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WO2018135464A1 - Procédé de criblage génétique rapide utilisant un séquenceur de nouvelle génération - Google Patents

Procédé de criblage génétique rapide utilisant un séquenceur de nouvelle génération Download PDF

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Publication number
WO2018135464A1
WO2018135464A1 PCT/JP2018/000952 JP2018000952W WO2018135464A1 WO 2018135464 A1 WO2018135464 A1 WO 2018135464A1 JP 2018000952 W JP2018000952 W JP 2018000952W WO 2018135464 A1 WO2018135464 A1 WO 2018135464A1
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mutation
gene
mutations
brca1
genetic
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Japanese (ja)
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博史 中岡
逸朗 井ノ上
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Inter University Research Institute Corp Research Organization of Information and Systems
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Inter University Research Institute Corp Research Organization of Information and Systems
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a genetic testing technique for detecting mutations occurring in germ line cells and somatic cells.
  • Cancer develops by accumulation of somatic mutations, but, like BRCA1 and BRCA2 related to breast cancer and ovarian cancer, it carries mutations in the germ line and genetically affects breast cancer and ovarian cancer. Some have genetic factors that are likely to develop.
  • the lifetime risk of suffering from breast cancer is said to be 56-85%.
  • Breast cancer also has a bilateral breast cancer rate of 40-65%, and the lifetime risk of developing ovarian cancer is more than 50% in people with mutations in BRCA1, and more than 20% in people with mutations in BRCA2. It has been broken.
  • the rate of suffering from male breast cancer or prostate cancer increases.
  • Genetic testing has become important not only for hereditary cancers but also for cancers caused by somatic mutations. This is because it is necessary to identify a gene mutation in order to examine the effectiveness for a specific drug in advance by developing a molecular target drug. By identifying genetic mutations, it is possible not only to select the most effective medicine for the patient, but also to avoid the treatment with the risk of side effects in patients who cannot expect the therapeutic effect. it can.
  • the genetic test for detecting a specific mutation is generally performed by the Sanger sequencing method (dideoxy method).
  • the Sanger sequencing method is accurate and is the standard method for genetic testing, but requires a lot of time and effort.
  • BRCA1 and BRCA2 are large genes having lengths of 81.1 and 84.7 kb and exons of 24 and 27, respectively. Therefore, genetic testing requires a lot of time and effort.
  • the Sanger sequencing method has a problem that a large amount of sample DNA is required.
  • next generation sequencing NGS
  • NGS next generation sequencing
  • Sequence information can be obtained in a very short time and at a low cost (Non-Patent Document 6).
  • Non-Patent Document 6 a large number of clinical sample DNA sequences can be determined by exon analysis that efficiently analyzes the sequence of a specific region of the genome sequence.
  • Non-Patent Documents 7 to 14 In order to identify BRCA1 / 2 gene mutations, NGS is used in combination with a target enrichment system that selectively concentrates a specific region (Non-Patent Documents 7 to 14). According to the report of EMQN, it is reported that 19% of research institutions use NGS technology for BRCA genetic testing (Non-patent Document 5).
  • NGS has been reported to give results comparable to the conventional Sanger sequencing method.
  • an inexpensive and highly accurate technique that can be applied not only to a group suspected of having familial tumors but also to the general population has not been reported.
  • the present invention relates to the following gene mutation test method.
  • a library in which a different index sequence for each sample is added to a specimen is prepared, the prepared library is pooled, the pooled DNA sample is hybridized to a probe set, and the captured DNA library pool is PCR amplified.
  • a gene mutation test method that detects a gene mutation by performing a next-generation sequencing method.
  • the genetic test method according to (1), wherein the gene mutation to be detected is a familial tumor.
  • the genetic test method according to (2), wherein the familial tumor is hereditary breast cancer ovarian cancer syndrome or Lynch syndrome.
  • the gene testing method according to (1), wherein the mutation of the gene to be examined is a somatic mutation.
  • the method of the present invention is rapid, highly accurate, and can be performed with a low cost and a small amount of DNA sample, it can be applied not only to a population suspected of having familial tumors but also to a general population. it can. Further, in the method of the present invention, by detecting somatic mutation, a drug that can be expected to have a high effect upon treatment with a molecular target drug can be selected and provided to a patient.
  • the present invention provides a method for performing a rapid and highly accurate genetic test at a low price by performing a target sequence using a large number of clinical specimens.
  • Table 1 shows a list of familial tumors and their causative genes. Examining patients with possible hereditary diseases to determine the causative gene. In addition, by examining a gene of a family who has developed a hereditary disease, it is possible to detect a person who may have the disease at an early stage. Furthermore, if it is possible to examine the general population as well as those who have a family member who has developed a genetic disease, it is very beneficial because it leads to early detection. In particular, BRCA1 and BRCA2 having a high mutation prevalence can detect a person who may develop a tumor in the future and perform preventive intervention if genetic testing can be performed on the general population. . For example, if it is found that there is a high possibility of suffering from breast cancer or ovarian cancer in the future, measures such as increasing the frequency of screening can be taken in order to detect cancer early.
  • Example 1 Outline of genetic testing method This testing method is roughly divided into three steps: a DNA library preparation step, a hybridization step, and a sequencing step (FIG. 1).
  • a DNA library preparation step a DNA library preparation step
  • a hybridization step a hybridization step
  • a sequencing step a sequencing step
  • each process is implemented using various kits, you may implement using the method and kit which can perform the same reaction.
  • Each kit uses a modified protocol attached to the kit so as to meet the purpose of genetic testing, and does not use the method of the kit as it is.
  • DNA samples are prepared from blood samples.
  • DNA was extracted from blood using MagNA Pure Compact (manufactured by Roche) and used.
  • MagNA Pure Compact manufactured by Roche
  • a DNA sample may be prepared from a diseased site such as a tumor tissue.
  • DNA library preparation step (1) First, the extracted DNA is quantified. The DNA concentration was measured with a FilterMax F5 multimode plate reader (manufactured by Molecular Devices) using a Qubit dsDNA BR Assay Kit (manufactured by Thermo Fisher). The DNA sample is diluted with nuclease-free water to a final concentration of 20 ng / ⁇ l. (2) On the PCR plate, 20 ng DNA is fragmented by transposase using SureSelectQXT Library Prep Kit (manufactured by Agilent Technologies), and then an adapter is added. (3) The adapter-added DNA is purified using AMPure XP beads (manufactured by Beckman Coulter).
  • Step 1 68 ° C for 2 minutes
  • Step 2 98 ° C for 2 minutes
  • Step 3 98 ° C for 30 seconds
  • Step 4 57 ° C for 30 seconds
  • Step 5 72 ° C for 1 minute
  • Step 6 72 ° C for 5 minutes
  • Step 7 Maintained at 4 ° C. Repeat steps 3-5 7 times.
  • (5) Purify DNA in the same manner as in step (3).
  • (6) The DNA concentration is measured using Qubit dsDNA BR Assay Kit in the same manner as in (1) above, and an equal amount of each DNA sample is pooled in a new tube so that the final amount is 1 ⁇ g.
  • library adjustment is performed at once for up to 96 different objects, samples are pooled, and the following steps are performed.
  • the number of samples to be pooled does not need to be limited to 96, and the number of samples convenient for performing the reaction, such as 384 samples, can be used.
  • the DNA library preparation step shown in this example is devised so that the index tag addition step for personal identification can be performed simultaneously with the DNA library amplification step by PCR. As a result, a large number of samples can be simultaneously performed in the following hybridization step.
  • the DNA library preparation step takes 1 hour each for steps (1), (2), and (6), and 1.5 hours each for steps (3) to (5).
  • the DNA obtained by the post capture pool method is purified using Dynabeads M-270 Streptavidin (manufactured by Thermo Fisher Scientific).
  • the purified DNA was amplified by PCR using TS-PCR oligos 1 and 2 by KAPA HiFi HotStart Ready Mix (manufactured by Kapa Biosystems). The sequence of the oligo DNA and the PCR cycle are as follows.
  • Oligo 1 AATGATACCGGCGACCACCGAGA (SEQ ID NO: 1)
  • Oligo 2 CAAGCAGAAGACGGCATACGAG (SEQ ID NO: 2)
  • Step 1 98 ° C for 30 seconds
  • Step 2 98 ° C for 10 seconds
  • Step 3 60 ° C for 30 seconds
  • Step 4 72 ° C for 30 seconds
  • Step 5 72 ° C for 5 minutes
  • Step 6 Maintain at 4 ° C. Repeat steps 2-4 12-12 times.
  • PCR amplified DNA is purified by AMPure XP beads.
  • the hybridization step takes 21 hours for the step (1), 3 hours for the step (2), and 1.5 hours for the step (3).
  • the steps from the DNA library preparation step to the hybridization with the probe in the hybridization step (1) may be performed on the first day, and the hybridization steps (2) and (3) may be performed on the second day.
  • (III) Sequencing step (1) The DNA library amplified by PCR is diluted and made single-stranded with alkali. (2) Sequencing is performed using MiSeq Reagent Kit v3 (manufactured by Illumina). Step (1) takes 0.5 hour and step (2) takes 55 hours. Therefore, all the processes are completed in about 5 days.
  • this genetic testing method is performed by using a small amount of DNA of 20 ng by combining a library preparation method that can be performed with SureSelectQXT Library Kit and a target enrichment system by hybridization that can be performed with SeqCap EZ Choice System. By pooling a large number of samples called samples and performing hybridization, genetic testing can be performed without cost and labor.
  • Example 2 Results of using a sample obtained from a breast cancer patient A blood sample was obtained from 11 patients whose diagnosis was confirmed pathologically at Chiba University and informed consent was obtained. Used for inspection.
  • Example 3 BRCA1 / BRCA2 gene test in a general population BRCA1 / 2 gene test was performed in the same manner as in Example 2 on a general population of 373 Japanese patients who received medical examinations.
  • a genetic test can be performed for screening a general population by the method of the present invention, a person having a gene mutation can be detected from a population with no family history.
  • VUS it is currently determined as VUS, and it is possible to evaluate by continually monitoring and accumulating information on unknown mutations that are meaningful or unknown, and accumulating important genetic information. Will be able to.
  • Example 4 Application to breast cancer patients Next, in order to verify the usefulness of this method, the results of analyzing samples of 288 breast cancer patients are shown. Familial tumors that can be considered as the risk of developing breast cancer as shown in Table 1 by extracting DNA from the cancer tissue of a breast cancer patient whose pathological diagnosis has been confirmed and informed consent has been obtained at the first clinic attached to Chongqing Medical University in China A multi-gene panel test for the causative gene was performed. Probes were designed to hybridize and capture the protein coding region of 54 genes shown in Table 1 and exon junction 50 bp. The mutations detected in breast cancer patients are classified, and single nucleotide substitutions determined to be causative are shown in Table 4, and insertion / deletion mutations are shown in Table 5.
  • Multi-gene panel testing which tests for a large number of genes, not only accumulates information such as the discovery of new mutations, but also treats with molecular targeted drugs when there is a mutation in the gene where the molecular targeted drug exists Expected to be applied in actual clinical practice. Proper treatment for patients who are expected to have an effect is considered to become more and more important from the viewpoint of medical economy, but the method shown in this example is a high-accuracy and rapid examination method. Further, since the inspection can be performed at a low cost, it is a very useful inspection method.

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Abstract

La présente invention concerne un procédé de criblage génétique qui est rapide, hautement précis et peu coûteux et qui consiste à regrouper des banques qui ont été créées, à les combiner avec un système d'enrichissement de cible basé sur l'hybridation et à mettre en œuvre un criblage génétique par séquençage de nouvelle génération.
PCT/JP2018/000952 2017-01-18 2018-01-16 Procédé de criblage génétique rapide utilisant un séquenceur de nouvelle génération Ceased WO2018135464A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020036537A (ja) * 2018-08-31 2020-03-12 シスメックス株式会社 解析方法、情報処理装置、遺伝子解析システム、プログラム、記録媒体
JP2020036536A (ja) * 2018-08-31 2020-03-12 シスメックス株式会社 解析方法、情報処理装置、遺伝子解析システム、プログラム、記録媒体
CN114438218A (zh) * 2022-04-01 2022-05-06 普瑞基准科技（北京）有限公司 一种检测多种肿瘤的基因Panel、试剂盒及应用

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
AHMADLOO, S. , ET AL.: "Rapid and cost-effective high-throughput sequencing for identification of germline mutations of BRCA1 and BRCA2", JOURNAL OF HUMAN GENETICS, vol. 62, no. 5, February 2017 (2017-02-01), pages 561 - 567, XP055519411 *
BOSDET IAN E. ET AL.: "A clinically validated diagnostic second-generation sequencing assay for detection of hereditary BRCA1 and BRCA2 mutations", JOURNAL OF MOLECULAR DIAGNOSTICS, vol. 15, November 2013 (2013-11-01), pages 796 - 809, XP055519408 *
CHONG HANSOOK KIM ET AL.: "The validation and clinical implementation of BRCAplus: A comprehensive high-risk breast cancer diagnostic assay", PLOS ONE, vol. 9, no. 5, 15 May 2014 (2014-05-15), XP055519416 *
HIGASHINO TOSHIHIDE ET AL.: "Multiple common and rare variants of ABCG2 cause gout", RMD OPEN, vol. 3, no. 2, August 2017 (2017-08-01), XP055519417 *
LINCOLN STEPHEN E. ET AL.: "A systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients", JOURNAL OF MOLECULAR DIAGNOSTICS, vol. 17, no. 5, September 2015 (2015-09-01), pages 533 - 544, XP055519415 *
STROM CHARLES M. ET AL.: "Development and validation of a next-generation sequencing assay for BRCA1 and BRCA2 variants for the clinical laboratory", PLOS ONE, vol. 10, no. 8, 21 August 2015 (2015-08-21), XP055519410 *
WALSH, TOM . ET AL.: "Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing", PNAS, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 107, no. 28, 28 June 2010 (2010-06-28), pages 12629 - 12633, XP055128035 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020036537A (ja) * 2018-08-31 2020-03-12 シスメックス株式会社 解析方法、情報処理装置、遺伝子解析システム、プログラム、記録媒体
JP2020036536A (ja) * 2018-08-31 2020-03-12 シスメックス株式会社 解析方法、情報処理装置、遺伝子解析システム、プログラム、記録媒体
CN114438218A (zh) * 2022-04-01 2022-05-06 普瑞基准科技（北京）有限公司 一种检测多种肿瘤的基因Panel、试剂盒及应用

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