 60 (-3279 T> G) existing in the enhancer region (Non-patent document 21). UGT1A1 * 6 [211 G> A (Gly71Arg)], UGT1A1 * 7 [1456 T> G (Tyr486Asp)] and UGT1A1 * 27 [686C> A (Pro229Gln)] with amino acid substitution are SN-38 glucuronic acid. Conjugation activity decreases (Non-patent Document 5, Non-patent Document 11).

  FIG. 1 shows a schematic diagram of the structure of the UGT1A1 gene (see FIG. 2 of Non-Patent Document 20 for the detailed structure of the UGT1A1 gene). * 28 exists in the promoter region of the UGT1A1 gene, and * 6 exists in exon 1.

  Sai et al. Revealed that * 6 [211 G> A (Gly71Arg)] and * 28 (-40_-39insTA) are independent of each other (on separate chromosomes) (Non-patent Document 18). Suggested that not only * 28 but also * 6 should be taken into account (Non-patent Document 20). Sai et al. Also found a decrease in the AUC ratio (see Non-Patent Document 10) depending on the number of haplotypes in patients with * 28 and * 6. There is a decrease (Non-patent Document 18), * 6 and * 28 bring about the same decrease in UGT activity, and it is independent (another haplotype), so in Japanese, “* 6 or * 28” Is a useful genetic polymorphism marker (Non-patent Document 20). * 6 A single effect also showed a significant association with an increase in the incidence of grade 3 or higher neutropenia (Non-patent Document 19). UGT1A1 * 6 is a polymorphism characteristic of East Asians. Therefore, it is taught that not only Japanese but also Koreans and Chinese need to consider * 6 as well as * 28 (Non-patent document 20, Non-patent document 15).

  Numerous methods such as the Invader method, DNA sequencing method, and SSCP method have been developed as methods for detecting single nucleotide substitution (SNP) or gene polymorphism (see Non-Patent Document 12). High-throughput methods are being researched day and night.

The TaqMan PCR method (also called 5'nuclease assay) was the method that Holland et al. Demonstrated for the first time in 1991 (Non-patent Document 7). They designed a complementary DNA probe labeled with ³² P at the 5 ′ end and protected at the 3 ′ end in the region amplified by PCR. When PCR is performed in the presence of this DNA probe, the probe is degraded with the progress of PCR due to the 5 ′ → 3 ′ exonuclease activity of Taq polymerase. It was confirmed that the labeled signal obtained from the decomposed probe was proportional to the amount of amplicon (Non-patent Document 7, Roche Diagnostics Corporation website). Furthermore, it has been shown that this method uses a fluorescently labeled probe to which FRET is applied, and the degradation of the probe can be monitored by measuring the change in fluorescence intensity with a fluorescence detector (Non-patent Document 13).

  Later, TaqMan PCR was applied to real-time quantitative PCR (Non-Patent Document 6), and a high-throughput detection method that simultaneously detects multiple gene polymorphisms at multiple locations (simultaneous) has been developed (Non-Patent Document 6). Patent Document 1, Non-Patent Document 4, Non-Patent Document 16, Non-Patent Document 22, Non-Patent Document 23, and Non-Patent Document 24) are not satisfactory in terms of simplicity and accuracy.

  Accordingly, the present inventors have intensively studied to develop a simpler, lower cost, accurate and high-throughput method, and as a result, the present invention has been completed.

Acommon 1317TC polymorphism in MTHFR can lead to erroneous 1298AC genotyping byPCR-RE and TaqMan probe assays.Allen RA, Gatalica Z, Knezetic J, HatcherL, Vogel JS, Dunn ST. Genet Test. 2007 Summer; 11 (2): 167-73 . Polymorphismsof UDP-glucuronosyltransferase gene and irinotecan toxicity: a pharmacogeneticanalysis.Ando Y, Saka H, Ando M, Sawa T, Muro K, Ueoka H, Yokoyama A, Saitoh S, Shimokata K, Hasegawa Y. Cancer Res. 2000 Dec 15; 60 (24): 6921-6. Racialvariability in the UDP-glucuronosyltransferase 1 (UGT1A1) promoter: a balancedpolymorphism for regulation of bilirubin metabolism? Beutler E, GelbartT, Demina A. Proc Natl Acad Sci U S A. 1998 Jul 7; 95 (14): 8170-4. Real-timePCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 issuperior to the standard PCR-SSP method.Ficko T, Galvani V, Rupreht R, Dovc T, Rozman P. Transfus Med. 2004 Dec; 14 (6): 425-32. Commonhuman UGT1A polymorphisms and the altered metabolism of irinotecan activemetabolite 7-ethyl-10-hydroxycamptothecin (SN-38) .Gagne JF, Montminy V, Belanger P, Journault K, Gaucher G, Guillemette C. Mol Pharmacol. 2002 Sep; 62 (3 ): 608-17. Realtime quantitative PCR.Heid CA, Stevens J, Livak KJ, Williams PM.Genome Res. 1996 Oct; 6 (10): 986-94. Detectionof specific polymerase chain reaction product by utilizing the 5 '---- 3'exonuclease activity of Thermus aquaticus DNA polymerase. Holland PM, Abramson RD, Watson R, GelfandDH.Proc Natl Acad Sci US A. 1991 Aug 15; 88 (16 ): 7276-80. Geneticvariants in the UDP-glucuronosyltransferase 1A1 gene predict the risk of severeneutropenia of irinotecan.Inocenti F, Undevia SD, Iyer L, Chen PX, DasS, Kocherginsky M, Karrison T, Janisch L, Ramirez J, Rudin CM, Vokes EE, RatainMJ. J Clin Oncol. 2004 Apr 15; 22 (8): 1382-8. Epub 2004 Mar 8. Genetic predisposition to the metabolism of irinotecan (CPT-11) .Role of uridinediphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of itsactive metabolite (SN-38) in human liver microsomes.Iyer L, King CD, Whitington PF, Green MD, Roy SK, Tephly TR, Coffman BL, Ratain MJ. J ClinInvest. 1998 Feb 15; 101 (4): 847-54. UGT1A1 * 28polymorphism as a determinant of irinotecan disposition and toxicity.Iyer L, Das S, Janisch L, Wen M, Ramirez J, Karrison T, Fleming GF, Vokes EE, Schilsky RL, Ratain MJ. Pharmacogenomics J. 2002; 2 (1 ): 43-7. Glucuronidationof 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan (CPT-11), by human UGT1A1 variants, G71R, P229Q, and Y486D.Jinno H, Tanaka-Kagawa T, Hanioka N, Saeki M , Ishida S, Nishimura T, Ando M, Saito Y, Ozawa S, Sawada J. Drug Metab Dispos. 2003 Jan; 31 (1): 108-13. Detectionof single nucleotide polymorphisms. Kwok PY, Chen X. Curr Issues Mol Biol. 2003 Apr; 5 (2): 43-60. Review. Allelicdiscrimination by nick-translation PCR with fluorogenic probes. Lee LG, Connell CR, Bloch W. Nucleic Acids Res. 1993 Aug 11; 21 (16): 3761-6. UGT1A1gene variations and irinotecan treatment in patients with metastatic colorectalcancer.Marcuello E, Altes A, Menoyo A, Del Rio E, Gomez-Pardo M, BaigetM. Br J Cancer. 2004 Aug 16; 91 (4): 678-82. Irinotecanpharmacokinetics / pharmacodynamics and UGT1A genetic polymorphisms in Japanese: roles of UGT1A1 * 6 and * 28.Minami H, Sai K, Saeki M, Saito Y, Ozawa S, Suzuki K, Kaniwa N, Sawada J, Hamaguchi T, Yamamoto N, Shirao K, Yamada Y, Ohmatsu H, Kubota K, Yoshida T, Ohtsu A, Saijo N. Pharmacogenet Genomics. 2007 Jul; 17 (7): 497-504. Four-colormultiplex reverse transcription polymerase chain reaction--overcoming itslimitations.Persson K, Hamby K, Ugozzoli LA. Anal Biochem. 2005 Sep 1; 344 (1): 33-42. Relevanceof different UGT1A1 polymorphisms in irinotecan-induced toxicity: a molecularand clinical study of 75 patients.Rouits E, Boisdron-Celle M, Dumont A, Guerin O, Morel A, Gamelin E. Clin Cancer Res. 2004 Aug 1; 10 (15) : 5151-9. UGT1A1haplotypes associated with reduced glucuronidation and increased serumbilirubin in irinotecan-administered Japanese patients with cancer.SaiK, Saeki M, Saito Y, Ozawa S, Katori N, Jinno H, Hasegawa R, Kaniwa N, SawadaJ, Komamura K, Ueno K, Kamakura S , Kitakaze M, Kitamura Y, Kamatani N, MinamiH, Ohtsu A, Shirao K, Yoshida T, Saijo N. Clin Pharmacol Ther. 2004 Jun; 75 (6): 501-15. Importanceof UDP-glucuronosyltransferase 1A1 * 6 for irinotecan toxicities in Japanesecancer patients.Sai K, Saito Y, Sakamoto H, Shirao K, Kurose K, Saeki M, Ozawa S, Kaniwa N, Hirohashi S, Saijo N, Sawada J, Yoshida T. CancerLett. 2008 Mar 18; 261 (2): 165-71. Epub 2007 Dec 20. Irinotecanpharmacogenetics in Japanese cancer patients: roles of UGT1A1 * 6 and * 28. Sai K, Sawada J, Minami H. Yakugaku Zasshi. 2008 Apr; 128 (4): 575-84.Review. Identification of a defect in the UGT1A1 gene promoter and its association withhyperbilirubinemia.Sugatani J, Yamakawa K, Yoshinari K, Machida T, Takagi H, Mori M, Kakizaki S, Sueyoshi T, Negishi M, Miwa M. BiochemBiophys Res Commun. 2002 Mar 29 ; 292 (2): 492-7. Four-colormultiplex 5 'nuclease assay for the simultaneous detection of the factor VLeiden and the prothrombin G20210A mutations.Ugozzoli LA, Hamby K. Mol Cell Probes. 2004 Jun; 18 (3): 161-6. Animproved real time PCR method for simultaneous detection of C282Y and H63Dmutations in the HFE gene associated with hereditary hemochromatosis.Walburger DK, Afonina IA, Wydro R. MutatRes. 2001 Jan; 432 (3-4): 69-78. Simultaneousgenotyping of coagulation factor XI type II and type III mutations by multiplexreal-time polymerase chain reaction to determine their prevalence in healthyand factor XI-deficient Italians.Zadra G, Asselta R, Tenchini ML, Castaman G, Seligsohn U, Mannucci PM, Duga S Haematologica. 2008 May; 93 (5): 715-21. Epub 2008 Apr 2.

  An object of the present invention is to detect a plurality of polymorphisms of the UGT1A1 gene related to the seriousness of side effects of irinotecan more easily, at lower cost, more accurately, and at higher throughput. Specifically, it is an object to detect UGT1A1 * 28 and * 6 polymorphisms more easily, at a lower cost, and more accurately and simultaneously.

  In order to solve the above-mentioned problems, the present invention provides a method for designing a primer used in the TaqMan PCR method, and a sequence of a primer and a probe used in the TaqMan PCR method.

  By using the present invention, a plurality of polymorphisms of the UGT1A1 gene related to the seriousness of side effects of irinotecan can be detected more easily, at lower cost, more accurately, and at higher throughput. Specifically, UGT1A1 * 28 and * 6 polymorphisms can be detected more easily, at lower cost, more accurately and simultaneously.

FIG. 1 is a schematic diagram of the structure of the UGT1A1 gene. Fig. 2-1A uses a plasmid control incorporating a wild type or mutant type sequence of UGT1A1 * 28 or UGT1A1 * 6 as a template DNA (the Hetero sample is a mixture of the wild type and the mutant type at the same concentration), and 4 Multiple types of primers (28F4, 28R4, 6F2 and 6R2) and four detection probes (UGT1A1 * 28 wild type, UGT1A1 * 28 mutant type, UGT1A1 * 6 wild type and UGT1A1 * 6 mutant type) FIG. 2-1B is a schematic diagram showing the detection result of the polymorphism by the ideal TaqMan PCR method. Figure 2-2 uses a plasmid control incorporating a wild type or mutant sequence of UGT1A1 * 28 or UGT1A1 * 6 as a template DNA (the Hetero sample is a mixture of the wild type and the mutant type at the same concentration), and 4 Multiple types of primers (28F4, 28R4, 6F7 and 6R7) and four detection probes (UGT1A1 * 28 wild type, UGT1A1 * 28 mutant type, UGT1A1 * 6 wild type and UGT1A1 * 6 mutant type) It is the result of performing type detection. Figure 3 shows a template control using a UGT1A1 * 28, UGT1A1 * 6 wild-type or mutant sequence as a template DNA (the Hetero sample is a mixture of wild-type and mutant types at the same concentration). Results of polymorphism detection using primers (28F4 and 6R2) and four detection probes (UGT1A1 * 28 wild type, UGT1A1 * 28 mutant, UGT1A1 * 6 wild type and UGT1A1 * 6 mutant) simultaneously It is. Figure 4 shows 20 ng or 10 ng of human genomic DNA as template DNA, and 2 types of primers (28F4 and 6R2) and 4 types of detection probes (UGT1A1 * 28 wild type, UGT1A1 * 28 mutant type, UGT1A1 * 6) This is a result of polymorphism detection using both wild type and UGT1A1 * 6 mutant type) simultaneously.

[Definition]
Simultaneously: In this specification, when “simultaneously” is detected, “simultaneously” is used, it does not mean “same time” in terms of time, for example, multiple wavelengths from the same PCR reaction solution in the same PCR tube. Is used to detect the fluorescence of the same, or to use a plurality of primers in the same PCR reaction solution in the same PCR tube.
Homo: Used in the normal sense of genetics, but in particular means that a particular polymorphism exists in both two homologous chromosomes.
Hetero: used in the normal sense of genetics, but in particular means that a particular polymorphism exists only in one of the two homologous chromosomes.
Wild type: Although all polymorphisms can also be referred to as wild type, in this specification the polymorphism most frequently observed in a particular population is referred to as wild type.
Variant: All polymorphisms can be referred to as wild-type, but in the present specification, polymorphisms other than the polymorphism most frequently observed in a specific population are referred to as variants.
Polymorphism: When used as a noun, it means an individual difference (variation) within a population in the genome base sequence or its sequence. When used as an adjective, it means that there is an individual difference (variation) within the population at a specific location in the genome base sequence.
Detecting polymorphism: In this specification, “detecting a polymorphism” means “detecting the presence of a polymorphic variant of interest in a sample from a specific individual”. In other cases, it may mean “determining the genotype of the polymorphism of interest”.
Genotype: An individual has a specific “genotype” means that the individual has a specific combination of polymorphisms (multiple polymorphic sites or two homologous chromosomes) for the polymorphic site (s) of interest It means having.

UGT1A1 * 28 polymorphism: A polymorphism in which the number of TA repeats in the promoter region of the UGT1A1 gene is increased from the usual 6 to 7 times. Regarding the position of the polymorphism, see Saiet al. Yakugaku Zasshi. 2008 Apr; 128 (4): 575-84. Review.
UGT1A1 * 6 polymorphism: A polymorphism (generally expressed as 211G> A) with an amino acid substitution in the UGT1A1 gene. Regarding the position of the polymorphism, see Saiet al. Yakugaku Zasshi. 2008 Apr; 128 (4): 575-84. Review.
UGT1A1 * 28 polymorphic site: Refers to the base or base pair location where the UGT1A1 * 28 polymorphism exists. It may be abbreviated as UGT1A1 * 28 site.
UGT1A1 * 6 polymorphic site: The base or base pair location where the UGT1A1 * 6 polymorphism exists. It may be abbreviated as UGT1A1 * 6 site.
UGT1A1 * 28 polymorphic region: A region on the genome sequence that includes the UGT1A1 * 28 site.
UGT1A1 * 6 polymorphic region: A region on the genome sequence that includes the UGT1A1 * 6 site.
UGT1A1 * 28 wild type: The genome sequence of an individual is "UGT1A1 * 28 wild type" means that it exists in the promoter region of both UGT1A1 genes of both of the two homologous chromosomes of the individual This means that the number of TA repeats is the usual 6 times.
UGT1A1 * 6 Wild type: The genome sequence of an individual is "UGT1A1 * 6 wild type" means that the UGT1A1 gene of both individuals or one of the target UGT1A1 genes has amino acid substitutions. This means that there is no type (generally expressed as 211G> A) (the base is G).
UGT1A1 * 28 variant: The genome sequence of an individual is "UGT1A1 * 28 variant" means that it exists in both of the two homologous chromosomes of the individual or in the promoter region of one UGT1A1 gene of interest. This means that the number of TA repeats is 7 instead of the usual 6.
UGT1A1 * 6 variant: The nucleotide sequence of a certain individual's genome is “UGT1A1 * 6 variant” means that there are many amino acid substitutions in both of the two homologous chromosomes of the individual or one UGT1A1 gene of interest. Means that there is a type (generally expressed as 211G> A) (the base is A).
UGT1A1 * 28: UGT1A1 * 28 polymorphism or UGT1A1 * 28 polymorphic site.
UGT1A1 * 6: UGT1A1 * 6 polymorphism or UGT1A1 * 6 polymorphic site.

Amplicon: A unit that is amplified as a continuous DNA in the PCR method.
Principle of TaqMan PCR method: In the present invention, the term “Principle of TaqMan PCR method” refers to the design of a complementary DNA probe labeled at the 5 ′ end in the region to be amplified by PCR, and in the presence of this DNA probe. The template is based on the fact that the PCR is performed and the 5 ′ → 3 ′ exonuclease activity of Taq polymerase degrades the probe as the PCR progresses, and the labeled signal obtained from the degraded probe is proportional to the amount of amplicon. The principle of DNA quantification.

[Experimental materials and methods]
<Human genomic DNA>
Extraction of human genomic DNA was performed using the QIAamp DNA Mini kit (Cat. No. 51304, QIAGEN). Blood for human genomic DNA extraction was obtained from Sekisui Medical Co., Ltd. in-house volunteers. The internal ethical rules were observed when collecting blood. Approximately 3 μg of genomic DNA was obtained from 200 μl of blood according to the protocol provided.

<Primer>
SIGMA's custom oligo DNA synthesis service was used to prepare the primers used for the PCR reaction. As the purification grade, cartridge purification (reverse phase cartridge (RP1)) was selected.

<Primer design>
Primer Express 3.0 (Applied Biosystems) was used as the primer design software. According to the provided manual, the primer probe tool Document Type TaqMan Allelic Discrimination was used as a design parameter. As selection criteria, Tm value: 58-60 ° C., base number: 20mer, GC content: 30-80% were used as a guide. Eleven primers satisfying the conditions were synthesized and tested for actual amplification efficiency and specificity, and excellent ones were selected from them.

  Table 1 shows the sequences of a pair of primer pairs (UGT-28 TprimerF4 and UGT-28 TprimerR4) that amplify the UGT1A1 * 28 polymorphic region. Table 1 shows the sequences of two pairs of primers (a pair of UGT-6TprimerF2 and UGT-6 TprimerR2 and a pair of UGT-6 TprimerF7 and UGT-6 TprimerR7) that amplify the UGT1A1 * 6 polymorphic region.

<TaqMan probe>
The TaqMan probe was created using the commissioned synthesis service of Operon Biotechnology Co., Ltd., and all the products were double-labeled with a fluorescent dye and a quencher. As the purification grade, HPLC purification was selected.

<TaqMan probe design>
Primer Express 3.0 (Applied Biosystems) was used as the probe design software. According to the provided manual, the primer probe tool Document Type TaqMan Allelic Discrimination was used as a design parameter. As selection criteria, Tm value: 65 to 68 ° C., base number: 30 to 40 mer, GC content: 30 to 80% was designed as a guide.

  Table 2 shows probes for determining whether the UGT1A1 * 28 polymorphic site is wild-type (UGT1A1 * 28 wild-type detection probe) or * 28 mutant type (UGT1A1 * 28 mutant-type detection probe). Show. Table 2 also shows a probe for determining whether the UGT1A1 * 6 polymorphic site is wild-type (UGT1A1 * 6 wild-type detection probe) or * 6 mutant type (UGT1A1 * 6 mutant-type detection probe) ).

<Real-Time PCR reaction>
Real-time PCR reaction (TaqMan PCR) was performed under the conditions shown in Table 3. The template DNA used was a plasmid control in which UGT1A1 * 28 and UGT1A1 * 6 wild-type or mutant sequences were incorporated (Hetero samples were mixed in an equal concentration of wild-type and mutant), or genomic DNA. The plasmid control was adjusted to 10 ⁵ to 10 ⁷ copies and the amount of water was adjusted so that the total volume was 50 μL. The amount of genomic DNA was adjusted to 10 to 20 ng, and the amount of water was adjusted so that the total amount was 50 μL. PreTex Ex Taq (TaKaRa) was used as ExTaq, and Multiplate PCR Plates Low96-wellclear (BIO RAD) was used as a PCR reaction tube. PTC-200 (Bio-Rad Laboratories, Inc.) was used as the thermal cycler. As shown in Table 4, the PCR reaction was performed in a two-step reaction at 98 ° C for 15 seconds and at 65 ° C for 1 minute.

<Fluorescence detection>
Chromo4 Real-Time PCR Detector (BIO RAD) was used for fluorescence detection of real-time PCR. Actually, fluorescence detection was performed simultaneously with the real-time PCR reaction. Data was analyzed using the OpticonMonitorVersion 3.1 program according to the provided manual. The conditions of fluorescence excitation and fluorescence detection are as follows; wavelength (fluorescence excitation range: 1 channel 450 to 490 nm, 2 channel 500 to 535 nm, 3 channel 555 to 585 nm, 4 channel 620 to 650 nm, fluorescence detection range: 1 channel 515 to 530 nm 2 channel 560 ~ 580nm 3 channel 610 ~ 650nm 4 channel 675 ~ 730nm).

  The value at which the amount of fluorescence was measured at the end of the experiment, that is, the maximum fluorescence value was expressed as Endpoint. The lowest fluorescence value measured in the well was defined as the baseline, and the relative fluorescence value of each dye was plotted on the vertical and horizontal axes.

[Results and discussion]
[Reference Example 1]
[Simultaneous detection of UGT1A1 * 28 and UGT1A1 * 6 polymorphisms using TaqMan PCR-1]
As a template DNA, a plasmid control incorporating a wild type or mutant sequence of UGT1A1 * 28 or UGT1A1 * 6 (the Hetero sample is a mixture of the wild type and the mutant type at the same concentration), and four types of primers (28F4 , 28R4, 6F2 and 6R2) and 4 detection probes (UGT1A1 * 28 wild type, UGT1A1 * 28 mutant type, UGT1A1 * 6 wild type and UGT1A1 * 6 mutant type), and UGT1A1 * by TaqMan PCR The results of simultaneous detection of 28 polymorphisms and UGT1A1 * 6 polymorphism are shown in FIG. 2-1A. The 28F4-28R4 primer pair amplifies a 149 bp amplicon of the UGT1A1 * 28 polymorphic region, and the 6F2-6R2 primer pair amplifies a 133 bp amplicon of the UGT1A1 * 6 polymorphic region.

  As a template DNA, a plasmid control incorporating a wild type or mutant sequence of UGT1A1 * 28 or UGT1A1 * 6 (the Hetero sample is a mixture of the wild type and the mutant type at the same concentration), and four types of primers (28F4 , 28R4, 6F7 and 6R7) and 4 detection probes (UGT1A1 * 28 wild type, UGT1A1 * 28 mutant type, UGT1A1 * 6 wild type and UGT1A1 * 6 mutant type), and UGT1A1 * by TaqMan PCR The results of simultaneous detection of 28 polymorphisms and UGT1A1 * 6 polymorphism are shown in Fig. 2-2.

  FIG. 2-1B schematically shows the detection result of the polymorphism by the ideal TaqMan PCR method. For example, each probe that detects UGT1A1 * 28 mutant type or UGT1A1 * 28 wild type is fluorescently labeled with HEX or FAM, respectively. Therefore, when both homologous genes are mutated (Homo), only HEX is detected. When fluorescence is strongly observed and both homologous genes are wild type (WT), fluorescence of only FAM is strongly observed, and when one homologous gene is mutant and the other homologous gene is wild type ( In the case of Hetero) both HEX and FAM fluorescence should be observed. Of course, when there is no DNA in the sample, ideally no fluorescence (background) is detected. The same is true for the UGT1A1 * 6 polymorphic site.

  Regarding the results of FIG. 2-1A, the background height of the HEX fluorescence signal (no DNA) in the detection of the UGT1A1 * 28 polymorphism or the deviation from the ideal pattern in the case of heterogeneity was not satisfactory. As for the results in FIG. 2-2, the weak fluorescence intensity of Cy5 (less than 0.125) and TXR (more than 0.06) in UGT1A1 * 6 polymorphism detection was not a satisfactory result.

[Simultaneous detection of UGT1A1 * 28 and UGT1A1 * 6 polymorphisms using TaqManPCR-2]
Since the result of the above Reference Example 1 was not satisfactory, the present inventors are able to detect the UGT1A1 * 28 and * 6 polymorphisms more easily, at lower cost, more accurately and simultaneously (simultaneous). Aiming at the development of a method that can do this, intensive study was repeated.

  Reference Example 1 was the best result among a number of primer pairs tested (data not shown). Therefore, the present inventors believe that the above problem cannot be solved only by optimizing the primer design, and tried a method of amplifying both the UGT1A1 * 28 polymorphic region and the UGT1A1 * 6 polymorphic region with a pair of primer pairs. It was. In the case of the 28F4-6R2 primer pair, a 424 bp amplicon is amplified.

  Usually, the efficiency of the PCR reaction is maximum when the amplicon is about 100 to 200 bp, and it is expected that the amplification efficiency and specificity decrease as the amplicon increases. Surprisingly, however, the 28F4-6R2 primer pair surpassed the results of the reference example in both fluorescence intensity level and low background, and more accurate polymorphism detection results were obtained. (Figure 3). The molecular mechanism underlying this phenomenon is not clear at this time. For example, when 4 primers 28F4, 28R4, 6F2 and 6R2 are used, the two intended amplicons (28F4-28R4 and In addition to 6F2-6R2), amplicons with 28F4-6R2 primer pairs can be generated, so it is possible that they may have an inhibitory effect on each other.

[Simultaneous detection of UGT1A1 * 28 and UGT1A1 * 6 polymorphisms using TaqManPCR-3]
Using 20 ng or 10 ng of human genomic DNA with known UGT1A1 * 28 and UGT1A1 * 6 site sequences as template DNA, and using the 28F4-6R2 primer pair and the four previously detected detection probes simultaneously, polymorphism detection The results are shown in FIG. Table 5 summarizes whether the sequences of the UGT1A1 * 28 and UGT1A1 * 6 sites were wild-type or mutant for each specimen.

  These results were in complete agreement with the already known polymorphisms by independent means. From this, it was found that if 20 ng or 10 ng of human genomic DNA was used, polymorphisms of UGT1A1 * 28 and * 6 could be detected easily, at low cost and accurately at the same time with good reproducibility.

  By using the method for detecting UGT1A1 * 28 polymorphism and * 6 polymorphism of the present invention, a reagent or kit for detecting UGT1A1 * 28 polymorphism and * 6 polymorphism can be obtained simply, at low cost, accurately and at high throughput. Can be provided. In addition, by using the UGT1A1 * 28 polymorphism and * 6 polymorphism detection method of the present invention, in order to predict the side effects of irinotecan and adjust the dose of the drug in a simple, low-cost, accurate and high-throughput manner. Necessary information can be provided to the doctor.

UGT1A1 UGT1A1 gene
Promoter promoter region
Exon area

28F4 UGT-28TprimerF4
28R4 UGT-28TprimerR4
6F2 UGT-6TprimerF2
6R2 UGT-6TprimerR2
6F7 UGT-6TprimerF7
6R7 UGT-6TprimerR7
Maximum fluorescence value of Endpoint, HEX HEX
Endpoint, FAM Maximum fluorescence value of FAM
Endpoint, Cy5 Cy5 Maximum fluorescence value
Endpoint, TXR Maximum fluorescence value of TXR
Homo homo
Hetero hetero
WT wild type

+ / + At the polymorphic site of interest, both homologous chromosomes must be mutated
+/- At the polymorphic site of interest, one homologous chromosome is mutated and the other homologous chromosome is wild-type
-/-At the polymorphic site of interest, both homologous chromosomes must be wild type
* 28 / * 28 UGT1A1 * 28 polymorphic site, both homologous chromosomes must be mutated
* 28 / * 6 One homologous chromosome is mutated at the UGT1A1 * 28 polymorphic site and the other homologous chromosome is wild-type, and one homologous chromosome is mutated at the UGT1A1 * 6 polymorphic site Note that the other homologous chromosome is wild type; note that the UGT1A1 * 28 polymorphism and the UGT1A1 * 6 polymorphism are on different chromosomes (different haplotypes).
* 6 / * 6 UGT1A1 * 6 polymorphic site, both homologous chromosomes must be mutated
* 28 /-At the UGT1A1 * 28 polymorphic site, one homologous chromosome is a mutant type and the other homologous chromosome is a wild type.
* 6 /-UGT1A1 * 6 polymorphic site, one homologous chromosome is mutated and the other homologous chromosome is wild type

Claims (4)

A method for simultaneously detecting two types of polymorphisms UGT1A1 * 28 and UGT1A1 * 6 that satisfy all the following conditions.
(a) Use the principle of TaqMan (registered trademark) PCR method
(b) TaqMan® PCR reaction solution contains a pair of primer pairs that amplify both the UGT1A1 * 28 polymorphic region and the UGT1A1 * 6 polymorphic region as a single amplicon.
(c) The TaqMan® PCR reaction solution does not contain a primer pair that amplifies only one of the UGT1A1 * 28 polymorphic region or UGT1A1 * 6 polymorphic region as an amplicon.
(d) a probe for detecting the UGT1A1 * 28 wild type, a probe for detecting the UGT1A1 * 28 mutant type, a probe for detecting the UGT1A1 * 6 wild type, wherein the TaqMan (registered trademark) PCR reaction solution is, And 4 types of probes for detecting UGT1A1 * 6 mutant
Here, a pair of primer pairs that amplify both the UGT1A1 * 28 polymorphic region and the UGT1A1 * 6 polymorphic region as one amplicon have the sequences of SEQ ID NO: 2 and SEQ ID NO: 5 The four probes, respectively, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and to have the sequence of SEQ ID NO: 11, The method of claim 1.   2. The method according to claim 1, wherein the four types of probes each have the sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. A kit for simultaneously detecting two types of polymorphisms UGT1A1 * 28 and UGT1A1 * 6, including all of the following:
(a) Two types of oligonucleotides for polymorphic region amplification having the sequences of SEQ ID NO: 2 and SEQ ID NO: 5
(b) Four types of polymorphism detection oligonucleotides having the sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, wherein the four types of polymorphism detection oligonucleotides are all , 5 'or 3' end is modified with a fluorescent dye and the other end is modified with a quencher