KR100706032B1 - A genotyping kit for surgical operation of paclitaxel eluting stent - Google Patents

Abstract

The present invention relates to a genotyping kit comprising a DNA chip and an electric DNA chip capable of analyzing the genetic polymorphisms of the MDR1 gene and the MRP2 gene. The kit for genotyping of the present invention comprises (i) a gene probe capable of specifically binding to the MDR1 gene and MRP2 gene of a human having a specific genetic polymorphism, and having a nucleotide sequence of SEQ ID NOs: 43 to 48; A linker comprising oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and an amine group sequentially, wherein the 5 ′ end of the electric gene probe is connected to the 3 ′ end of oligo (dT) ₁₅ ; And a aldehyde bonded to the surface, and a solid-state DNA chip comprising a solid in which an amine group of the electric linker is linked through a aldehyde group on the surface and a Schiff's base reaction; And, (ii) fluorescently labeled deoxy nucleotides for labeling the cDNA synthesized from RNA taken from the sample. If the kit for genotyping of the present invention can predict in advance whether vascular restenosis will occur in a patient who has undergone a paclitaxel release stent, the kit may be widely used for a safer stent procedure.

MDR1 gene, MRP2 gene, DNA chip, genotyping kit

Description

Genotyping Kit for Surgical Operation of Paclitaxel Eluting Stent

The present invention relates to a genotyping kit for determining the suitability of a procedure for paclitaxel release stent. More specifically, the present invention relates to a genotyping kit comprising a DNA chip and an electric DNA chip capable of analyzing the genetic polymorphisms of the MDR1 gene and the MRP2 gene.

Stenting is known to be the most effective treatment for patients with obstructive coronary artery disease. The stent procedure is a method of opening a blocked blood vessel by inserting a stent, which is a metal mesh form, into a blood vessel region in which a disease occurs, and has a characteristic of leaving a metallic stent at the procedure unlike a conventional balloon pottery procedure. However, due to the stent inserted into the blood vessel, the vessel wall is continuously damaged, and the abnormality of the vessel wall at the site where the stent is occluded is more likely to occur because of the abnormal cell proliferation of the damaged vessel wall. Efforts to overcome this are ongoing.

As a result of this effort, by coating a specific drug on the stent, after the stent procedure, a drug eluting stent is released in which a specific drug is released into the blood vessel, and by performing the drug release stent thus developed, The rate of stent restenosis due to stents has been significantly reduced (Windecker S. et al., N. Engl. J. Med., 353: 653-662, 2005).

However, for some specific drug coated stents, the rate of vascular restenosis due to the stent has not been reduced, a representative of which is paclitaxel releasing stent. Paclitaxel, which has a specific therapeutic effect on breast and ovarian cancer, has the effect of inhibiting cell proliferation by forming abnormal tubulin polymers in the process of cell proliferation. The advantage is that it can last. In order to utilize these characteristics, it was expected that paclitaxel-coated paclitaxel-releasing stents could prevent abnormal growth of vascular cells, which causes stenosis due to stents. The average rate of stent restenosis still ranges from 11.7-13.1%, and the results of the study have not been reported.

If the cause of restenosis occurring during the procedure of paclitaxel releasing stent can be identified, it is predicted that paclitaxel releasing stent can be cured with higher probability of coronary artery disease.

Therefore, the necessity of developing a method for safer procedure of paclitaxel-releasing stent has been steadily emerging by identifying restenosis occurring during the procedure of paclitaxel-releasing stent.

Therefore, the present inventors have investigated the restenosis occurring during the procedure of paclitaxel-releasing stent, and as a result of intensive research to develop a method that can be used to safely process paclitaxel-releasing stent, the genes in specific parts of human MDR1 gene and MRP2 gene In patients with genetic polymorphism, we found that the probability of restenosis during the procedure of paclitaxel-releasing stents maintained a high level. When using a diagnostic kit including a DNA chip to confirm whether a patient exhibits a specific genetic polymorphism prior to the procedure of paclitaxel releasing stent, it has been confirmed that the paclitaxel releasing stent can be safely performed, thereby completing the present invention. Was done.

After all, the main object of the present invention is to provide a DNA chip capable of detecting genetic polymorphisms in specific portions of human MDR1 gene and MRP2 gene.

It is another object of the present invention to provide a diagnostic kit comprising an electric DNA chip.

The kit for genotyping of the present invention comprises (i) a gene probe capable of specifically binding to the MDR1 gene and MRP2 gene of a human having a specific genetic polymorphism, and having a nucleotide sequence of SEQ ID NOs: 43 to 48; A linker comprising oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and an amine group sequentially, wherein the 5 ′ end of the electric gene probe is connected to the 3 ′ end of oligo (dT) ₁₅ ; And a aldehyde bonded to the surface, and a solid-state DNA chip comprising a solid in which an amine group of the electric linker is linked through a aldehyde group on the surface and a Schiff's base reaction; And, (ii) fluorescently labeled deoxy nucleotides for labeling the cDNA synthesized from RNA taken from the sample. In this case, the fluorescently labeled deoxy nucleotides are not particularly limited thereto, but are not limited thereto, such as Texas Red dATP, cyanine 3 dCTP, cyanine 5 dGTP, and fluorescein-12 dUTP. It is preferable to use such as.

In order to determine the cause of restenosis occurring during the procedure of paclitaxel releasing stent, the present inventors have identified multiple drug resistance gene-1 (MDR1) and its expression product P-glycoprotein, and Attention was directed to MRP2 (multidrug resistance associated protein 2).

MDR1 and its expression product, P-glycoprotein, are known to confer resistance to various drugs in cancer cells and normal cells. The MDR gene is a 100 kb gene present on chromosome 7 in humans, consisting of 28 axons spliced with 4.5 kb of mRNA, and the P-glycoprotein expressed therefrom is a type of ATP binding cassette subfamily B. (ATP binding cassette sub-family B member 1, ABCB1), which has been reported to have a broad substrate specificity capable of capturing a variety of drugs (Fojo AT et al., Proc. Natl. Acad. Sci., USA. 84: 265-9, 1987; Thiebaut F. et al., Proc. Natl. Acad. Sci., USA., 84: 7735-8, 1987; Pauli-Magnus C et al., Pharmaceutical Research, 21 (6 ): 904-913, 2004).

In addition, recent studies have shown that genetic polymorphism of MDR1 can express altered function of P-glycoprotein, and that the response to drugs may change depending on the genotype of MDR1. (Kim RB et al., Clin. Pharmacol. Ther., 70: 189-199, 2001; Kurata Y. et al., Clin. Pharmacol. Ther., 72: 209-219, 2002; Sakaeda T. et. al., Pharm. Res., 18: 1400-1404, 2001; Horinouchi M. et al., Pharm.Res., 19: 1581-1585, 2002; Siddiqui A. et al., N. Engl. J. Med , 348: 1442-1448, 2003).

In addition, the MRP2 gene, which encodes a protein belonging to ABCB1, is known to be involved in the formation of resistance to various drugs, similar to the MDR1 gene. It is known that the response to the drug may be different from that of other individuals (Yi SY et al., Clin. Pharmacol. Ther., 76: 418-427, 2004).

On the other hand, since the cell proliferation inhibitory activity of paclitaxel is known to be affected by the activity of the P-glycoprotein and MRP2 protein, the rate of 11.7-13.1% stent restenosis after the paclitaxel releasing stent was performed was MDR1 and MRP2 genes. It is assumed that it may be influenced by the genetic polymorphism of. To confirm this hypothesis, genomic DNA of patients undergoing paclitaxel release stents and vascular restenosis has been detected, and genetic polymorphisms are detected in genes encoding various MDR1 and MRP2 proteins present in the electrical genomic DNA. We attempted to screen for genetic polymorphisms that were found to be statistically related to the development of vascular restenosis. As a result, it was found that the 1774th base was statistically related to the occurrence of vascular restenosis, especially based on the transcription start points of the G2677T / A of the 21st axon of MDR1 and the C3435T and MRP2 genes of the 26th axon. In the case of MDR1 gene, it was confirmed that the genetic polymorphism, which is homozygous or heterozygous for the T trait, is statistically closely related to the development of vascular restenosis.

Therefore, the present inventors analyzed the genotype of the 1774th base on the basis of the transcription start points of the G2677T / A of the MDR1 electrolysis 21 axon and the C3435T and MRP2 genes of the 26 axon prior to performing the paclitaxel release stent. In patients with homozygous or heterozygous T-transformation and patients with homozygous 1774 bases on the basis of the transcription start of MRP2 gene, paclitaxel release stents are more likely to cause vascular restenosis. It can be recognized in advance that it is possible to prevent the occurrence of vascular restenosis caused by the procedure of paclitaxel release stent.

To demonstrate this, the genotype includes a DNA chip capable of detecting the 1774th base on the basis of the transcription start point of the G2677T / A of the 21st axon and the C3435T and MRP2 genes of the 26th axon representing the T trait of the MDR1 gene. An analysis kit was produced. Subsequently, after the paclitaxel releasing stent, each cDNA was obtained from a patient in which vascular restenosis occurred and a patient not in vascular restenosis, and then applied to the DNA chip. As a result, after the paclitaxel releasing stent, cDNA obtained from the patient who developed vascular restenosis was detected by binding specifically to the electric DNA chip, while cDNA obtained from the patient who did not develop vascular restenosis was It was confirmed that the non-specific binding to the DNA chip.

Therefore, a genotyping kit including a DNA chip capable of detecting the 1774th base on the basis of the transcription start point of the G2677T / A of the 21st axon and the C3435T and MRP2 genes of the 26th axon, which represents the T trait of MDR1, can be used. In this case, it was found that paclitaxel releasing stent can be confirmed in advance.

If the kit for genotyping of the present invention can predict in advance whether vascular restenosis will occur in a patient who has undergone a paclitaxel release stent, the kit may be widely used for a safer stent procedure.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Determination of Genotype in Patients with Coronary Artery Disease Treated with Paclitaxel Release Stent

Example 1-1 Determination of MDR1 Genotype in Patients with Coronary Artery Disease Treated with Paclitaxel Release Stent

A total of 106 patients with coronary artery disease who underwent a paclitaxel releasing stent (Taxus, Boston Scientific, USA) and who had been diagnosed with vascular restenosis by coronary angiography for 9 months, were given genomic DNA from them. MDR1 genotype was determined.

Specifically, 8 ml of blood collected from the patient was applied to a DNA extraction kit (QIAGEN, Hilden, Germany) to obtain genomic DNA of the patient. Using each of the genomic DNA obtained as a template as a template, using the following primers, PCR reaction solution (10.0ng of DNA, 1X PCR Buffer, 0.125units of AmpliTaq) Gold PCR was performed using DNA polymerase (ABI, USA), 3.0 mM MgCl ₂ , 0.25 mM of each dNTP, and 0.5 mmol of each primer in 10 μl reaction volume (10 minutes at 95 ° C., 30 cycles (95 ° C.) 30 seconds, 1 minute at 60 ° C. and 1 minute at 72 ° C.), and 5 minutes at 72 ° C.) to obtain amplified sections.

MDR1_-1459G / AF: 5'-ggagcaaagaaatggaatacaat-3 '(SEQ ID NO: 1), MDR1_-1459G / AR: 5'-ttctcccgtgaagaccaagtt-3' (SEQ ID NO: 2), MDR1_-935A / GF: 5'-caggaaacagctatgaccgcatgctgaagaa '(SEQ ID NO: 3), MDR1_-935A / GR: 5'-tgtaaaacgacggccagtccttctcccgtgaagac-3' (SEQ ID NO: 4), MDR1_-709C / GF: 5'-caggaaacagctatgaccgcatgctgaagaaagacc-3 (SEQ ID NO: 5) ', MDR1_-709C / GR : 5'-tgtaaaacgacggccagtccttctcccgtgaagac-3 '(SEQ ID NO: 6), MDR1_-693T / GF: 5'-ggagcaaagaaatggaatacaat-3' (SEQ ID NO: 7), MDR1_-693T / GR: 5'-ttctcccgtgaagaccaagtt-3 '(SEQ ID NO: 8 ), MDR1_-A61G-F: 5'-gaccgcaatggaggagc-3 '(SEQ ID NO: 9), MDR1_-A61G-R: 5'-actatgtaaactatgaaaatgaaacaag-3' (SEQ ID NO: 10), MDR1_-C1236T-F: 5'-caggaaacagc tgtacc -3 '(SEQ ID NO: 11), MDR1_-C1236T-R: 5'-tgtaaaacgacggccagttccatcaacactgacctgg-3' (SEQ ID NO: 12), MDR1_G2677T / AF: 5'-caggaaacagctatgacctcagcattctgaagtcatgg-3 '(SEQ ID NO: 13), MDR1_26 '-tgtaaaacgacggccagttccaagaactggctttgc-3' (SEQ ID NO: 14), MDR1_C3435T- F: 5'-tgtttgactgcagcattg-3 '(SEQ ID NO: 15), MDR1_C3435T-R: 5'-tttatttgaagagagacttacattagg-3' (SEQ ID NO: 16), MDR1_G3751A-F: 5'-acctgcattgtgattgct-3 '(SEQ ID NO: 17), MDR1_375 -R: 5'-actgaccattgaaaaatagatg- 3 '(SEQ ID NO: 18)

Subsequently, 1U of alkaline phosphatase and exonuclease I were treated with the previously obtained amplified sections, reacted at 37 ° C for 1 hour, and then treated at 72 ° C for 15 minutes to obtain purified sections. .

1 μl of the electrically purified fragments were applied to a genotyping kit (SNaPShot assay kit, ABI, USA) to obtain their respective sequencing data, and then the electrical data were transferred to the Gene Mapper software (ABI, USA). 9 SNP positions (Promoter-1459G / A (Experimental Group 1), Promoter-935A / G (Experimental Group 2), Promoter-709C / G / T (Experimental Group 3), Promoter-693T / C (Experimental Group 4)) , A61G of Axon No. 2 (Exp. 5), C1236T of Axon No. 12 (Exp. 6), G2677T / A of Axon No. 21 (Exp. 7), C3435T (Exp. 8) of Axon No. 26, and G3751A of Axon No. 28 (Exp. Genetic polymorphisms were analyzed in 9)) to compare their genotype distribution and vascular restenosis (see Table 1a).

Comparison of genotype distribution and vascular restenosis rate according to SNP position of MDR1 gene in patients undergoing paclitaxel release stent Experimental group Genotype distribution Vascular restenosis P value ^* Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 Experiment 8 Experiment 9 GG: GA: AA = 53 (52.0%): 40 (39.2%): 9 (8.8%) AA: AG: GG = 89 (84.0%): 16 (15.1%): 1 (0.9%) CC: CG, CT: GG, TT = 101 (95.3%): 5 (4.7%): 0 TT: TC: CC = 95 (90.5%): 10 (9.5%): 0 (0%) AA: AG: GG = 106 ( 100%): 0 (0%): 0 (0%) CC: CT: TT = 17 (16.0%): 49 (46.2%): 40 (37.7%) GG: GT: TT = 42 (40.8%): 48 (46.6%): 13 (12.6%) ^b CC: CT: TT = 42 (40.0%): 51 (48.6%): 12 (11.4%) TT: TC: CC = 95 (90.5%): 10 (9.5 %): 0 (0%) GG: GA: AA = 9/53: 6/40: 0/9 GG: GA: AA = 14/89: 2/16: 0/1 CC: CG, CT: GG, TT = 16/101: 0 / 5: 0/0 TT: TC: CC = 15/95: 1/10: 0/0 AA: AG: GG = 16/106: 0/0: 0/0 CC: CT: TT = 1/17: 9 / 49: 6/40 GG: GT: TT = 2/44: 12/47: 2/13 CC: CT: TT = 1/44: 13/50: 2/12 GG: GA: AA = 16/104: 0/2: 0/0 0.412 0.865 1.00 1.00- ^a 0.464 0.008 0.002 1.00

*: Significant for P <0.05

a: no gene polymorphism

b: G and A traits have the same function

As shown in Table 1a, the results of Experimental Group 7 (G2677T / A of Axon No. 21) and Experimental Group 8 (C3435T of Axon No. 26) were significant. G2677T / A and Axon No. 26 of Axon No. 21 were significant. C3435T was predicted to be associated with vascular restenosis by paclitaxel releasing stent procedure.

Example 1-2 Determination of MRP2 Genotype in Patients with Coronary Artery Disease Treated with Paclitaxel Release Stent

A total of 106 patients with coronary artery disease who underwent a paclitaxel releasing stent (Taxus, Boston Scientific, USA) and who had been diagnosed with vascular restenosis by coronary angiography for 9 months, were given genomic DNA from them. MRP2 genotype was determined. That is, except that the following primer was used, PCR was performed in the same manner as in Example 1-1, to obtain respective fragments, and their nucleotide sequences were determined.

1774-F: 5'-caggaaacagctatgaccgattctccaccctctctttt-3 '(SEQ ID NO: 19), 1774-R: 5'-tgtaaaacgacggccagtcattcagtgtgggagaaaat-3' (SEQ ID NO: 20), 1549-F: 5'-caggaaacagctatgacccccactttttaatt't 21 , 1549-R: 5'-tgtaaaacgacggccagtctgggactacaggcacat-3 '(SEQ ID NO: 22), 24-F: 5'-caggaaacagctatgacctaggctcacactggataagc-3' (SEQ ID NO: 23), 24-R: 5'-tgtaaaacgacggccagttgcacatctaacatttctgg-3 '(SEQ ID NO: 24) ), 23-F: 5'-caggaaacagctatgacctaggctcacactggataagc-3 '(SEQ ID NO: 25), 23-R: 5'-tgtaaaacgacggccagttgcacatctaacatttctgg-3' (SEQ ID NO: 26), 31-F: 5'-caggaaacagctatgacccatgggtcct (SEQ ID NO: 3) 27), 31-R: 5'-tgtaaaacgacggccagtccccatggtacctcctcat-3 '(SEQ ID NO: 28), 1249-F: 5'-tttgtccatgggtcctaattt-3' (SEQ ID NO: 29), 1249-R: 5'-atgaagttggtcacatccatg-3 '(SEQ ID NO: 27) Number 30), 1457-F: 5'-atggtgcttgtaatcccaatt-3 '(SEQ ID NO: 31), 1457-R: 5'-ttgcccaaactcccatta-3' (SEQ ID NO: 32), 19-F: 5'-aaaaaaggagagtttgctaagaatc-3 '( SEQ ID NO: 33), 19-R: 5'-atactgagcagttcaggaattagatatt-3 '(SEQ ID NO: 33) 34), 2934-F: 5'-agttctactaatattgaggtgggga-3 '(SEQ ID NO: 35), 2934-R: 5'-aataaaagccacagaattcatca-3' (SEQ ID NO: 36), 3972-F: 5'-taccgacctgagctggatc-3 '( SEQ ID NO: 37), 3972-R: 5'-catccaggccttccttca-3 '(SEQ ID NO: 38), 30-F: 5'-caggaaacagctatgaccgtagccactccgagccttag-3' (SEQ ID NO: 39), 30-R: 5'-tgtaaaacgacggccagtggaatcagacctggatgaaaa-3 ' (SEQ ID NO: 40), 12-F: 5'-cccacaggctgcacaccat-3 '(SEQ ID NO: 41), 12-R: 5'-tgttactgttgagcaagggtta-3' (SEQ ID NO: 42)

Then, input it into the genotyping program and place 12 SNP positions (the 1774 base at the top based on the transcription start point of the MRP2 gene (experimental group 11), the 1549 base at the top based on the transcription start point (experimental group 12), transcription 24th base (experimental group 13) to the top based on the starting point, 23rd base (experimental group 14) to the top based on the starting point of transcription, 49th base (experimental group 15) to the top based on the axon 4, and 1249th base of the axon 10 (experimental) 16), 1457th base of axon 10 (experimental group 17), 3rd base downward (experimental group 18) based on axon 19, 2934th base of axon 22 (experimental group 19), 3972th base of axon 28 (experimental group 20) Genetic polymorphisms were analyzed at the 35th base (Experimental Group 21) and 12th base (Experimental Group 22) at the bottom based on Axon 31, and the results of the analysis were compared. 1b).

Comparison of genotype distribution and vascular restenosis rate according to SNP position of MRP2 gene in patients undergoing paclitaxel release stent Experimental group genotype characteristics restenosis (-) ^a n = 91 restenosis (+) ^b n = 15 P value ^* Experimental Group 11 Wild type G / G 41 7 0.0010 Heterojunction G / Del 45 3 Variant Del / Del 5 5 Experimental Group 12 Wild type AA 4 One 0.0110 Heterojunction AG 42 One Variant GG 42 13 Experimental Group 13 Wild type CC 43 13 0.0100 Heterojunction CT 43 One Variant TT 3 One Experimental group 14 Wild type GG 90 14 0.1420 Heterojunction GA One One Variant AA 0 0 Experimental group 15 Wild type CC 42 13 0.0080 Heterojunction CT 45 One Variant TT 4 One Experimental Group 16 Wild type GG 73 11 0.5180 Heterojunction GA 15 4 Variant AA 3 0 Experimental Group 17 Wild type CC 90 15 0.6830 Heterojunction CT One 0 Variant TT 0 0 Experimental Group 18 Wild type AA 91 15 . Heterojunction AG 0 0 Variant GG 0 0 Experimental Group 19 Wild type GG 84 15 0.5880 Heterojunction GA 5 0 Variant AA One 0 Experimental Group 20 Wild type CC 42 13 0.0080 Heterojunction CT 45 One Variant TT 4 One Experimental group 21 Wild type GG 89 15 0.8570 Heterojunction GA One 0 Variant AA 0 0 Experimental Group 22 Wild type GG 88 17 . Heterojunction GA 0 0 Variant AA 0 0

a: no vascular restenosis occurs

b: vascular restenosis occurs

*: Significant for P <0.05

As shown in Table 1b, it was predicted that the 1774th base on the basis of MRP2 gene transcription start point may be related to vascular restenosis by paclitaxel releasing stent procedure.

Example 2 Haplotype Analysis

In the results of Example 1-1, the haplotypes of G2677T / A of the 21st axon and the C3435T of the 26th axon of the MDR1 gene predicted to be related to vascular restenosis by paclitaxel release stent procedure The analysis was performed.

That is, in each genomic DNA obtained from 106 patients with coronary artery disease of Example 1, sequence data of 21 axon and 26 axon were obtained from a haploid program based on a Bayesian algorithm (Haploview 3.2, Broad Institute of Harvard and MIT, USA) program and the results were analyzed (Table 2).

Haplotype Analysis and Comparison of MDR1 Genes Haflow type Restenosis (+) ^a Restenosis (-) ^b P value ^* GC 13 (43.1%) 120 (65.8%) 0.0173 TT 16 (53.1%) 54 (29.6%) 0.0113 TC 0 (0.0%) 4 (2.3%) 0.4628 GT 1 (0.3%) 4 (2.3%) 0.6775 Sum 30 182

a: no vascular restenosis occurs

b: vascular restenosis occurs

*: significant for p <0.05

As shown in Table 2 above, as a result of performing haplotype analysis of the MDR1 gene, four haplotypes were detected. Among these, considering the P value, the TT haplotype was predicted to be closely related to vascular restenosis by paclitaxel release stent procedure (P = 0.0113).

Example 3 Basic Physiological Data Analysis of Patients

Example 3-1 Basic Physiological Data Analysis of G2677T / A of MDR1 Gene 21 Axon and C3435T of 26 Axon 26

Among the patients with genetic polymorphism at G2677T / A of 21 axon selected in Example 1-1 above, there were 42 GG / AA polymorphisms and 61 GT / TT polymorphisms and genetic polymorphisms at C3435T of 26 axon The 44 CC polymorphisms and 62 CT / TT polymorphisms among the patients were analyzed for their age (A), sex ratio (S), diabetes rate (D), hypertension rate (H), smoker rate (Sk), and transfer. Basic physiological data such as the percentage of patients with a history of acute myocardial infarction (Hm) were compared (see Table 3a).

Comparison of Basic Physiological Data of Patients According to Genotype of MDR1 Gene G2677T / A P value ^* C3435T P value GG / AA (42) GT / TT (61) CC (44) CT / TT (62) A S (M: F) D (%) H (%) Sk (%) Hm (%) 60.3 ± 10.2 29:13 14 (33.3%) 25 (59.5%) 8 (19.0%) 8 (19.0%) 60.2 ± 8.8 49:12 13 (21.3%) 28 (45.9%) 16 (26.2%) 7 (11.5%) 0.955 0.189 0.173 0.174 0.350 0.255 60.0 ± 8.9 30:14 14 (32.6%) 24 (55.8%) 8 (18.6%) 7 (15.9%) 59.8 ± 9.8 49:13 14 (22.6%) 30 (48.4%) 17 (27.4%) 14 (22.2%) 0.903 0.360 0.271 0.552 0.356 0.492

*: Significant for P <0.05

As shown in Table 3a, the basic physiological data of the patients did not show a significant difference depending on the genotype.

Example 3-2 Basic Physiological Data Analysis of the 1774th Base from Above Based on MRP2 Gene Transcription Start Point

Basic physiological studies of the patients in the method of Example 3-1, except that patients with a genetic polymorph at the 1774th base up were based on the MRP2 gene transcription start point selected in the above Examples 1-2. The data were analyzed (see Table 3b).

Comparison of Basic Physiological Data of Patients According to Genotype of MRP2 MRP2_-1774G / Del P value ^* GG (48) G / Del (48) Del / Del (10) A S (M: F) D (%) H (%) Sk (%) Hm (%) 60.0 ± 8.9 37:11 15 30 9 8 60.3 ± 10.2 36:12 10 21 14 7 59.8 ± 9.8 7: 3 3 4 3 0 0.903 0.889 0.828 0.135 0.453 0.386

*: Significant for P <0.05

As shown in Table 3b, the basic physiological data of the patients did not show a significant difference according to the genotype.

Example 4 Quantitative Analysis of Coronary Angiography Data of Patients

Example 4-1 Quantitative Analysis of Coronary Angiography Data for G2677T / A of Axon 21 and C3435T of Axon 26

Among the patients with genetic polymorphism at G2677T / A of 21 axon selected in Example 1-1 above, there were 42 GG / AA polymorphisms and 61 GT / TT polymorphisms and genetic polymorphisms at C3435T of 26 axon Among 44 patients with CC polymorphism and 62 CT / TT polymorphisms, coronary angiography data (multi-stenting rate (MS), incidence of type B2 / C lesions (BC), average baseline diameter before pre-RD) ), Mean post-MLD postoperatively, pre-LL lesion length, acute gain (AG), restenosis rate within the stent (STR), restenosis rate around the stent (SGR), 9 months After the mean (FU) mean reference diameter (FU-RD), after 9 months the mean minimum inner diameter (FU-MLD), late loss (LL) and loss index (LI) were measured, and the measured values were measured by the quantitative analysis system ( Quantitative analysis was performed by typing in a quantitative coronary angiography system, Inturis DICOM Recorder, ModuleInfo, Philips, Netherlands (see Table 4a). In this case, AG means the difference between the minimum inner diameter (post-MLD) after the last stent procedure and the minimum inner diameter before the stent procedure, LL means the difference between the post-MLD and FU-MLD, LI is the LL value The result of dividing by AG.

Comparison of Coronary Angiography Data of Patients According to Genotype in MDR1 Gene G2677T / A P value C3435T P value ^* GG / AA (42) GT / TT (61) CC (44) CT / TT (62) MS (%) BC (%) pre-RD (mm) post-MLD (mm) pre-LL (mm) AG (mm) STR (%) SGR (%) FU-RD (mm) FU-MLD (mm) LL (mm) LI 8 (19.0%) 31 (73.8%) 2.86 ± 0.44 2.65 ± 0.49 25.8 ± 14.4 2.00 ± 0.47 1 (2.4%) 2 (4.8%) 2.88 ± 0.43 2.48 ± 0.59 0.16 ± 0.58 0.07 ± 0.29 10 (16.4%) 41 (67.2%) 2.86 ± 0.45 2.72 ± 0.44 21.3 ± 12.2 2.00 ± 0.53 14 (22.9%) 14 (22.9%) 2.84 ± 0.47 2.23 ± 0.76 0.48 ± 0.73 0.23 ± 0.45 0.727 0.473 0.950 0.420 0.089 0.956 0.004 0.012 0.690 0.087 0.022 0.047 9 (20.5%) 31 (72.1%) 2.82 ± 0.41 0.62 ± 0.47 25.8 ± 14.9 2.00 ± 0.48 0 (0%) 1 (2.3%) 2.87 ± 0.39 2.48 ± 0.52 0.14 ± 0.54 0.05 ± 0.26 9 (14.5%) 41 (66.1%) 2.87 ± 0.46 0.76 ± 0.47 20.9 ± 11.5 1.98 ± 0.51 14 (22.6%) 14 (22.6%) 2.87 ± 0.50 2.23 ± 0.81 0.49 ± 0.73 0.25 ± 0.46 0.341 0.345 0.586 0.122 0.064 0.768 0.001 0.004 0.990 0.072 0.009 0.017

*: significant for p <0.05

As shown in Table 4a, the T traits present in the two genotypes were analyzed to have a significant correlation with restenosis after stent surgery.

In particular, the LI value is a value directly indicating the degree of restenosis occurring after the stent procedure, and it may be considered that the restenosis rate increases as the LI value increases. Therefore, referring to the LI value of Table 4a, it was confirmed that the rate of restenosis increased by about 3 to 5 times in patients with genetic polymorphisms having at least one T trait than in patients without T traits.

Example 4-2 Quantitative Analysis of Coronary Angiography Data of the 1774th Base on the Top of MRP2 Gene Transcription Start Point

Coronary vessels of the patients by the method of Example 4-1, except for targeting patients with a genetic polymorph at base 1774 based on the MRP2 gene transcription start point selected in Examples 1-2 above The imaging data (LL and LI) were obtained and quantified (see Table 4b).

Comparison of Coronary Angiographic Data of Patients with Genotypes in MRP2 Gene MRP2_-1774G / Del P value ^* GG (48) G / Del (48) Del / Del (10) LL (mm) LI 0.508 0.254 0.108 0.015 0.981 0.598 0.001 <0.001

*: significant for p <0.05

As shown in Table 4b, when the 1774th base was removed based on the MRP2 gene transcription start point, it was analyzed that the incidence of restenosis after the stent procedure was significantly increased.

In particular, when referring to the LI value, when the 1774th base is removed from the top of the MRP2 gene transcription start point, it was confirmed that the restenosis rate is increased by about two times or more than the patient without the base removed.

Example 5 : Preparation of Genotyping Kits

As shown in the results of the above Examples 1 to 4, by analyzing the genetic polymorphisms of specific regions of the MDR1 gene and the MRP2 gene, it was possible to predict the occurrence of vascular restenosis during the procedure of paclitaxel releasing stent. In order to be able to analyze the polymorphisms, genotyping kits were prepared comprising DNA chips capable of detecting the electrical polymorphisms and fluorescently labeled deoxy nucleotides used to obtain cDNA from mRNA obtained from a sample.

First, an analytical probe was constructed to analyze the genetic polymorphism of the 1774th base on the basis of the transcription start points of the G2677T / A of the 21st axon of MDR1 and the C3435T and MRP2 genes of the 26th axon. The sequence is as follows.

2677F: 5'-caggaaacagctatgacctattcgaagagtgggcacaa-3 '(SEQ ID NO: 43)

2677R: 5'-tgtaaaacgacggccagttccaagaactggctttgct-3 '(SEQ ID NO: 44)

3435F: 5'-tgtttgactgcag-3 '(SEQ ID NO: 45)

3435R: 5'-tttatttgaagagagacttacattaggc-3 '(SEQ ID NO: 46)

1774F: 5'-agattcatgacttcctggctcctt-3 '(SEQ ID NO 47)

1774R: 5'-acaacaattctccttcctcacacg-3 '(SEQ ID NO 48)

Then, a linker in which oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and an amine group were sequentially formed was constructed, and the 5 'end of each probe obtained before was connected to the thymine portion of the linker.

Subsequently, the probes connected to the electric linker were dissolved at 50 μM in buffer (350 mM sodium bicarbonate, pH 9.0), respectively, and the arrayer (MicroGrid II, BioRobotics, USA) was used to drop the size 150㎛, 400㎛ interval, and then the seed base reaction. Subsequently, the mixture was washed sequentially with 0.2% (w / v) SDS solution and tertiary distilled water, and immersed in NaBH ₄ solution (0.1 g NaBH ₄ , 30 ml PBS, 10 ml ethanol) for 15 minutes. The non-aldehyde residue was reduced, washed with tertiary distilled water and dried to prepare a DNA chip.

Meanwhile, cyanine 5 dGTP (Cyanine 5 dGTP, Amersham, GB), which is a fluorescently labeled deoxy nucleotide, was prepared to label cDNA synthesized from RNA collected from a sample.

Example 6 Screening of Patients with Vascular Stenosis Using Genotyping Kit

40 patients (experimental group 31) who performed the paclitaxel-releasing stent of Example 1 and showed vascular restenosis symptoms and 40 patients who performed the paclitaxel-releasing stent of Example 1 and did not show vascular restenosis symptoms (Experimental group 32) Blood was collected to obtain blood, and each obtained blood was subjected to an RNA extraction kit (Micro-to-Midi Total RNA purification system, Life Technologies, USA) to separate each mRNA. Each cDNA was fluorescently labeled from the mRNA isolated from the electro-isolated mRNA using cyanine 5 dGTP included in the reverse transcription kit (Platinum Biochip Reagent Kit, Genocheck, Korea) and the kit prepared in Example 5 above. .

Subsequently, each of the cDNAs electrosynthesized was added to the DNA chip prepared in Example 5, hybridization reaction was performed for 12 hours, and the hybridization terminated DNA chip was subjected to a flat plate scanner (GenePix Scanner 4000A, Axon Inc, USA). The number of probes showing fluorescence reaction among the probes included in the electric DNA chip was counted, and the average ratio of the color reaction probes was measured therefrom (see Table 5).

Average color reaction rate (%) of each sample detected by DNA chip Experimental group Average color reaction rate Experimental group 31 80.6 Experimental group 32 13.4

As shown in Table 5, the DNA chip included in the genotyping kit prepared in Example 6 showed a high rate of color reaction for the samples obtained from patients undergoing paclitaxel release stents and showing vascular restenosis. In addition, the samples obtained from patients undergoing paclitaxel release stents and not showing vascular restenosis showed a very low rate of color reaction.

Therefore, using the genotyping kit of the present invention prior to the operation of the stent, if the genotype of the patient is confirmed in advance, it was analyzed whether the patient is suitable for the procedure of paclitaxel release stent.

As described and demonstrated in detail above, the present invention provides a genotyping kit comprising a DNA chip and an electric DNA chip capable of analyzing the genetic polymorphisms of the MDR1 gene and the MRP2 gene. If the kit for genotyping of the present invention can predict in advance whether vascular restenosis will occur in a patient who has undergone a paclitaxel release stent, the kit may be widely used for a safer stent procedure.

Attach sequence list electronic file

Claims (3)

(Iii) a gene probe capable of specifically binding to a human MDR1 gene and an MRP2 gene and having a nucleotide sequence of SEQ ID NOs: 43 to 48; (Ii) a linker comprising oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and amine groups sequentially, wherein the 5 'end of the electric gene probe is connected to the 3' end of oligo (dT) ₁₅ ; And, (Iii) A DNA chip for genotyping of MDR1 and MRP2 genes, in which an aldehyde is bonded to a surface and an amine group of an electric linker is linked to a surface aldehyde and a Schiff's base reaction. The genotyping kit for genotyping of MDR1 gene and MRP2 gene, comprising a fluorescently labeled deoxy nucleotide for labeling cDNA synthesized from the DNA chip for genotyping of claim 1 and RNA collected from a sample. The method of claim 2, The fluorescently labeled deoxy nucleotides are Texas Red dATP, cyanine 3 dCTP, cyanine 5 dGTP or fluorescein-12 dUTP. Kit for genotyping.