WO2020153617A1 - Il6 gene single nucleotide polymorphism marker for predicting risk of developing drug-induced leukopenia and method for predicting risk of leukopenia using same - Google Patents

Il6 gene single nucleotide polymorphism marker for predicting risk of developing drug-induced leukopenia and method for predicting risk of leukopenia using same Download PDF

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WO2020153617A1
WO2020153617A1 PCT/KR2019/018715 KR2019018715W WO2020153617A1 WO 2020153617 A1 WO2020153617 A1 WO 2020153617A1 KR 2019018715 W KR2019018715 W KR 2019018715W WO 2020153617 A1 WO2020153617 A1 WO 2020153617A1
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leukopenia
risk
predicting
single nucleotide
nucleotide polymorphism
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김주한
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서울대학교 산학협력단
서울대학교병원
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a composition for predicting the risk of leukopenia including a monobasic polymorphic marker in the IL6 gene and a method for predicting the risk of leukopenia using the same.
  • Thiopurine-based drugs such as 6-mercaptopurine, azathiopurine, and thioguanine are widely used in the treatment of patients such as leukemia, Crohn's disease, and ulcerative colitis. It is an immunosuppressive drug.
  • the main problem with the use of cheopurine-based drugs is that even when patients start taking the prescribed doses calculated according to BSA, side effects such as leukopenia or liver toxicity occur in about 6% of patients.
  • Thiopurine-based drug activity is associated with thiopurine s-methyltransferase (TPMT), in which case the TPMT gene has a mutation, TPMT enzyme activity decreases and TGN metabolite increases As a result, serious bone marrow suppression may be reported.
  • TPMT thiopurine s-methyltransferase
  • the U.S. Food and Drug Administration recommends measuring TPMT enzyme activity before using cheopurine-based drugs.
  • the incidence of TPMT mutations in Asians is 2 to 3%, which is found to be significantly lower than in whites (10%), it is questionable whether it is useful to measure the enzyme activity of TPMTs in Asians in advance.
  • previous studies have shown that the NUDT15 mutation, which has been reported to be the genetic cause of leukopenia induced by thiopurine, is observed at a relatively high frequency in 42.5% of Korean leukopenia patients, but the remaining patients are not explained by the NUDT15 mutation. Considering, the understanding of the genetic causes of leukopenia in Asians is still lacking.
  • Patent Document 0001 Korean Registered Patent Publication No. 10-1704143
  • the present inventors conducted a study to develop a marker capable of predicting the risk of developing leukopenia, which is one of the side effects caused by cheopurine-based drugs, and as a result, the significance of specific monobasic polymorphic sites in the IL6 gene and leukopenia
  • the present invention was completed by confirming the correlation.
  • An object of the present invention is to provide a composition for predicting the risk of leukopenia including a monobasic polymorphic marker in the IL6 gene.
  • Another object of the present invention is to provide a kit for predicting the risk of leukopenia, comprising a polynucleotide specifically hybridizing to a polynucleotide comprising the single-basic polymorphic marker site.
  • Another object of the present invention is to provide a method for providing information on predicting the risk of leukopenia including the step of identifying the genotype of the monobasic polymorphic marker.
  • the present invention is a polynucleotide consisting of 10 to 100 consecutive DNA sequences comprising a single nucleotide polymorphism (SNP) region of the 101st nucleotide of SEQ ID NO: 1 or complementary thereof. It provides a composition for predicting the risk of leukopenia, comprising a polynucleotide.
  • SNP single nucleotide polymorphism
  • the present invention includes a polynucleotide composed of 10 to 100 contiguous DNA sequences or a complementary polynucleotide thereof, which specifically hybridizes with the single-nucleotide polymorphic site of the 101 nucleotide of SEQ ID NO: 1.
  • the present invention provides a kit for predicting the risk of leukopenia.
  • the present invention provides a method for providing information on predicting the risk of leukopenia including the step of identifying the genotype of the 101 nucleotide of SEQ ID NO: 1 from sample DNA.
  • the monobasic polymorphic marker in the IL6 gene according to the present invention has a high correlation with the risk of leukopenia induced by drugs, in particular, chipurin-based drugs, and thus, leukemia, Crohn's disease, ulcerative colitis, or Customized patient who can effectively predict or diagnose patients with high susceptibility to leukopenia occurring during chemopurine-based drug treatment in organ transplantation, etc., and achieve optimal treatment effects by administering the appropriate drugs to patients. Treatment can be performed efficiently. Furthermore, the monobasic polymorphic marker according to the present invention can be used in drug development studies for the treatment of drug-induced leukopenia.
  • FIG. 1 is a view showing a process for selecting a patient group to be used as an analysis object in one embodiment of the present invention among leukemia patients using 6-mercaptopurine.
  • FIG. 2 is a diagram showing specific results confirming a correlation between a single nucleotide polymorphism site (rs13306435) on a IL6 gene and a dose percentage decrease (Trend test).
  • FIG. 3 is a diagram showing specific results confirming a correlation between a single nucleotide polymorphism site on the IL6 gene (rs13306435) and G4N (Grade 4 Neutropenia).
  • the present invention comprises a single nucleotide polymorphism (SNP) region of the 101 nucleotide of SEQ ID NO: 1, polynucleotide consisting of 10 to 100 consecutive DNA sequence or a complementary polynucleotide thereof It provides a composition for predicting the risk of leukopenia, comprising.
  • SNP single nucleotide polymorphism
  • 'polymorphism' means a case in which two or more alleles exist in a single locus
  • 'polymorphic site' means a locus in which the allele exists.
  • a single base differs from person to person is referred to as'single nucleotide polymorphism', that is, single nucleotide polymorphism (SNP).
  • the term'allele' refers to several types of a gene present in the same locus of the homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.
  • the "SEQ ID NO: 1" is a polymorphic sequence comprising a polymorphic site.
  • the polymorphic sequence means a sequence comprising a polymorphic site containing SNP in the polynucleotide sequence.
  • the 101 nucleotide of SEQ ID NO: 1 is present at the IL locus on 7p15.3, and can be represented by rs13306435.
  • the allele of SEQ ID NO: 101, rs13306435, is T.
  • the allele of A at the time of mutation is A. Therefore, since the 101st nucleotide of SEQ ID NO: 1 may be A or T(U), it was described as “w” according to the multi-base description method.
  • the polynucleotide or the complementary polynucleotide thereof according to the present invention may be composed of 10 or more, preferably 10 to 100, more preferably 20 to 80, even more preferably 40 to 60 consecutive bases And is not limited thereto.
  • leukopenia includes those induced by thiopurine-based drugs, for example, when treating leukemia, Crohn's disease, ulcerative colitis, or organ transplant drugs in patients with organ transplants. It may be induced.
  • the cheopurine-based drug includes, but is not limited to, 6-mercaptopurine, azathioprine, or thioguanine.
  • the monobasic polymorphic marker can be used for predicting the risk of leukopenia, a specific base at the site of a monobasic polymorphism as a result of genetic analysis of a group in which leukopenia, a side effect that occurs with the administration of cheopurine-based drugs It is based on the high probability of existence.
  • the monobasic polymorphic marker in the IL6 gene according to the present invention has a high correlation with leukopenia induced by drugs, especially cheopurine-based drugs, and the resulting dose reduction, thereby using leukemia, Crohn's disease, ulcerative colitis Or, it can effectively predict or diagnose patients with high sensitivity to leukopenia, which occurs during chemopurine-based drug treatment, such as organ transplantation, and can achieve optimal treatment effects by administering appropriate drugs to patients. Patient-specific treatment can be efficiently performed. Furthermore, the monobasic polymorphic marker according to the present invention can be used in drug development studies for the treatment of drug-induced leukopenia.
  • the present invention comprises a polynucleotide consisting of 10 to 100 consecutive DNA sequences or a complementary polynucleotide thereof, comprising a single nucleotide polymorphism (SNP) region of the 101 nucleotide of SEQ ID NO: 1
  • a kit for predicting the risk of leukopenia comprising polynucleotides that specifically hybridize.
  • the polynucleotide that specifically hybridizes with the polynucleotide or its complementary polynucleotide is an allele-specific polynucleotide.
  • An allele-specific polynucleotide means to hybridize specifically to each allele. That is, it refers to hybridization so that the base of the polymorphic site present in the polymorphic sequence can be specifically distinguished.
  • It may be a probe or a primer that specifically hybridizes with the polynucleotide or a complementary polynucleotide thereof, but is not limited thereto.
  • the "probe” refers to nucleic acid fragments such as RNA or DNA corresponding to a few bases to hundreds of bases, which are capable of specifically binding other than mRNA, and are labeled to confirm the presence or absence of a specific mRNA and expression level.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. The appropriate probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.
  • the "primer” is a nucleic acid sequence having a short free 3-terminal hydroxyl group, capable of forming complementary templates and base pairs, and acting as a starting point for template strand copying. Speak.
  • the primer can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer lengths can be appropriately selected according to techniques known in the art.
  • the kit may be a kit of various types depending on the method using the polynucleotide, and includes, but is not limited to, PCR kit, DNA chip kit, microarray, and the like.
  • the present invention provides a method for providing information on predicting the risk of leukopenia, comprising the step of identifying the genotype of the 101 nucleotide of SEQ ID NO: 1 from sample DNA.
  • the allele of the 101st nucleotide of SEQ ID NO: 1 is TA or AA, it can be predicted that the probability of developing leukopenia is higher than when the allele is TT.
  • leukopenia includes those induced by thiopurine-based drugs, for example, when treating leukemia, Crohn's disease, ulcerative colitis, or organ transplant drugs in patients with organ transplants. It may be induced.
  • the cheopurine-based drug includes, but is not limited to, 6-mercaptopurine, azathioprine, or thioguanine.
  • the method for providing information according to the present invention may further include isolating DNA from a biological sample in order to obtain sample DNA.
  • DNA separation phenol/chloroform extraction method, SDS extraction method, CTAB separation method (Cetyl Trimethyl AmmoniumBromide; Murray et al., Nuc. Res., 4321-4325, 1980) commonly used in the art, or commercially available It can be performed using a DNA extraction kit, but is not limited thereto.
  • the biological sample includes, but is not limited to, blood, saliva, urine, skin cells, mucosal cells, and hair tissue of the subject.
  • Gene sequence analysis may be performed to perform the step of identifying the genotype.
  • any method known in the art can be used, for example, using an automatic base sequencer, pyrosequencing, restriction fragment length polymorphism (PCR-RELP), PCR-SSCP method ( Single strand conformation polymorphism), PCR-SSO method (specific sequence oligonucleotide), PCR-SSO method and dot hybridization method (allele specific oligonucleotide) hybridization method, TaqMan-PCR method, MALDI-TOF/MS method, RCA method (Rolling circle amplification), HRM (high resolution melting) method, primer extension method, Southern blot hybridization method and a known method such as dot hybridization method can be used, but is not limited thereto.
  • PCR-RELP restriction fragment length polymorphism
  • PCR-SSCP method Single strand conformation polymorphism
  • PCR-SSO method specific sequence oligonucleotide
  • NUDT15 Normal Metabolizer (*1/*1) NUDT15 Intermediate Metabolizer (*1/*2, *1/*3, *1/*4, *1/*5, *1/*6, *1/*9) NUDT15 Poor Metabolizer (*3/*3) total TPMT Normal Metabolizer (*1/*1, *1/*1S, *1S/*1S) 188 48 One 237 TPMT Intermediate Metabolizer (*1/*3C, *1S/*3C, *1/*6) 6 One 7 TPMT Poor Metabolizer total 194 49 One 244
  • the initial recommended dose of 6-mercaptopurine is 75 mg/m 2 .
  • the initial dose was adjusted from 75 mg/m 2 to 50 mg/m 2 .
  • the protocol for treatment was determined on a case-by-case basis by the responsible physician for the modification or discontinuation of the drug dose due to adverse events.
  • Past medical records for each patient were reviewed, and information on 6-mercaptopurine, including dose, duration of treatment, and the timing and extent of leukopenia was independently assessed by physicians with unknown genotyping results.
  • 6-Mercaptopurine was prescribed with a schedule of 6-10 repetitions of the same anticancer treatment for one cycle lasting a total of 12 weeks.
  • the first cycle he started taking a prescribed dose of the drug based on the body surface area (BSA), and continued to reduce the dose of 6-mercaptopurine according to adverse events such as leukopenia or hepatotoxicity.
  • BSA body surface area
  • the dose percentage is the ratio of the BSA-based and actual doses in the last cycle of maintenance therapy and is defined as follows.
  • G4N Gram 4 Neutropenia incidence
  • candidate genes were selected by performing multiple covariate linear regression analysis using GVB as a dose percentage and G4N as dependent variables.
  • GVB Genetic Variant Burden
  • the term GVB means the geometric mean of SIFT scores less than 0.7 among SIFT scores, which are indicators of deteriousness of mutation.
  • a multi-covariate linear regression analysis using dose percentage and G4N as dependent variables was performed for all variations of the candidate genes, and three candidate variations shown in Table 2 were selected. Among them, the rs13306435 mutation of the IL6 gene corresponding to the nonsynonymous variant was selected as the final mutation.
  • a single nucleotide polymorphism site was performed by performing a regression analysis among 188 persons selected in Example 1 The effect of (rs13306435) on the dose percentage was statistically confirmed.
  • Correction factors for regression analysis include gender, age at the start of the last cycle, BSA, and genotypes of mutations (0 for wild type, 1 for heterozygous mutant, 1 for homozygous mutant) 2).
  • the dose percentage criteria are 10%, 15%, 25%, 35%, 45%, 60%, 80%, 100%, and the G4N standard is 0%, 4%, 6%. , 8%, 10%, 15%, 20%, 25%.
  • Table 3 shows the results of the regression analysis. Statistically significant (p-value ⁇ 0.05) results are underlined.
  • the p-value of logistic regression was significant at 10%, 15%, 25%, 35%, 45%, and 60% on a dose percentage basis.
  • the p-value of logistic regression was significant at 15%, 20%, and 25% in the G4N criterion.
  • the p-value of linear regression was significant in both criteria.
  • the rs13306435 mutation of the IL6 gene selected as the final candidate in Example 2 was analyzed using Fisher's Exact Test (variant aspect) and Cochran Armitage Trend Test. In Fisher's exact test, the specific single base polymorphism site and leukopenia incidence and odds ratio (OR) were calculated with 95% confidence intervals (CI), and statistical significance was determined to be p ⁇ 0.05.

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Abstract

The present invention relates to a composition for predicting the risk of developing leukopenia comprising a single nucleotide polymorphism marker in an IL6 gene and a method for predicting the risk of developing leukopenia using same. As the single nucleotide polymorphism marker in an IL6 gene according to the present invention has a high correlation with the risk of developing leukopenia induced by drugs, in particular thiopurine-based drugs, patients with high susceptibility to leukopenia occurring during thiopurine-based drug treatment from leukemia, Crohn's disease, ulcerative colitis, or organ transplantation etc. can be effectively predicted or diagnosed by using the single nucleotide polymorphism marker at an early stage, and by administering a drug suitable for the patient through such prediction and diagnosis, it is possible to efficiently perform patient-specific treatment that can achieve an optimal treatment effect. Furthermore, the single nucleotide polymorphism marker according to the present invention can be utilized in drug development studies for the treatment of drug-induced leukopenia.

Description

약물 유도 백혈구 감소증 발병 위험 예측용 IL6 유전자 단일염기다형성 마커 및 이를 이용한 백혈구 감소증 발병 위험 예측 방법 IL6 gene monobasic polymorphic marker for predicting the risk of developing drug-induced leukopenia and a method for predicting the risk of leukopenia using the same
본 발명은 IL6 유전자 내의 단일염기다형성 마커를 포함하는 백혈구 감소증 발병 위험 예측용 조성물 및 이를 이용한 백혈구 감소증 발병 위험 예측 방법에 관한 것이다. The present invention relates to a composition for predicting the risk of leukopenia including a monobasic polymorphic marker in the IL6 gene and a method for predicting the risk of leukopenia using the same.
치오퓨린(thiopurine)계열 약물인 6-머캅토퓨린(6-mercaptopurine), 아자치오퓨린(azathiopurine), 치오구아닌(thioguanine) 등은 백혈병, 크론병, 궤양성 대장염과 같은 환자의 치료에 널리 사용되는 면역 억제성(immunosuppressive) 약물이다. 치오퓨린 계열 약물 사용에 있어서 주요 문제점은 BSA에 따라 계산된 예정된 용량의 약물을 복용하기 시작하더라도 약 6%의 환자에서 백혈구 감소증이나 간독성 등의 부작용이 나타난다는 것이다. 치오퓨린 계열 약물의 활성은 S-메틸트랜스퍼라제(thiopurine S-methyltransferase; TPMT)와 연관되어 있는데, 이와 관련하여 TPMT 유전자에 돌연변이를 가지고 있는 경우 TPMT 효소 활성이 감소하고 TGN 대사물질(metabolite)이 증가하여 심각한 골수억제가 발생할 수 있다는 결과가 보고되었다. 따라서 이러한 위험을 줄이기 위해 미국 식품 의약안전청은 치오퓨린 계열 약물을 사용하기 전에 TPMT 효소 활성을 측정할 것을 권고하고 있다. 하지만 아시아인에서 TPMT 돌연변이 발생 빈도는 2 내지 3%로 백인에서(10%) 보다 현저히 낮은 빈도로 발견되는 것을 고려해 볼 때, 아시아인에서 TPMT의 효소 활성을 미리 측정하는 것이 유용성이 있을지는 의문이다. 또한 이전의 연구에 의하면, 치오퓨린에 의해 유도되는 백혈구 감소증의 유전적 원인으로 보고되었던 NUDT15 돌연변이의 경우 한국인 백혈구 감소증 환자의 42.5%에서 비교적 높은 빈도로 관찰되긴 하나, NUDT15 돌연변이로 설명되지 않는 나머지 환자를 고려해 볼 때, 아시아인에서 발병하는 백혈구 감소증의 유전적 원인에 대한 이해는 여전히 부족하다.Thiopurine-based drugs such as 6-mercaptopurine, azathiopurine, and thioguanine are widely used in the treatment of patients such as leukemia, Crohn's disease, and ulcerative colitis. It is an immunosuppressive drug. The main problem with the use of cheopurine-based drugs is that even when patients start taking the prescribed doses calculated according to BSA, side effects such as leukopenia or liver toxicity occur in about 6% of patients. Thiopurine-based drug activity is associated with thiopurine s-methyltransferase (TPMT), in which case the TPMT gene has a mutation, TPMT enzyme activity decreases and TGN metabolite increases As a result, serious bone marrow suppression may be reported. Therefore, in order to reduce this risk, the U.S. Food and Drug Administration recommends measuring TPMT enzyme activity before using cheopurine-based drugs. However, considering that the incidence of TPMT mutations in Asians is 2 to 3%, which is found to be significantly lower than in whites (10%), it is questionable whether it is useful to measure the enzyme activity of TPMTs in Asians in advance. . In addition, previous studies have shown that the NUDT15 mutation, which has been reported to be the genetic cause of leukopenia induced by thiopurine, is observed at a relatively high frequency in 42.5% of Korean leukopenia patients, but the remaining patients are not explained by the NUDT15 mutation. Considering, the understanding of the genetic causes of leukopenia in Asians is still lacking.
[선행기술문헌][Advanced technical literature]
[특허문헌][Patent Document]
(특허문헌 0001) 한국 등록특허공보 제10-1704143호(Patent Document 0001) Korean Registered Patent Publication No. 10-1704143
이에 본 발명자들은 치오퓨린 계열 약물에 의해 발생하는 부작용 중 하나인 백혈구 감소증의 발병 위험성을 예측할 수 있는 마커를 개발하고자 연구를 수행한 결과, IL6 유전자 내의 특정 단일염기다형성 부위와 백혈구 감소증 발병과의 유의성 있는 상관관계를 확인함으로써, 본 발명을 완성하였다. Accordingly, the present inventors conducted a study to develop a marker capable of predicting the risk of developing leukopenia, which is one of the side effects caused by cheopurine-based drugs, and as a result, the significance of specific monobasic polymorphic sites in the IL6 gene and leukopenia The present invention was completed by confirming the correlation.
본 발명의 목적은 IL6 유전자 내의 단일염기다형성 마커를 포함하는 백혈구 감소증 발병 위험 예측용 조성물을 제공하는 것이다. An object of the present invention is to provide a composition for predicting the risk of leukopenia including a monobasic polymorphic marker in the IL6 gene.
본 발명의 다른 목적은, 상기 단일염기다형성 마커 부위를 포함하는 폴리뉴클레오티드에 특이적으로 혼성화하는 폴리뉴클레오티드를 포함하는 백혈구 감소증 발병 위험 예측 키트를 제공하는 것이다. Another object of the present invention is to provide a kit for predicting the risk of leukopenia, comprising a polynucleotide specifically hybridizing to a polynucleotide comprising the single-basic polymorphic marker site.
또한, 본 발명의 또 다른 목적은 상기 단일염기다형성 마커의 유전자형을 확인하는 단계를 포함하는 백혈구 감소증 발병 위험 예측에 대한 정보제공방법을 제공하는 것이다.In addition, another object of the present invention is to provide a method for providing information on predicting the risk of leukopenia including the step of identifying the genotype of the monobasic polymorphic marker.
상기 과제를 해결하기 위하여, 본 발명은 서열번호 1의 101번째 뉴클레오티드의 단일염기다형성(single nucleotide polymorphism, SNP) 부위를 포함하는, 10 내지 100개의 연속적인 DNA 서열로 구성되는 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드를 포함하는, 백혈구 감소증 발병 위험 예측용 조성물을 제공한다. In order to solve the above problems, the present invention is a polynucleotide consisting of 10 to 100 consecutive DNA sequences comprising a single nucleotide polymorphism (SNP) region of the 101st nucleotide of SEQ ID NO: 1 or complementary thereof. It provides a composition for predicting the risk of leukopenia, comprising a polynucleotide.
또한, 본 발명은 서열번호 1의 101번째 뉴클레오티드의 단일염기다형성 부위를 포함하는, 10 내지 100개의 연속적인 DNA 서열로 구성되는 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드와 특이적으로 혼성화하는 폴리뉴클레오티드를 포함하는, 백혈구 감소증 발병 위험 예측 키트를 제공한다. In addition, the present invention includes a polynucleotide composed of 10 to 100 contiguous DNA sequences or a complementary polynucleotide thereof, which specifically hybridizes with the single-nucleotide polymorphic site of the 101 nucleotide of SEQ ID NO: 1. The present invention provides a kit for predicting the risk of leukopenia.
또한, 본 발명은 시료 DNA로부터 서열번호 1의 101번째 뉴클레오티드의 유전자형을 확인하는 단계를 포함하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법을 제공한다. In addition, the present invention provides a method for providing information on predicting the risk of leukopenia including the step of identifying the genotype of the 101 nucleotide of SEQ ID NO: 1 from sample DNA.
본 발명에 따른 IL6 유전자 내의 단일염기다형성 마커는 약물, 특히 치오퓨린 계열 약물에 의해 유도되는 백혈구 감소증의 발병 위험과 높은 상관관계를 가지고 있는바, 이를 이용하여 백혈병, 크론병, 궤양성 대장염, 또는 장기이식 등에서 치오퓨린 계열 약물 치료 중 발생하는 백혈구 감소증에 대해 감수성이 높은 환자를 조기에 효과적으로 예측 또는 진단할 수 있고, 이를 통해 환자에게 적합한 약물을 투여하여 최적의 치료 효과를 달성할 수 있는 환자 맞춤형 치료를 효율적으로 수행할 수 있다. 나아가 본 발명에 따른 단일염기다형성 마커를 약물 유도 백혈구 감소증의 치료를 위한 약물 개발 연구에 활용할 수 있다.The monobasic polymorphic marker in the IL6 gene according to the present invention has a high correlation with the risk of leukopenia induced by drugs, in particular, chipurin-based drugs, and thus, leukemia, Crohn's disease, ulcerative colitis, or Customized patient who can effectively predict or diagnose patients with high susceptibility to leukopenia occurring during chemopurine-based drug treatment in organ transplantation, etc., and achieve optimal treatment effects by administering the appropriate drugs to patients. Treatment can be performed efficiently. Furthermore, the monobasic polymorphic marker according to the present invention can be used in drug development studies for the treatment of drug-induced leukopenia.
도 1은 6-머캅토퓨린을 사용한 백혈병 환자들 중 본 발명의 일 실시예에서 분석 대상으로 이용할 환자군을 선정하기 위한 과정을 나타낸 도이다.1 is a view showing a process for selecting a patient group to be used as an analysis object in one embodiment of the present invention among leukemia patients using 6-mercaptopurine.
도 2는 IL6 유전자상의 단일염기다형성 부위(rs13306435)와 복용량 비율(dose percentage) 감소와의 상관관계(Trend test)를 확인한 구체적인 결과를 나타낸 도이다. FIG. 2 is a diagram showing specific results confirming a correlation between a single nucleotide polymorphism site (rs13306435) on a IL6 gene and a dose percentage decrease (Trend test).
도 3은 IL6 유전자상의 단일염기다형성 부위(rs13306435)와 G4N(Grade 4 Neutropenia)와의 상관관계(Trend test)를 확인한 구체적인 결과를 나타낸 도이다. FIG. 3 is a diagram showing specific results confirming a correlation between a single nucleotide polymorphism site on the IL6 gene (rs13306435) and G4N (Grade 4 Neutropenia).
이하 본 발명에 대하여 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
일 양태로, 본 발명은 서열번호 1의 101번째 뉴클레오티드의 단일염기다형성(single nucleotide polymorphism, SNP) 부위를 포함하는, 10 내지 100개의 연속적인 DNA 서열로 구성되는 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드를 포함하는, 백혈구 감소증 발병 위험 예측용 조성물을 제공한다. In one aspect, the present invention comprises a single nucleotide polymorphism (SNP) region of the 101 nucleotide of SEQ ID NO: 1, polynucleotide consisting of 10 to 100 consecutive DNA sequence or a complementary polynucleotide thereof It provides a composition for predicting the risk of leukopenia, comprising.
본 발명에 있어서, '다형성(polymorphism)'은 단일 유전자 좌위에 두 가지 이상의 대립 유전자가 존재하는 경우를 의미하며, '다형성 부위(polymorphic site)'란 상기 대립 유전자가 존재하는 유전자 좌위를 의미한다. 다형성 부위 중에서, 사람에 따라 단일 염기만이 상이한 것을 '단일염기다형성', 즉 SNP(single nucleotide polymorphism)라고 한다.In the present invention,'polymorphism' means a case in which two or more alleles exist in a single locus, and'polymorphic site' means a locus in which the allele exists. Among polymorphic sites, a single base differs from person to person is referred to as'single nucleotide polymorphism', that is, single nucleotide polymorphism (SNP).
본 발명에 있어서, '대립유전자(allele)'는 상동염색체의 동일한 유전자 좌위에 존재하는 한 유전자의 여러 타입을 말한다. 대립유전자는 다형성을 나타내는데 사용되기도 하며, 예컨대, SNP는 두 종류의 대립인자(biallele)를 갖는다.In the present invention, the term'allele' refers to several types of a gene present in the same locus of the homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.
본 발명에 있어서, 상기 "서열번호 1"은 다형성 부위를 포함하는 다형성 서열(polymorphic sequence)이다. 다형성 서열이란 폴리뉴클레오티드 서열 중에 SNP를 포함하는 다형성 부위(polymorphic site)를 포함하는 서열을 의미한다.In the present invention, the "SEQ ID NO: 1" is a polymorphic sequence comprising a polymorphic site. The polymorphic sequence means a sequence comprising a polymorphic site containing SNP in the polynucleotide sequence.
구체적으로, 서열번호 1의 101번째 뉴클레오티드는 7p15.3 상의 IL 유전자 좌에 존재하는 것으로, rs13306435로 표시할 수 있다. 상기 서열번호 1의 101번째 뉴클레오티드, 즉 rs13306435 의 대립유전자형은 T이다. 변이가 일어났을 시의 대립유전자형은 A이다. 따라서 서열번호 1의 101 번째 뉴클레오티드는 A 또는 T(U)일 수 있으므로 다중염기 기재 방식에 따라 “w”로 기재하였다.Specifically, the 101 nucleotide of SEQ ID NO: 1 is present at the IL locus on 7p15.3, and can be represented by rs13306435. The allele of SEQ ID NO: 101, rs13306435, is T. The allele of A at the time of mutation is A. Therefore, since the 101st nucleotide of SEQ ID NO: 1 may be A or T(U), it was described as “w” according to the multi-base description method.
본 발명에 따른 상기 폴리뉴클레오티드 또는 그의 상보적 폴리뉴클레오티드는 10개 이상, 바람직하게는 10 내지 100개, 보다 바람직하게는 20 내지 80개, 보다 더 바람직하게는 40 내지 60개의 연속 염기로 구성될 수 있으며, 이에 제한되지 않는다. The polynucleotide or the complementary polynucleotide thereof according to the present invention may be composed of 10 or more, preferably 10 to 100, more preferably 20 to 80, even more preferably 40 to 60 consecutive bases And is not limited thereto.
본 발명에 있어서, 백혈구 감소증은 치오퓨린(thiopurine) 계열 약물에 의해 유도되는 것을 포함하며, 예를 들어, 백혈병, 크론병, 궤양성 대장염, 또는 장기이식 환자에서 치오퓨린(thiopurine) 계열 약물 치료시 유도되는 것일 수 있다. In the present invention, leukopenia includes those induced by thiopurine-based drugs, for example, when treating leukemia, Crohn's disease, ulcerative colitis, or organ transplant drugs in patients with organ transplants. It may be induced.
상기 치오퓨린 계열 약물에는 6-머캅토퓨린 (6-mercaptopurine), 아자치오프린(azathioprine) 또는 치오구아닌(thioguanine) 등이 포함되나 이에 제한되지 않는다. The cheopurine-based drug includes, but is not limited to, 6-mercaptopurine, azathioprine, or thioguanine.
본 발명에서 상기 단일염기다형성 마커가 백혈구 감소증 발병 위험 예측에 이용될 수 있다는 것은, 치오퓨린계 약물 투여에 따라 발생하는 부작용인 백혈구 감소증이 발병된 군의 유전자 분석 결과 단일염기다형성 부위에서 특정 염기가 존재할 확률이 높다는 것에 근거한 것이다. In the present invention, the monobasic polymorphic marker can be used for predicting the risk of leukopenia, a specific base at the site of a monobasic polymorphism as a result of genetic analysis of a group in which leukopenia, a side effect that occurs with the administration of cheopurine-based drugs It is based on the high probability of existence.
본 발명에 따른 IL6 유전자 내의 단일염기다형성 마커는 약물, 특히 치오퓨린 계열 약물에 의해 유도되는 백혈구 감소증 및 이로 인한 복용량 감소와 높은 상관관계를 가지고 있는바, 이를 이용하여 백혈병, 크론병, 궤양성 대장염, 또는 장기이식 등 치오퓨린 계열 약물 치료 중 발생하는 백혈구 감소증에 대해 감수성이 높은 환자를 조기에 효과적으로 예측 또는 진단할 수 있고, 이를 통해 환자에게 적합한 약물을 투여하여 최적의 치료 효과를 달성할 수 있는 환자 맞춤형 치료를 효율적으로 수행할 수 있다. 나아가 본 발명에 따른 단일염기다형성 마커를 약물 유도 백혈구 감소증의 치료를 위한 약물 개발 연구에 활용할 수 있다.The monobasic polymorphic marker in the IL6 gene according to the present invention has a high correlation with leukopenia induced by drugs, especially cheopurine-based drugs, and the resulting dose reduction, thereby using leukemia, Crohn's disease, ulcerative colitis Or, it can effectively predict or diagnose patients with high sensitivity to leukopenia, which occurs during chemopurine-based drug treatment, such as organ transplantation, and can achieve optimal treatment effects by administering appropriate drugs to patients. Patient-specific treatment can be efficiently performed. Furthermore, the monobasic polymorphic marker according to the present invention can be used in drug development studies for the treatment of drug-induced leukopenia.
다른 양태로, 본 발명은 서열번호 1의 101번째 뉴클레오티드의 단일염기다형성(single nucleotide polymorphism, SNP) 부위를 포함하는, 10 내지 100개의 연속적인 DNA 서열로 구성되는 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드와 특이적으로 혼성화하는 폴리뉴클레오티드를 포함하는, 백혈구 감소증 발병 위험 예측 키트를 제공한다. In another aspect, the present invention comprises a polynucleotide consisting of 10 to 100 consecutive DNA sequences or a complementary polynucleotide thereof, comprising a single nucleotide polymorphism (SNP) region of the 101 nucleotide of SEQ ID NO: 1 Provided is a kit for predicting the risk of leukopenia, comprising polynucleotides that specifically hybridize.
상기 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드와 특이적으로 혼성화하는 폴리뉴클레오티드는 대립형질 특이적(allele-specific) 폴리뉴클레오티드이다. 대립형질 특이적 폴리뉴클레오티드는 각 대립형질에 특이적으로 혼성화하는 것을 의미한다. 즉, 다형성 서열 중에 존재하는 다형성 부위의 염기를 특이적으로 구별할 수 있도록 혼성화하는 것을 말한다. The polynucleotide that specifically hybridizes with the polynucleotide or its complementary polynucleotide is an allele-specific polynucleotide. An allele-specific polynucleotide means to hybridize specifically to each allele. That is, it refers to hybridization so that the base of the polymorphic site present in the polymorphic sequence can be specifically distinguished.
상기 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드와 특이적으로 혼성화하는 프로브 또는 프라이머일 수 있으며, 이에 제한되지 않는다. It may be a probe or a primer that specifically hybridizes with the polynucleotide or a complementary polynucleotide thereof, but is not limited thereto.
상기 "프로브"는 mRNA외 특이적으로 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어서 특정 mRNA의 존재 유무, 발현양을 확인할 수 있다. 프로브는 올리고뉴클레오티드(oligonucleotide) 프로브, 단쇄 DNA(single strand DNA) 프로브, 이중쇄 DNA(double strand DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적절한 프로브의 선택 및 혼성화 조건은 당해 기술 분야에 공지된 기술에 따라 적절히 선택할 수 있다.The "probe" refers to nucleic acid fragments such as RNA or DNA corresponding to a few bases to hundreds of bases, which are capable of specifically binding other than mRNA, and are labeled to confirm the presence or absence of a specific mRNA and expression level. Can. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. The appropriate probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.
상기 "프라이머"는 짧은 자유 3-말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로서 작용하는 짧은 핵산서열을 말한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(즉, DNA 폴리머라제 또는 역전사효소) 및 상이한 4 가지의 뉴클레오사이드 트리포스페이트의 존재 하에서 DNA 합성을 개시할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 기술에 따라 적절히 선택될 수 있다.The "primer" is a nucleic acid sequence having a short free 3-terminal hydroxyl group, capable of forming complementary templates and base pairs, and acting as a starting point for template strand copying. Speak. The primer can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer lengths can be appropriately selected according to techniques known in the art.
상기 키트는 상기 폴리뉴클레오티드를 사용하는 방법에 따라 다양한 형태의 키트일 수 있으며, 예를 들어, PCR 키트, DNA 칩 키트, 마이크로어레이 등을 포함하나 이에 제한되는 것은 아니다.The kit may be a kit of various types depending on the method using the polynucleotide, and includes, but is not limited to, PCR kit, DNA chip kit, microarray, and the like.
또 다른 양태로, 본 발명은 시료 DNA로부터 서열번호 1의 101번째 뉴클레오티드의 유전자형을 확인하는 단계를 포함하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법을 제공한다. In another aspect, the present invention provides a method for providing information on predicting the risk of leukopenia, comprising the step of identifying the genotype of the 101 nucleotide of SEQ ID NO: 1 from sample DNA.
이때 상기 서열번호 1의 101번째 뉴클레오티드의 유전자형이 A 인 경우, 백혈구 감소증 발병 위험 확률이 높은 것으로 예측할 수 있다. At this time, when the genotype of the 101st nucleotide of SEQ ID NO: 1 is A, it can be predicted that the probability of developing leukopenia is high.
보다 구체적으로, 상기 서열번호 1의 101번째 뉴클레오티드의 대립유전자형이 TA 또는 AA인 경우, 대립유전자형이 TT인 경우에 비해 백혈구 감소증 발병할 확률이 높은 것으로 예측할 수 있다. More specifically, when the allele of the 101st nucleotide of SEQ ID NO: 1 is TA or AA, it can be predicted that the probability of developing leukopenia is higher than when the allele is TT.
본 발명에 있어서, 백혈구 감소증은 치오퓨린(thiopurine) 계열 약물에 의해 유도되는 것을 포함하며, 예를 들어, 백혈병, 크론병, 궤양성 대장염, 또는 장기이식 환자에서 치오퓨린(thiopurine) 계열 약물 치료시 유도되는 것일 수 있다. In the present invention, leukopenia includes those induced by thiopurine-based drugs, for example, when treating leukemia, Crohn's disease, ulcerative colitis, or organ transplant drugs in patients with organ transplants. It may be induced.
상기 치오퓨린 계열 약물에는 6-머캅토퓨린 (6-mercaptopurine), 아자치오프린(azathioprine) 또는 치오구아닌(thioguanine) 등이 포함되나 이에 제한되지 않는다. The cheopurine-based drug includes, but is not limited to, 6-mercaptopurine, azathioprine, or thioguanine.
본 발명에 따른 정보제공방법은 시료 DNA를 수득하기 위하여, 생물학적 시료로부터 DNA를 분리하는 단계를 더 포함할 수 있다. 상기 DNA 분리는 당업계에서 통상적으로 사용되는 페놀/클로로포름 추출법, SDS 추출법, CTAB 분리법(Cetyl Trimethyl AmmoniumBromide; Murray et al., Nuc. Res., 4321-4325, 1980) 등을 이용하거나, 상업적으로 판매되는 DNA 추출 키트를 이용하여 수행할 수 있으며, 이에 제한되지 않는다. 상기 생물학적 시료에는 대상의 혈액, 타액, 뇨, 피부세포, 점막 세포 및 모발 등 모든 조직이 포함되며, 이에 제한되지 않는다. The method for providing information according to the present invention may further include isolating DNA from a biological sample in order to obtain sample DNA. For the DNA separation, phenol/chloroform extraction method, SDS extraction method, CTAB separation method (Cetyl Trimethyl AmmoniumBromide; Murray et al., Nuc. Res., 4321-4325, 1980) commonly used in the art, or commercially available It can be performed using a DNA extraction kit, but is not limited thereto. The biological sample includes, but is not limited to, blood, saliva, urine, skin cells, mucosal cells, and hair tissue of the subject.
상기 유전자형을 확인하는 단계를 수행하기 위하여 유전자 서열 분석이 수행될 수 있다. 서열 분석은 당업계에 공지된 방법을 모두 이용할 수 있으며, 예를 들어, 자동염기서열분석기를 사용하거나, 파이로시퀀싱(pyrosequencing), PCR-RELP법(restriction fragment length polymorphism), PCR-SSCP법(single strand conformation polymorphism), PCR-SSO법(specific sequence oligonucleotide), PCR-SSO법과 도트 하이브리드화 법을 조합한 ASO(allele specific oligonucleotide) 하이브리드화법, TaqMan-PCR법, MALDI-TOF/MS법, RCA법 (rolling circle amplification), HRM(high resolution melting)법, 프라이머 신장법, 서던 블롯 하이브리드화 법 및 도트 하이브리드화법 등의 공지의 방법을 이용할 수 있고, 이에 제한되지 않는다. Gene sequence analysis may be performed to perform the step of identifying the genotype. For the sequence analysis, any method known in the art can be used, for example, using an automatic base sequencer, pyrosequencing, restriction fragment length polymorphism (PCR-RELP), PCR-SSCP method ( Single strand conformation polymorphism), PCR-SSO method (specific sequence oligonucleotide), PCR-SSO method and dot hybridization method (allele specific oligonucleotide) hybridization method, TaqMan-PCR method, MALDI-TOF/MS method, RCA method (Rolling circle amplification), HRM (high resolution melting) method, primer extension method, Southern blot hybridization method and a known method such as dot hybridization method can be used, but is not limited thereto.
이하, 본 발명을 실시예에 의거하여 보다 구체적으로 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명을 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail based on examples. However, the following examples are only intended to illustrate the invention and do not limit the invention.
실시예 1. 실험 대상의 선정Example 1. Selection of test subjects
한국 서울대병원과 서울아산병원에서 6-머캅토퓨린을 사용하여 유지요법을 진행한 298명의 소아 ALL 환자들을 1차 선별하였다. 이 중 재발, 버킷림프종, 임상 데이터 부재 등의 이유로 총 54명을 본 실험에서 제외하였다. 또한 244명 중에서도, star allele 개념에 근거하여 NUDT15, TPMT 유전자가 정상적인 활성(normal activity)인 188명을 분석대상으로 설정하였다. 본 실험에 참가한 모든 참가자는 한국인이었으며, 구체적인 실험 대상의 선정 과정을 하기 도 1 및 표 1에 나타내었다.The primary screening of all 298 pediatric ALL patients who underwent maintenance therapy using 6-mercaptopurine at Seoul National University Hospital and Seoul Asan Hospital in Korea. Of these, 54 patients were excluded from this study for reasons such as recurrence, Burkitt's lymphoma, and absence of clinical data. In addition, among 244 people, based on the concept of star allele, 188 people whose NUDT15 and TPMT genes are normal activities were set as an analysis target. All participants who participated in this experiment were Koreans, and the process of selecting a specific test subject is shown in FIG. 1 and Table 1 below.
NUDT15 NUDT15 Normal MetabolizerNormal Metabolizer (*1/*1)(*1/*1) NUDT15 NUDT15 Intermediate MetabolizerIntermediate Metabolizer (*1/*2, *1/*3, *1/*4, *1/*5, *1/*6, *1/*9)(*1/*2, *1/*3, *1/*4, *1/*5, *1/*6, *1/*9) NUDT15 NUDT15 Poor MetabolizerPoor Metabolizer (*3/*3)(*3/*3) totaltotal
TPMTTPMT Normal MetabolizerNormal Metabolizer (*1/*1, *1/*1S, *1S/*1S)(*1/*1, *1/*1S, *1S/*1S) 188188 4848 1One 237237
TPMTTPMT Intermediate MetabolizerIntermediate Metabolizer (*1/*3C, *1S/*3C, *1/*6)(*1/*3C, *1S/*3C, *1/*6) 66 1One 77
TPMTTPMT Poor MetabolizerPoor Metabolizer
totaltotal 194194 4949 1One 244244
소아 암 그룹(Children's Cancer Group; CCG)의 치료요법(treatment protocols)에 의하면, 6-머캅토퓨린의 초기 권장 용량은 75mg/m 2이다. 하지만 서울아산병원에서 해당 용량으로 치료를 진행한 상당수의 환자에서 심각한 약물 독성을 보여 화학요법을 중단하는 결과를 보이는 바, 초기 투여 용량을 75mg/m 2에서 50mg/m 2으로 조정하였다. 치료를 위한 프로토콜은 이상반응(adverse events)에 따른 약물 용량 수정 또는 중단에 대해서는 책임 있는 의사가 케이스 별로 결정하였다. 각 환자에 대한 과거의 의료기록은 검토되었고, 용량, 치료기간 및 백혈구 감소증의 발병 시기 및 정도를 포함하는 6-머캅토퓨린에 대한 정보는 유전자형 분석 결과를 알지 못하는 의사에 의해 독립적으로 평가되었다.According to the treatment protocols of the Children's Cancer Group (CCG), the initial recommended dose of 6-mercaptopurine is 75 mg/m 2 . However, as a significant number of patients who were treated with the corresponding dose at Seoul Asan Hospital showed severe drug toxicity and stopped chemotherapy, the initial dose was adjusted from 75 mg/m 2 to 50 mg/m 2 . The protocol for treatment was determined on a case-by-case basis by the responsible physician for the modification or discontinuation of the drug dose due to adverse events. Past medical records for each patient were reviewed, and information on 6-mercaptopurine, including dose, duration of treatment, and the timing and extent of leukopenia was independently assessed by physicians with unknown genotyping results.
6-머캅토퓨린은 총 12주 동안 지속되는 한 싸이클(cycle)의 동일 항암 치료를 6-10회 반복하는 스케줄로 처방되었다. 첫 싸이클에서는 체표면적(Body Surface Area; BSA)에 기초한 예정된 용량의 약물 복용을 시작하였고, 백혈구 감소증이나 간독성 등의 이상반응(adverse events)에 따라 6-머캅토퓨린 용량을 계속적으로 감량하면서 싸이클을 지속하였다. 6-Mercaptopurine was prescribed with a schedule of 6-10 repetitions of the same anticancer treatment for one cycle lasting a total of 12 weeks. In the first cycle, he started taking a prescribed dose of the drug based on the body surface area (BSA), and continued to reduce the dose of 6-mercaptopurine according to adverse events such as leukopenia or hepatotoxicity. Continued.
본 발명에서는 복용량 비율(dose percentage)이라는 개념을 도입하여 환자가 부작용 없이 복용할 수 있는 치오퓨린의 최대 복용량을 정의하였다. 복용량 비율(dose percentage)은 유지요법 마지막 사이클의 BSA에 기반한 복용량과 실제 복용량의 비율이며, 다음과 같이 정의된다.In the present invention, the concept of a dose percentage was introduced to define the maximum dose of cheopurine that a patient can take without side effects. The dose percentage is the ratio of the BSA-based and actual doses in the last cycle of maintenance therapy and is defined as follows.
Figure PCTKR2019018715-appb-img-000001
Figure PCTKR2019018715-appb-img-000001
또한, G4N(Grade 4 Neutropenia incidence)라는 개념을 도입하여, ANC(Absolute Neutrophil Count)<500로 정의되는 4단계 호중구 감소증의 상대적인 발생빈도를 표현하였다.In addition, the concept of G4N (Grade 4 Neutropenia incidence) was introduced to express the relative incidence of stage 4 neutropenia defined by ANC (Absolute Neutrophil Count) <500.
Figure PCTKR2019018715-appb-img-000002
Figure PCTKR2019018715-appb-img-000002
본 분석에서는 표 1에서 정의된 전체 244명의 환자 중, NUDT15와 TPMT가 모두 정상적인 대사자(normal metabolizer)인 188명의 환자를 대상으로 복용량 비율(dose percentage)에 대한 분석을 진행하였다. 또한 상기 188명 중 G4N 임상정보가 있는 185명을 대상으로 G4N에 대한 분석을 진행하였다.In this analysis, among the total of 244 patients defined in Table 1, a dose percentage was analyzed for 188 patients, both of which are normal metabolizers, both NUDT15 and TPMT. In addition, among the 188 patients, 185 patients with G4N clinical information were analyzed for G4N.
실시예 2. 전체 엑솜 시퀀싱(Whole Exome Sequencing)을 통한 유전자, 변이 분석 Example 2. Analysis of genes and mutations through whole exome sequencing
실시예 1에서 선별한 188명의 환자들을 대상으로 골수, 혈액에서 DNA를 추출한 후 전체 엑솜 시퀀싱을 진행하였다. 도1에 나온 바와 같이, GVB를 이용하여 복용량 비율(dose percentage), G4N을 종속변수로 하는 다중 공변량 선형 회귀 분석(multiple covariate linear regression analysis)을 실행하여 후보 유전자들을 선정하였다. GVB(Genetic Variant Burden)라 함은 변이의 독성(deleteriousness)을 나타내는 지표인 SIFT 점수 중 0.7 미만인 SIFT 점수들의 geometric mean을 의미한다. 다음으로 후보 유전자들의 모든 변이에 대하여 dose percentage, G4N을 종속변수로 하는 다중 공변량 선형 회귀 분석을 실행하였고, 표 2에 표시된 후보 변이 3개를 선정하였다. 이 중 nonsynonymous variant에 해당하는 IL6 유전자의 rs13306435 변이를 최종 변이로 선정하였다.After extracting DNA from bone marrow and blood for 188 patients selected in Example 1, whole exome sequencing was performed. As shown in FIG. 1, candidate genes were selected by performing multiple covariate linear regression analysis using GVB as a dose percentage and G4N as dependent variables. The term GVB (Genetic Variant Burden) means the geometric mean of SIFT scores less than 0.7 among SIFT scores, which are indicators of deteriousness of mutation. Next, a multi-covariate linear regression analysis using dose percentage and G4N as dependent variables was performed for all variations of the candidate genes, and three candidate variations shown in Table 2 were selected. Among them, the rs13306435 mutation of the IL6 gene corresponding to the nonsynonymous variant was selected as the final mutation.
CHRCHR BPBP REFREF ALTALT Gene NameGene Name 1KG AF1KG AF 1KG AFR AF1KG AFR AF 1KG AMR AF1KG AMR AF 1KG EAS AF1KG EAS AF 1KG EUR AF1KG EUR AF 1KG SAS AF1KG SAS AF ConsequenceConsequence SIFTSIFT CADDCADD dbSNPdbSNP
77 2277103922771039 TT AA IL6IL6 0.02040.0204 0.00150.0015 0.07780.0778 0.02580.0258 0.01690.0169 0.00310.0031 missense_variantmissense_variant 0.410.41 3.5343.534 rs13306435rs13306435
1212 5788374157883741 CC TT MARSMARS 0.00640.0064 00 00 0.03080.0308 00 0.0010.001 synonymous_variantsynonymous_variant 0.010.01 14.0814.08 rs117101415rs117101415
1212 5790659257906592 GG AA MARSMARS 0.00640.0064 00 00 0.03080.0308 00 0.0010.001 synonymous_variantsynonymous_variant 1One 17.8717.87 rs117633211rs117633211
실시예 3. 회귀분석(Regression analysis)을 통한 유전자, 변이 분석 Example 3. Analysis of genes and mutations through regression analysis
실시예 2에서 최종 후보로 선정된 변이의 복용량 비율(dose percentage), G4N에 대한 영향력을 추가적으로 검증하기 위하여, 실시예 1에서 선별된 188명 중 회귀분석(Regression analysis)을 수행하여 단일염기다형성 부위(rs13306435)가 복용량 비율(dose percentage)에 미치는 영향력을 통계적으로 확인하였다. 회귀분석 시 보정요소로는 성별, 마지막 사이클 시작 시 나이, BSA, 그리고 변이의 유전형(야생형(wild type)이면 0, 이형접합 돌연변이체(heterozygous mutant)면 1, 동형접합 돌연변이체(homozygous mutant)면 2)을 포함하였다. 또한 로지스틱 회귀분석 시에는 복용량 비율(dose percentage) 기준을 10%, 15%, 25%, 35%, 45%, 60%, 80%, 100%로, G4N 기준을 0%, 4%, 6%, 8%, 10%, 15%, 20%, 25%로 하여 분석하였다.In order to further verify the dose percentage of the mutation selected as the final candidate in Example 2 and its influence on G4N, a single nucleotide polymorphism site was performed by performing a regression analysis among 188 persons selected in Example 1 The effect of (rs13306435) on the dose percentage was statistically confirmed. Correction factors for regression analysis include gender, age at the start of the last cycle, BSA, and genotypes of mutations (0 for wild type, 1 for heterozygous mutant, 1 for homozygous mutant) 2). Also, in logistic regression, the dose percentage criteria are 10%, 15%, 25%, 35%, 45%, 60%, 80%, 100%, and the G4N standard is 0%, 4%, 6%. , 8%, 10%, 15%, 20%, 25%.
회귀분석 결과를 하기 표 3에 나타내었다. 통계적으로 유의한(p-value<0.05) 결과에 대해서는 밑줄로 표시하였다. Table 3 shows the results of the regression analysis. Statistically significant (p-value<0.05) results are underlined.
Gene (variant)Gene (variant) IndexIndex TypeType CriteriaCriteria CaseCase ControlControl PP Coefficient(linear regression) Coefficient(linear regression) / OR(logistic regression)/ OR (logistic regression)
IL6 (rs13306435) IL6 (rs13306435) DIP(%)DIP(%) Linear regressionLinear regression 0.00150.0015 (-)0.2323(-) 0.2323
Logistic regressionLogistic regression <10<10 33 185185 0.02570.0257 38 (2.04-2382.37)38 (2.04-2382.37)
<15<15 44 184184 0.02640.0264 11.84 (1.18-121.28)11.84 (1.18-121.28)
<25<25 99 179179 0.00070.0007 12.61 (2.76-56.05)12.61 (2.76-56.05)
<35<35 2525 163163 0.00050.0005 7.7 (2.41-24.61)7.7 (2.41-24.61)
<45<45 3333 155155 0.00440.0044 5.01 (1.62-15.34)5.01 (1.62-15.34)
<60<60 7474 114114 0.03080.0308 3.46 (1.16-11.61)3.46 (1.16-11.61)
<80<80 129129 5959 0.35380.3538 1.86 (0.56-8.44)1.86 (0.56-8.44)
<100<100 162162 2626 0.41020.4102 2.39 (0.45-44.49)2.39 (0.45-44.49)
G4N(%)G4N (%) Linear regressionLinear regression 0.00200.0020 0.00530.0053
Logistic regressionLogistic regression >0>0 9595 9393 0.19350.1935 2.11 (0.71-7.1)2.11 (0.71-7.1)
>4>4 6363 125125 0.59950.5995 1.34 (0.43-3.91)1.34 (0.43-3.91)
>6>6 4848 140140 0.47370.4737 1.51 (0.45-4.54)1.51 (0.45-4.54)
>8>8 3030 158158 0.63320.6332 1.39 (0.3-4.79)1.39 (0.3-4.79)
>10>10 2222 166166 0.29030.2903 2.09 (0.44-7.45)2.09 (0.44-7.45)
>15>15 1111 177177 0.03480.0348 4.83 (0.96-19.54)4.83 (0.96-19.54)
>20>20 55 183183 0.00260.0026 20.57 (2.9-183.55)20.57 (2.9-183.55)
>25>25 33 185185 0.020.02 25.23 (1.82-699.41)25.23 (1.82-699.41)
표 3에 나타낸 바와 같이, 복용량 비율(dose percentage) 기준에서는 10%, 15%, 25%, 35%, 45%, 60%에서 로지스틱 회귀(logistic regression)의 p-값이 유의하였다. 또한 G4N 기준에서는 15%, 20%, 25%에서 로지스틱 회귀(logistic regression)의 p-값이 유의하였다. 두 기준 모두에서 선형 회귀(linear regression)의 p-값이 유의하였다.As shown in Table 3, the p-value of logistic regression was significant at 10%, 15%, 25%, 35%, 45%, and 60% on a dose percentage basis. In addition, the p-value of logistic regression was significant at 15%, 20%, and 25% in the G4N criterion. The p-value of linear regression was significant in both criteria.
실시예 4. T-test(Trend test)를 통한 유전자, 변이 분석 Example 4. Analysis of genes and mutations through T-test (Trend test)
실시예 2에서 최종 후보로 선정된 IL6 유전자의 rs13306435 변이에 대해 Fisher's Exact Test(variant 측면)와 Cochran Armitage Trend Test를 이용한 분석을 수행하였다. Fisher's exact test에 있어서, 특정 단일염기다형성 부위와 백혈구 감소증 발병 상태 및 위험률(odds ratio, OR)은 95% 신뢰구간(confidence intervals, CI)으로 산출하였으며, 통계학적 유의성은 p<0.05로 판정하였다. The rs13306435 mutation of the IL6 gene selected as the final candidate in Example 2 was analyzed using Fisher's Exact Test (variant aspect) and Cochran Armitage Trend Test. In Fisher's exact test, the specific single base polymorphism site and leukopenia incidence and odds ratio (OR) were calculated with 95% confidence intervals (CI), and statistical significance was determined to be p<0.05.
그 결과, IL6 유전자상의 단일염기다형성 부위(rs13306435, 서열번호 1의 101번째 염기에 해당)가 백혈구 감소증과 유의한 연관성을 보임을 확인하였다. rs13306435와 복용량 비율(dose percentage) 감소와의 상관관계(Trend test)를 확인한 구체적인 결과를 하기 표 4 및 표 5에 나타내었다. 통계적으로 유의성 있는 p값을 밑줄로 표시하였다.As a result, it was confirmed that the single nucleotide polymorphism site on the IL6 gene (rs13306435, corresponding to the 101st base of SEQ ID NO: 1) showed a significant association with leukopenia. Table 4 and Table 5 below show specific results confirming the correlation between rs13306435 and dose percentage reduction. Statistically significant p-values are underlined.
Gene (variant)Gene (variant) IndexIndex CriteriaCriteria REFREF HETHET HOMHOM FET(Dominant)FET(Dominant) FET(Recessive)FET(Recessive) CATTCATT
PP OR OR PP OR OR PP
(95% CI)(95% CI) (95% CI)(95% CI)
IL6 (rs13306435) IL6 (rs13306435) DIP(%)DIP(%) <10<10 1One 22 00 0.01710.0171 25.26(1.24-1549.88)25.26 (1.24-1549.88) 1One 0 (0-Inf)0 (0-Inf) 0.00020.0002
≥10≥10 172172 1313 00
<15<15 22 22 00 0.03260.0326 12.73(0.86-188.75)12.73 (0.86-188.75) 1One 0 (0-Inf)0 (0-Inf) 0.00170.0017
≥15≥15 171171 1313 00
<25<25 55 44 00 0.00270.0027 11.85(2.06-64.49)11.85 (2.06-64.49) 1One 0 (0-Inf)0 (0-Inf) 0.00000.0000
≥25≥25 168168 1111 00
<35<35 1818 77 00 0.0010.001 7.4(2.03-26.62)7.4 (2.03-26.62) 1One 0 (0-Inf)0 (0-Inf) 0.00010.0001
≥35≥35 155155 88 00
<45<45 2626 77 00 0.00630.0063 4.89(1.38-16.96)4.89 (1.38-16.96) 1One 0 (0-Inf)0 (0-Inf) 0.0020.002
≥45≥45 147147 88 00
<60<60 6464 1010 00 0.02950.0295 3.38(1-13.19)3.38 (1-13.19) 1One 0 (0-Inf)0 (0-Inf) 0.0240.024
≥60≥60 109109 55 00
<80<80 117117 1212 00 0.39720.3972 1.91(0.49-10.96)1.91 (0.49-10.96) 1One 0 (0-Inf)0 (0-Inf) 0.3220.322
≥80≥80 5656 33 00
<100<100 148148 1414 00 0.69820.6982 2.36(0.33-103.9)2.36 (0.33-103.9) 1One 0 (0-Inf)0 (0-Inf) 0.40220.4022
≥100≥100 2525 1One 00
IndexIndex CriteriaCriteria REFREF HETHET HOMHOM FET(Dominant)FET(Dominant) FET(Recessive)FET(Recessive) CATTCATT
PP OR OR PP OR OR PP
(95% CI)(95% CI) (95% CI)(95% CI)
IL6 (rs13306435) IL6 (rs13306435) G4N(%)G4N (%) >0>0 8585 1010 00 0.2840.284 1.99(0.59-7.75)1.99 (0.59-7.75) 1One 0 (0-Inf)0 (0-Inf) 0.21570.2157
≤0≤0 8585 55 00
>4>4 5757 66 00 0.58460.5846 1.32(0.37-4.39)1.32 (0.37-4.39) 1One 0 (0-Inf)0 (0-Inf) 0.61220.6122
≤4≤4 113113 99 00
>6>6 4343 55 00 0.54150.5415 1.47(0.37-5.06)1.47 (0.37-5.06) 1One 0 (0-Inf)0 (0-Inf) 0.49590.4959
≤6≤6 127127 1010 00
>8>8 2727 33 00 0.71430.7143 1.32(0.22-5.37)1.32 (0.22-5.37) 1One 0 (0-Inf)0 (0-Inf) 0.67830.6783
≤8≤8 143143 1212 00
>10>10 1919 33 00 0.39470.3947 1.98(0.33-8.3)1.98 (0.33-8.3) 1One 0 (0-Inf)0 (0-Inf) 0.13350.1335
≤10≤10 151151 1212 00
>15>15 88 33 00 0.04810.0481 4.99(0.76-24.59)4.99 (0.76-24.59) 1One 0 (0-Inf)0 (0-Inf) 0.01630.0163
≤15≤15 162162 1313 00
>20>20 22 33 00 0.0040.004 20.12(2.1-261.65)20.12 (2.1-261.65) 1One 0 (0-Inf)0 (0-Inf) 00
≤20≤20 168168 1212 00
>25>25 1One 22 00 0.01760.0176 24.82(1.22-1523.35)24.82 (1.22-1523.35) 1One 0 (0-Inf)0 (0-Inf) 0.00020.0002
≤25≤25 169169 1313 00
상기 표 4, 표 5에 나타낸 바와 같이, 복용량 비율(dose percentage) 10%, 15%, 25%, 35%, 45%, 60% 기준과 G4N 15%, 20%, 25%에서 IL6 유전자상의 단일염기다형성 부위(rs13306435)에 대한 대립 유전자 빈도(Allelic Frequency)는 복용량 비율(dose percentage)의 감소와 높은 상관을 보였다.As shown in Tables 4 and 5 above, a single dose on the IL6 gene at 10%, 15%, 25%, 35%, 45%, and 60% dose percentage and G4N 15%, 20%, 25% The allelic frequency for the nucleotide polymorphism site (rs13306435) showed a high correlation with a decrease in the dose percentage.
또한, 유전형(Genotype)과, 복용량 비율(dose percentage) 또는 ANC 빈도의 관계를 Wilcoxon rank sum test를 통해 브록스 플롯(blox plot)으로 나타내었다(도 2 및 3).In addition, the relationship between the genotype (Genotype), the dose percentage (dose percentage), or the ANC frequency was shown as a Brock plot (blox plot) through the Wilcoxon rank sum test (FIGS. 2 and 3 ).
도 2 및 3에 나타낸 바와 같이, Wilcoxon rank sum test를 실시한 결과, 복용량 비율(dose percentage) 기준에서 p-값은 0.0013으로 유의함을 확인하였다. 반면, G4N(Grade 4 Neutropenia incidence) 기준에서 p-값은 0.2803이었다.2 and 3, as a result of the Wilcoxon rank sum test, it was confirmed that the p-value was significant at a dose percentage criterion of 0.0013. On the other hand, in the G4N (Grade 4 Neutropenia incidence) standard, the p-value was 0.2803.
이상의 실험 결과를 통해, IL6 유전자상의 단일염기다형성 부위(rs13306435, 서열번호 1의 101번째 염기에 해당)가 백혈구 감소증에 대한 민감도에 영향을 주는 것을 확인하였다.Through the above experimental results, it was confirmed that a single nucleotide polymorphism site on the IL6 gene (rs13306435, corresponding to the 101st base of SEQ ID NO: 1) affects sensitivity to leukopenia.
이상으로 본 발명의 특정한 부분을 상세히 기술한 바, 당 업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific parts of the present invention in detail above, it will be apparent to those of ordinary skill in the art that this specific technique is only a preferred embodiment, whereby the scope of the present invention is not limited. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

  1. 서열번호 1의 101번째 뉴클레오티드의 단일염기다형성(single nucleotide polymorphism, SNP) 부위를 포함하는, 10 내지 100개의 연속적인 DNA 서열로 구성되는 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드를 포함하는, 백혈구 감소증 발병 위험 예측용 조성물.Risk of leukopenia, comprising a polynucleotide consisting of 10 to 100 contiguous DNA sequences or a complementary polynucleotide thereof, comprising a single nucleotide polymorphism (SNP) site of the 101 nucleotide of SEQ ID NO: 1 Prediction composition.
  2. 제1항에 있어서, 상기 서열번호 1의 101번째 뉴클레오티드는 IL6 유전자 좌에 위치하는 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측용 조성물.The composition for predicting the risk of leukopenia according to claim 1, wherein the 101th nucleotide of SEQ ID NO: 1 is located at the IL6 locus.
  3. 제1항에 있어서, 상기 백혈구 감소증은 치오퓨린(thiopurine) 계열 약물에 의해 유도되는 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측용 조성물. According to claim 1, The leukopenia is characterized in that is induced by a thiopurine (thiopurine)-based drug, the composition for predicting the risk of leukopenia.
  4. 제3항에 있어서, 상기 치오퓨린 계열 약물은 6-머캅토퓨린 (6-mercaptopurine), 아자치오프린(azathioprine) 및 치오구아닌(thioguanine)으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측용 조성물.The leukopenia according to claim 3, wherein the chemopurine-based drug is at least one selected from the group consisting of 6-mercaptopurine, azathioprine, and thioguanine. Composition for predicting the risk of development.
  5. 서열번호 1의 101번째 뉴클레오티드의 단일염기다형성(single nucleotide polymorphism, SNP) 부위를 포함하는, 10 내지 100개의 연속적인 DNA 서열로 구성되는 폴리뉴클레오티드 또는 이의 상보적인 폴리뉴클레오티드와 특이적으로 혼성화하는 폴리뉴클레오티드를 포함하는, 백혈구 감소증 발병 위험 예측 키트. A polynucleotide consisting of 10 to 100 consecutive DNA sequences comprising a single nucleotide polymorphism (SNP) region of the 101 nucleotide of SEQ ID NO: 1 or a polynucleotide specifically hybridizing to a complementary polynucleotide thereof Containing, leukopenia risk development kit.
  6. 제5항에 있어서, 상기 특이적으로 혼성화하는 폴리뉴클레오티드는 프로브 또는 프라이머인 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측 키트.The kit for predicting the risk of leukopenia according to claim 5, wherein the specifically hybridizing polynucleotide is a probe or a primer.
  7. 시료 DNA로부터 서열번호 1의 101번째 뉴클레오티드의 유전자형을 확인하는 단계를 포함하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법. A method of providing information on predicting the risk of leukopenia, comprising the step of identifying the genotype of the 101 nucleotide of SEQ ID NO: 1 from sample DNA.
  8. 제7항에 있어서, 상기 방법은 상기 서열번호 1의 101번째 뉴클레오티드의 유전자형이 A인 경우, 백혈구 감소증 발병 위험이 높은 것으로 예측하는 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법.The method of claim 7, wherein when the genotype of the 101st nucleotide of SEQ ID NO: 1 is A, the risk of leukopenia is predicted to be high.
  9. 제7항에 있어서, 상기 방법은 상기 서열번호 1의 101번째 뉴클레오티드의 대립유전자형이 TA 또는 AA인 경우, 백혈구 감소증 발병 위험이 높은 것으로 예측하는 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법.The method according to claim 7, wherein the method is characterized in that if the allele of the 101 nucleotide of SEQ ID NO: 1 is TA or AA, it is characterized by predicting a high risk of leukopenia, providing information on the risk of leukopenia. Way.
  10. 제7항에 있어서, 상기 백혈구 감소증은 치오퓨린(thiopurine) 계열 약물에 의해 유도되는 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법.The method of claim 7, wherein the leukopenia is induced by a thiopurine-based drug.
  11. 제10항에 있어서, 상기 치오퓨린 계열 약물은 6-머캅토퓨린 (6-mercaptopurine), 아자치오프린(azathioprine) 및 치오구아닌(thioguanine)으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 백혈구 감소증 발병 위험 예측에 대한 정보제공방법.The leukopenia according to claim 10, wherein the cheopurine-based drug is at least one selected from the group consisting of 6-mercaptopurine, azathioprine, and thioguanine. Method of providing information on the prediction of outbreak risk.
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