WO2022149575A1 - Kit de détection de modification de méthylation mlh1 et de mutation braf - Google Patents

Kit de détection de modification de méthylation mlh1 et de mutation braf Download PDF

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WO2022149575A1
WO2022149575A1 PCT/JP2022/000095 JP2022000095W WO2022149575A1 WO 2022149575 A1 WO2022149575 A1 WO 2022149575A1 JP 2022000095 W JP2022000095 W JP 2022000095W WO 2022149575 A1 WO2022149575 A1 WO 2022149575A1
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probe
methylation
gene
region
base sequence
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憲章 中村
光芳 大場
岳司 永坂
彰一 硲
浩昭 永野
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東洋鋼鈑株式会社
学校法人 川崎学園
国立大学法人山口大学
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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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a kit for detecting methylation modification of the MLH1 gene encoding a DNA mismatch repair protein and a V600E gene mutation in BRAF (v-raf murine sarcoma viral oncogene homolog B1).
  • colorectal cancers are thought to develop due to the accumulation of gene mutations in colorectal mucosal cells, mainly due to exposure to environmental factors (sporadic colorectal cancer). Therefore, the prevalence of sporadic colorectal cancer increases with age.
  • some colorectal cancers are familial and are collectively called hereditary colorectal cancer.
  • hereditary colorectal cancer the germline mutation that causes it is found to be a mutation in the tumor suppressor gene or mismatch repair gene, and it is a premature onset, and has the characteristic of developing multiple colorectal cancer and cancer in other organs at a high rate.
  • Lynch syndrome (sometimes referred to as hereditary non-polyposis colorectal cancer: HNPCC), which frequently develops colorectal cancer in an autosomal dominant manner, Includes familial adenomatous polyposis (FAP).
  • FAP familial adenomatous polyposis
  • Lynch syndrome which has no difference in clinical characteristics from sporadic colorectal cancer, is used in daily practice. It is highly likely that many have been overlooked. Reliable identification of hereditary tumors reliably aids in cancer prevention and early detection in the family.
  • Lynch syndrome is an autosomal dominant disorder mainly caused by germline mutations in mismatch repair genes.
  • Lynch syndrome is diagnosed according to the following procedure. First, for patients with suspected Lynch syndrome, we will collect clinical information such as family history, age of onset, related cancers, and histopathological features, and consider whether they meet the so-called Amsterdam Standard II or the revised Zebesda Guidelines. If these Amsterdam Standard II or revised Zebesda Guidelines are met, a microsatellite instability (MSI) test of the tumor tissue or an immunohistological test for the causative mismatch repair protein is performed, and the frequency is high. Confirm the disappearance of mismatch repair proteins by MSI (high-frequency MSI: MSI-H) or immunostaining.
  • MSI microsatellite instability
  • microsite instability means that microsatellite, which is a repeating sequence of one to several bases existing in the genome due to an abnormality in the mismatch repair mechanism, has a different number of repetitions from normal cells.
  • MSI-H Frequent microsatellite instability
  • the Amsterdam Standard II may miss Lynch Syndrome due to its strict standards, and the revised Zebesda Guidelines have been devised to make up for its shortcomings.
  • the revised Zebesda Guidelines recommend that MSI tests be performed when 1 to 5 items of clinical information and findings are met. And if the result of the MSI test on the tumor cells is MSI-H, Lynch syndrome is strongly suspected.
  • a definitive diagnosis of Lynch syndrome is made by identifying pathogenic mutations in the germline of the mismatch repair gene. Specifically, the patient's blood is used to directly test for mutations in the germline of the mismatch repair gene (eg, the MLH1 gene). If a pathogenic variant is identified, it is a definitive diagnosis of Lynch syndrome.
  • the mismatch repair gene eg, the MLH1 gene
  • MSI-H cancer is found not only in Lynch syndrome but also in sporadic solid tumors (reported to be found in ⁇ 15% of sporadic colorectal cancers). Therefore, by distinguishing between Lynch syndrome and sporadic MSI-H cancer, it is possible to narrow down Lynch syndrome more accurately.
  • Patent Document 1 describes a method for detecting the methylation of CpG islands in the MLH1 gene by a methylation-sensitive enzyme, and simultaneously amplifying the CpG islands in the MLH1 gene and the region containing V600E in the BRAF gene to simultaneously amplify the CpG islands.
  • a method for detecting methylation by a methylation-sensitive enzyme and V600E in the BRAF gene by sequence analysis is disclosed.
  • the V600E mutation in the BRAF gene is found in hairy cell leukemia, malignant melanoma, thyroid cancer, ovarian cancer, colorectal cancer, etc., and about 10% in colorectal cancer.
  • 35 to 43% of sporadic colorectal cancers showing MSI-H have V600E mutations in the BRAF gene in tumor tissue
  • most colorectal cancers in Lynch syndrome show MSI-H but V600E in the BRAF gene. Almost no mutations are found. Therefore, the presence or absence of the V600E mutation in the BRAF gene may be used to distinguish between sporadic colorectal cancer and Lynch syndrome.
  • Patent Document 1 discloses a specific region for detecting methylation of the MLH1 gene, and when methylation cytosine in the region is detected by a methylation sensitivity restriction enzyme cleavage method and methylation is detected. Discloses that the subject is determined not to have Lynch syndrome or Lynch-like. Further, Patent Document 1 discloses that a predetermined region of the BRAF gene is amplified at the same time as the above-mentioned methylation of the MLH1 gene, and the presence or absence of a V600E mutation in the BRAF gene is investigated.
  • Patent Document 2 discloses a cancer-specific variant for detecting a mutation (V600E) in the BRAF gene, a method for diagnosing cancer, and a method for developing a therapeutic agent for treating cancer. ..
  • the present inventors have obtained a probe for detecting a region encoding a mutation (V600E) in the BRAF gene based on a sequence in which cytosine in the region is replaced with thymine.
  • V600E a mutation in the BRAF gene
  • methylation of the MLH1 gene and mutation (V600E) in the BRAF gene can be detected simultaneously with high accuracy, and the present invention was completed.
  • the present invention includes the following. (1) A step of treating genomic DNA prepared from a sample with hydrogen sulfite, a region for detecting methylation of the MLH1 gene using genomic DNA treated with hydrogen sulfite as a template, and a V600E mutation site in the BRAF gene.
  • the step of amplifying the region containing the A method for simultaneously detecting methylation of the MLH1 gene and V600E mutation of the BRAF gene in a sample including the steps of (2) Regarding the region for detecting methylation of the MLH1 gene, a methylation-compatible probe corresponding to the amplicon when the region is methylated and a demethylation corresponding to the amplicon when the region is not methylated.
  • the method according to (1) wherein a probe set including a corresponding probe is used.
  • (4) Regarding the region containing the V600E mutation site in the BRAF gene a wild-type probe corresponding to the case where the V600E mutation site is a wild type and a mutant probe corresponding to the case where the V600E mutation site is a mutant type.
  • the method according to (1) wherein a probe set consisting of the above is used.
  • the method according to (6), wherein the blocking nucleic acid contains the base sequence of SEQ ID NO: 15.
  • a region for detecting methylation of the MLH1 gene and containing a probe for the MLH1 gene corresponding to the base sequence of the region after hydrogen sulfite treatment and a V600E mutation site in the BRAF gene A kit that simultaneously detects methylation of the MLH1 gene and V600E mutation of the BRAF gene in a sample, including a probe set, including a probe for the BRAF gene corresponding to the base sequence of the region after hydrogen sulfite treatment.
  • the probe for the MLH1 gene includes a methylation-compatible probe corresponding to an amplicon when the region is methylated and a non-methylation-compatible probe corresponding to an amplicon when the region is not methylated.
  • the BRAF gene probe is characterized by including a wild-type probe corresponding to the case where the V600E mutation site is a wild type and a mutant probe corresponding to the case where the V600E mutation site is a mutant type.
  • the blocking nucleic acid contains the base sequence of SEQ ID NO: 15.
  • the probe for the MLH1 gene and the probe for the BRAF gene include a microarray immobilized on a substrate.
  • the probe for detecting the region containing the V600E mutation site in the BRAF gene is designed based on the base sequence after bisulfite treatment, the methylation of the MLH1 gene and the sudden V600E in the BRAF gene are designed. Mutations can be detected simultaneously with high accuracy. Therefore, by applying the present invention, it is possible to quickly and efficiently distinguish between sporadic colorectal cancer and Lynch syndrome.
  • A shows the result of determining the methylation of the MLH1 gene
  • B shows the result of determining the V600E mutation in the BRAF gene. It is a characteristic diagram which shows the result of having performed PCR by applying the PCR program shown in Table 4 or the PCR program shown in Table 9, and measuring the fluorescence intensity from each probe.
  • the MLH1 gene (MutL homolog 1 gene) is a gene located on chromosome 3 and encoding a type of mismatch repair protein. By methylation of the MLH1 gene, the expression of the MLH1 gene is suppressed. Methylation of the MLH1 gene can result in cancers such as colon cancer, small bowel cancer, uterine body cancer, ovarian cancer, gastric cancer, renal pelvis / urinary tract cancer, pancreatic cancer, biliary tract cancer, brain tumor, Muir-Torre syndrome, malignant melanoma, non-malignant melanoma. It is also found in other cancers such as small cell lung cancer, prostate cancer, and thyroid cancer.
  • cancers such as colon cancer, small bowel cancer, uterine body cancer, ovarian cancer, gastric cancer, renal pelvis / urinary tract cancer, pancreatic cancer, biliary tract cancer, brain tumor, Muir-Torre syndrome, malignant melanoma, non-
  • Methylation that suppresses the expression of the MLH1 gene is determined by hydrogen sulfite treatment (also referred to as bisulfite treatment), which will be described in detail later.
  • hydrogen sulfite treatment also referred to as bisulfite treatment
  • CpG islands are defined as regions where CG sequences (dinucleotides consisting of 5'-CG-3') frequently appear in specific regions.
  • the probability of appearance of a dinucleotide consisting of 5'-CG-3' is 1/16. Therefore, the region in which the dinucleotide consisting of 5'-CG-3'appears frequently is the region in which the appearance probability of the dinucleotide consisting of 5'-CG-3'appearing in the region exceeds 1/16. Can be defined. Further, the region in which the dinucleotide consisting of 5'-CG-3'appears frequently is not limited to the above definition, and the GC content contained in the region exceeds 50% and is present in 5'. -The CG-3'ratio may be defined as 60% or more (CpG observed / expected> 0.6) of the amount expected from the GC content.
  • methylation that suppresses the expression of the MLH1 gene methylation in the region disclosed in Herman JG. Et al., Proc. Natl. Acad. Sci., 95, 6870-6875, 1998 was discriminated.
  • the methylation of the CpG sequence in the marker region for determining the MLH1 methylation group disclosed in JP-A-2019-135928 may be discriminated.
  • the base sequence of the marker region for determining the MLH1 methylation group disclosed in JP-A-2019-135928 is shown in SEQ ID NO: 1.
  • cytosine For methylation that suppresses the expression of the MLH1 gene, it is particularly preferable to detect the methylation of cytosine in the CpG sequence contained in the nucleotide sequence shown in SEQ ID NO: 1.
  • the cytosine to be detected is not particularly limited as long as it is cytosine in the CpG sequence of the base sequence shown in SEQ ID NO: 1.
  • the positions of cytosine that detect methylation include, for example, the 1st, 7th, 38th, 47th, 61st, 78th, 85th, 95th, 97th, 103rd, and 122nd positions of the base sequence of SEQ ID NO: 1.
  • the cytosine for detecting methylation may be one of these cytosines, two cytosines, 3 or more, 4 or more, 5 or more, 10 or more cytosines, or all cytosines. May be.
  • the position of the cytosine that detects methylation can be preferably the cytosine at position 264 or the cytosine at position 272 in SEQ ID NO: 1, and more preferably the cytosine at position 264 and position 272 in SEQ ID NO: 1.
  • the BRAF gene is a gene encoding a BRAF (v-raf murine sarcoma viral oncogene homolog B1) protein, and BRAF activated by a mutation in which valine at position 600 becomes glutamic acid (V600E mutation) activates the MAPK pathway. It is known to mutate and excite abnormal cell proliferation.
  • the V600E mutation in the BRAF gene is a gene mutation found in lung cancer, colon cancer, thyroid cancer, biliary tract cancer and the like.
  • the base sequence of a part of the BRAF gene containing the V600E gene mutation is shown in SEQ ID NO: 2.
  • A is the first codon (ATG) (not shown in SEQ ID NO: 2) and T at the 1799th position is A, so that the 600th valine counted from the N-terminal of the BRAF protein is It will mutate to glutamic acid.
  • w (T or A) at position 267 is the position of the V600E gene mutation.
  • the genomic DNA prepared from the sample is treated with bisulfite.
  • the sample means a biological sample obtained from a test subject such as a patient suspected of having a disease.
  • the biological sample is not particularly limited, but is limited to sheep water containing fetal or embryo-derived cells, fetal-derived cell samples such as navel cord, body fluid samples such as blood, ascites, and saliva, cancer tissues or tissues suspected of having cancer, and oral mucosal cells. , Hair, skin can be mentioned.
  • the method for extracting genomic DNA is not particularly limited, and a conventionally known method or a method using a commercially available genomic DNA extraction reagent kit can be appropriately used.
  • Bisulfite treatment is also called bisulfite treatment, and unmethylated cytosine residues contained in genomic DNA are converted to uracil. On the other hand, methylated cytosine residues contained in genomic DNA are not converted. For example, in the CpG island contained in genomic DNA, if the cytosine of the 5'-CG-3'dinucleotide is not methylated, the dinucleotide is converted to 5'-UG-3'. Also, if cytosine, a dinucleotide consisting of 5'-CG-3', is methylated, the dinucleotide remains 5'-CG-3'.
  • the concentration of bisulfite added during treatment with bisulfite is not particularly limited as long as it can sufficiently convert unmethylated cytosine in genomic DNA, but is not particularly limited, for example, in a solution containing genomic DNA.
  • the final concentration is 1 M or more, preferably 1 to 15 M, and more preferably 3 to 10 M.
  • the incubation conditions (temperature and time) after the addition of bisulfite can be appropriately set according to the amount of bisulfite added. For example, when bisulfite is added at a final concentration of 6 M, 50 to Incubate at 80 ° C for 10 minutes to 7 hours.
  • the region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are each amplified by a pair of primers.
  • the base length of the region to be amplified is not particularly limited, but can be several tens to several thousand bases, particularly preferably 50 to 1000 bases, and 50 to 700 bases. It is more preferably 50 to 500 bases long, further preferably 50 to 300 bases, and even more preferably 50 to 100 bases.
  • the region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are not particularly limited, but are preferably substantially the same base length.
  • the region for detecting methylation of the MLH1 gene and the region containing the V600E mutation site in the BRAF gene are preferably the same base length, but may have different base lengths.
  • the different base length is preferably, for example, 20 bases or less, preferably 15 bases or less. It is more preferably 10 bases or less, and most preferably 5 bases or less.
  • the region for detecting methylation of the MLH1 gene is not particularly limited, but may be, for example, a region consisting of the above-mentioned nucleotide sequence of SEQ ID NO: 1 or a part of the region contained in the nucleotide sequence of SEQ ID NO: 1.
  • the base sequence of SEQ ID NO: 1 includes the 1st, 7th, 38th, 47th, 61st, 78th, 85th, 95th, 97th, 103rd, 122nd, and 133rd positions.
  • Cytosine which constitutes CpG, exists at positions, 605, 631, 638, 653, and 663.
  • a region containing one or more cytosines selected from these can be a region for detecting methylation of the MLH1 gene. More specifically, when detecting the methylation of cytosine at position 264 and cytosine at position 272 in SEQ ID NO: 1, in order to detect the methylation of the MLH1 gene in the region containing these cytosine at position 264 and cytosine at position 272. It can be an area.
  • a pair of primers for amplifying the region for detecting methylation of the MLH1 gene can be appropriately designed according to a conventional method.
  • the region for detecting methylation of the MLH1 gene is a predetermined partial region in SEQ ID NO: 1
  • the pair of primers can be designed based on the base sequence of SEQ ID NO: 1.
  • the forward primer is 5'more than cytosine at position 264 in the base sequence of SEQ ID NO: 1.
  • It can be designed at a position separated by several bases in the side (upstream) direction, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, and 1 to 30 bases.
  • several bases for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, 1 to 1 to 3'side (downstream) from cytosine at position 272 in the base sequence of SEQ ID NO: 1. It can be designed at a position 30 bases away.
  • a pair of primers for amplifying the region for detecting methylation of the MLH1 gene hybridize to a region containing no dinucleotide consisting of 5'-CG-3'constituting CpG islands. It is preferable to design in. This is because the region for detecting methylation of the MLH1 gene is amplified using the genomic DNA after hydrogen sulfite treatment as a template. That is, since cytosine other than the dinucleotide consisting of 5'-CG-3'is not methylated, it is converted to uracil by hydrogen sulfite treatment.
  • a pair of primers for amplifying the region for detecting methylation of the MLH1 gene is from 5'-CG-3'in the base sequence of SEQ ID NO: 1. It will be designed based on the base sequence in which cytosine other than the dinucleotide is replaced with uracil (or thymine).
  • the region containing the V600E mutation site in the BRAF gene is not particularly limited, but may be, for example, a region consisting of the above-mentioned nucleotide sequence of SEQ ID NO: 2 or a partial region contained in the nucleotide sequence of SEQ ID NO: 2. ..
  • the V600E mutation site is a mutation in which A is the first (not shown in SEQ ID NO: 2) of the start codon (ATG) and T at the 1799th position is A. Therefore, the region containing the V600E mutation site in the BRAF gene can be rephrased as the region containing the 1799th base with A as the first codon (ATG).
  • the region containing the V600E mutation site in the BRAF gene can be paraphrased as the region containing the 267th base in SEQ ID NO: 2.
  • a pair of primers for amplifying the region containing the V600E mutation site in the BRAF gene can also be appropriately designed according to a conventional method.
  • a pair of primers for amplifying the region containing the V600E mutation site in the BRAF gene can be designed based on the base sequence of SEQ ID NO: 2. That is, the pair of primers is designed as a forward primer and a reverse primer so as to amplify the region of the start codon (ATG) containing the 1799th base (267th base in SEQ ID NO: 2) with A as the first. ..
  • a forward primer for example, several bases, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, or 1 to 30 bases in the 5'side (upstream) direction from the 267th base in the base sequence of SEQ ID NO: 2. It can be designed at a position separated from the base.
  • the reverse primer several bases, for example, 1 to 5 bases, 1 to 10 bases, 1 to 20 bases, or 1 to 1 to 3'side (downstream) from the 267th base in the base sequence of SEQ ID NO: 2. It can be designed at a position 30 bases away.
  • the region containing the V600E mutation site in the BRAF gene is also amplified using the genomic DNA after hydrogen sulfite treatment as a template. Since cytosine contained in the BRAF gene present in genomic DNA is not methylated, it is converted to uracil by bisulfite treatment. Therefore, using the genomic DNA after hydrogen sulfite treatment as a template, the pair of primers for amplifying the region containing the V600E mutation site in the BRAF gene replaces cytosine in the nucleotide sequence of SEQ ID NO: 2 with uracil (or thymine). It will be designed based on the base sequence.
  • a polymerase chain reaction PCR
  • LAMP Loop-Mediated Isothermal Amplification
  • ICAN Isothermal and Chimeric primer-initiated Amplification of Nucleic acids
  • the method for labeling the amplified amplicon is not particularly limited, but for example, a method in which a primer used for the amplification reaction is labeled in advance may be used, or a labeled nucleotide may be used as a substrate for the amplification reaction. You may use the method you use.
  • the labeling substance is not particularly limited, but a radioisotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin can be used.
  • this reaction system includes a buffer required for nucleic acid amplification / labeling, a heat-resistant DNA polymerase, a primer specific to the amplification region, and a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate to which a fluorescent label or the like is added). , Nucleotide triphosphate, magnesium chloride and the like.
  • the region for detecting the methylation of the amplified MLH1 gene and the region containing the V600E mutation site in the BRAF gene are detected using the probe for the MLH1 gene and the probe for the BRAF gene, respectively.
  • the regions for detecting methylation of the MLH1 gene include a methylation-compatible probe corresponding to the case where the methylation site is methylated and a non-methylation-compatible probe corresponding to the case where the methylation site is not methylated. It can be detected using a probe for the MLH1 gene that contains it. That is, the methylation-compatible probe corresponds to the case where the cytosine of the dinucleotide composed of 5'-CG-3'is methylated, and the cytosine of 5'-CG-3'is maintained even by the above-mentioned bisulfite treatment. Therefore, it has a sequence corresponding to the dinucleotide consisting of 5'-CG-3'.
  • the non-methylation-compatible probe corresponds to the case where the dinucleotide consisting of 5'-CG-3'is not methylated, and the cytosine of 5'-CG-3'is converted to uracil by the above-mentioned hydrogen sulfite treatment. It will have a sequence corresponding to the dinucleotide consisting of 5'-UG-3'(5'-TG-3' as an amplicon).
  • a pair of probes consisting of a methylation-compatible probe and a non-methylation-compatible probe may be simply referred to as a probe set or a pair of probe sets. These probe sets preferably have the same sequence except for the methylation site to be detected (cytosine of the dinucleotide consisting of 5'-CG-3').
  • the region for detecting methylation of the MLH1 gene contains multiple methylation sites (5'-CG-3'dinucleotides), methylation of one or more of these multiple methylation sites.
  • Methylation in the partial region containing the site can be detected with a pair of probe sets.
  • the pair of probe sets may have a sequence corresponding to one methylation site contained in the region for detecting methylation of the MLH1 gene, or may correspond to two or more methylation sites. It may have a sequence. More specifically, a pair of probe sets can have sequences corresponding to 2-7 methylation sites and can also have sequences corresponding to 2-4 methylation sites. Probe specificity can be enhanced by using probe sets that correspond to multiple methylation sites.
  • a pair of probe sets that detect methylation can be designed for each of multiple methylation sites contained in the region for detecting methylation of the MLH1 gene. With these multiple probe sets, multiple methylation sites can also be detected independently. By preparing a plurality of probe sets, all the methylation sites contained in the amplicon can be targeted for detection, but some methylation sites can be selected and targeted for detection.
  • the region containing the V600E mutation site in the BRAF gene includes a wild-type probe corresponding to the case where the V600E mutation site is wild-type and a mutant probe corresponding to the case where the site is mutant type. It can be detected using a probe for. That is, the wild-type probe is designed to hybridize to the base sequence in which the 1799th base (267th base in SEQ ID NO: 2) is T among the start codons (ATG) in the BRAF gene, with A as the first. To. In addition, the mutant probe is designed to hybridize to the base sequence in which the 1799th base (267th base in SEQ ID NO: 2) is A among the start codons (ATG) in the BRAF gene. To.
  • the region containing the V600E mutation site in the BRAF gene is amplified using the genomic DNA after hydrogen sulfite treatment as a template, as described above. Since cytosine contained in the BRAF gene present in genomic DNA is not methylated, it is converted to uracil by bisulfite treatment. Therefore, in the region containing the V600E mutation site in the BRAF gene, which was amplified using the genomic DNA after bisulfite treatment as a template, the position originally cytosine is amplified as thymine. Therefore, the wild-type probe and the mutant probe are designed based on the base sequence in which cytosine in SEQ ID NO: 2 is replaced with thymine, respectively.
  • the base length of these probes is not particularly limited, but can be, for example, 10 to 30 base lengths, preferably 15 to 25 base lengths.
  • the probe designed as described above is preferably nucleic acid, more preferably DNA.
  • the DNA includes both double-stranded and single-stranded DNA, but is preferably single-stranded DNA.
  • the probe can be obtained, for example, by chemically synthesizing it with a nucleic acid synthesizer.
  • a nucleic acid synthesizer a device called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer, or the like can be used.
  • the probe designed as described above is preferably used in the form of a microarray (as an example, a DNA chip) by immobilizing its 5'end on a carrier.
  • the microarray is provided with a probe set for detecting methylation or unmethylation at a plurality of methylation sites contained in the above-mentioned amplicon. Further, a plurality of probe sets corresponding to different partial regions may be provided.
  • the material of the carrier those known in the art can be used and are not particularly limited.
  • precious metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductive materials such as carbon represented by graphite and carbon fiber; single crystal silicon, amorphous.
  • Silicon materials typified by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials typified by SOI (silicon on insulator); glass, quartz glass, alumina, sapphire, ceramics, fol Inorganic materials such as sterite and photosensitive glass; polyethylene, ethylene, polyprovylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinylacetate, polyvinyl alcohol, polyvinyl acetal , Acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and organic such as
  • the carrier it is preferable to use a carrier having a carbon layer and a chemically modifying group on the surface.
  • the carrier having a carbon layer and a chemical modification group on the surface includes a carrier having a carbon layer and a chemical modification group on the surface of the substrate and a carrier having a chemical modification group on the surface of the substrate composed of the carbon layer.
  • the material of the substrate those known in the art can be used, and the same materials as those mentioned above as the carrier materials can be used without particular limitation.
  • a carrier having a fine flat plate-like structure is preferably used.
  • the shape is not limited to rectangular, square and round, but usually 1 to 75 mm square, preferably 1 to 10 mm square, and more preferably 3 to 5 mm square are used. Since it is easy to produce a carrier having a fine flat plate-like structure, it is preferable to use a substrate made of a silicon material or a resin material, and in particular, a carrier having a carbon layer and a chemically modifying group on the surface of a substrate made of single crystal silicon is more preferable. preferable.
  • Single crystal silicon contains a slight change in the orientation of the crystal axis in a partial part (sometimes called a mosaic crystal) or an atomic scale disorder (lattice defect). Is also included.
  • the carbon layer formed on the substrate is not particularly limited, but is limited to synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, and carbon-based material (for example, graphite, fullerene, carbon nanotube). ), A mixture thereof, or a laminate thereof is preferable.
  • carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like may be used.
  • soft diamond is a general term for incomplete diamond structures that are a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC: Diamond Like Carbon), and the mixing ratio thereof is not particularly limited.
  • the carbon layer has excellent chemical stability and can withstand subsequent reactions in the introduction of chemically modifying groups and the bond with the substance to be analyzed, and the bond is flexible because it is bonded to the substance to be analyzed by electrostatic bonding. It is advantageous in that it has the property, it is transparent to the detection system UV because it does not absorb UV, and it can be energized during electroblotting. It is also advantageous in that there is little non-specific adsorption in the binding reaction with the substance to be analyzed. As described above, a carrier whose substrate itself is made of a carbon layer may be used.
  • the carbon layer can be formed by a known method.
  • microwave plasma CVD Chemical vapor deposit
  • ECRCVD Electro cyclotron resonance chemical vapor deposit
  • ICP Inductive coupled plasma
  • DC sputtering method ECR (Electric cyclotron resonance) sputtering method
  • ionized vapor deposition method arc.
  • Examples include a formula vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.
  • the raw material gas (methane) is decomposed by the glow discharge generated between the electrodes by the high frequency, and a carbon layer is synthesized on the substrate.
  • the ionization vapor deposition method thermions generated by the tungsten filament are used to decompose and ionize the raw material gas (benzene), and a carbon layer is formed on the substrate by the bias voltage.
  • a carbon layer may be formed by an ionization vapor deposition method in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.
  • an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate carbon atom plasma from the cathode and evaporate source.
  • a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate carbon atom plasma from the cathode and evaporate source.
  • a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light to melt it and depositing carbon atoms on a glass substrate.
  • Nd: YAG laser pulse oscillation
  • the thickness of the carbon layer is usually about a monolayer to 100 ⁇ m, and if it is too thin, the surface of the underlying substrate may be locally exposed, and conversely, it is thick. In this case, the productivity is deteriorated, so it is preferably 2 nm to 1 ⁇ m, more preferably 5 nm to 500 nm.
  • the oligonucleotide probe By introducing a chemically modifying group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier.
  • the chemically modifying group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group and an active ester group.
  • the introduction of the amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas or by plasma treatment. Alternatively, it can be carried out by irradiating the carbon layer with ultraviolet rays in chlorine gas to chlorinate it, and then irradiating it with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyhydric amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.
  • the introduction of the carboxyl group can be carried out, for example, by reacting the amino acidized carbon layer as described above with an appropriate compound.
  • Examples of the compound used for introducing a carboxyl group are represented by the formula: X-R1-COOH (in the formula, X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms).
  • Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid;
  • R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid.
  • Polycarboxylic acids such as polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; formula: R3-CO-R4-COOH (in the formula, R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms.
  • R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms); ketoic acid or aldehyde acid; formula: X-OC-R5-COOH (in the formula, X is a halogen atom, R5 is a single bond or Represents a divalent hydrocarbon group having 1 to 12 carbon atoms.)
  • Monohalides of dicarboxylic acids such as succinic acid monoclonal acid, malonic acid monoclonal acid; phthalic acid anhydride, succinic acid anhydride, oxalic acid anhydride, maleine anhydride. Examples thereof include acid anhydrides and acid anhydrides such as butanetetracarboxylic acid anhydride.
  • the introduction of the epoxy group can be carried out, for example, by reacting the amino acidized carbon layer as described above with an appropriate polyvalent epoxy compound. Alternatively, it can be obtained by reacting an organic peracid with a carbon-carbon double bond contained in the carbon layer.
  • organic peracetic acid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, trifluoroperacetic acid and the like.
  • the introduction of the formyl group can be carried out, for example, by reacting the amino acidized carbon layer as described above with glutaraldehyde.
  • the introduction of the hydroxyl group can be carried out, for example, by reacting the chlorinated carbon layer with water as described above.
  • the active ester group means an ester group that has an electron-attracting group with high acidity on the alcohol side of the ester group and activates the nucleophilic reaction, that is, an ester group with high reaction activity. It is an ester group that has an electron-withdrawing group on the alcohol side of the ester group and is more activated than the alkyl ester.
  • the active ester group has reactivity with a group such as an amino group, a thiol group and a hydroxyl group. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. are active ester groups having much higher activity than alkyl esters and the like. Known as.
  • examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalateimide group, 5-norbornen-2,3-dicarboxyimide group and the like.
  • an N-hydroxysuccinimide group is preferably used.
  • the carboxyl group introduced as described above is combined with a dehydration condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-. It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group to which an active ester group such as an N-hydroxysuccinimide group is bonded can be formed at the end of the hydrocarbon group via an amide bond (Japanese Patent Laid-Open No. 2001-139532).
  • a dehydration condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-.
  • the probe is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted onto a carrier by a spotter device or the like.
  • a spotter device or the like Can produce microarrays immobilized on a carrier.
  • the spotting solution may be manually spotted with a micropipettor.
  • Incubation After spotting, it is preferable to carry out incubation because the reaction in which the probe binds to the carrier proceeds. Incubation is usually carried out at a temperature of ⁇ 20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed in a high humidity atmosphere, for example, in a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing with a washing solution (for example, 50 mM TBS / 0.05% Tween20, 2 ⁇ SSC / 0.2% SDS solution, ultrapure water, etc.) in order to remove DNA that is not bound to the carrier. ..
  • a washing solution for example, 50 mM TBS / 0.05% Tween20, 2 ⁇ SSC / 0.2% SDS solution, ultrapure water, etc.
  • Hybridization reaction is performed between the amplicon obtained as described above and a probe immobilized on a carrier (methylation-compatible probe and non-methylation-compatible probe, and wild-type probe and mutant probe). Methylation of the MLH1 gene can be detected based on hybrids to methylated and non-methylated probes, and V600E mutations in the BRAF gene can be detected based on hybrids to wild-type and mutant probes. be able to.
  • the solution containing the amplicon preferably contains a blocking nucleic acid that hybridizes to a base sequence (that is, wild type) that does not have a V600E mutation in the BRAF gene.
  • the blocking nucleic acid has a base sequence complementary to a nucleic acid fragment having a wild-type sequence in the region containing the V600E mutation site in the BRAF gene amplified by a pair of primers.
  • the genomic DNA after treatment with bisulfite is used as a template to amplify the region containing the V600E mutation in the BRAF gene, and the amplified region contains a large amount of adenine and thymine (AT). rich).
  • AT adenine and thymine
  • the hybridization between the probe fixed on the carrier and the amplicon can be detected based on the signal from the label attached to the amplicon.
  • the signal from the label for example, when a fluorescent label is used, the fluorescent signal is detected by using a fluorescent scanner, and the signal intensity can be quantified by analyzing this with image analysis software.
  • the hybridization reaction is preferably carried out under stringent conditions.
  • the stringent condition is a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, after a hybridization reaction at 50 ° C. for 16 hours, 2 ⁇ SSC / 0.2% SDS, 25 Refers to the conditions for cleaning at °C, 10 minutes and 2 ⁇ SSC, 25 °C, 5 minutes.
  • the hybridization temperature can be 45 to 60 ° C. when the salt concentration is 0.5 ⁇ SSC, and it is more preferable to lower the hybridization temperature when the chain length of the probe is short. When the chain length is long, it is more preferable to set the hybridization temperature higher than this. Needless to say, the higher the salt concentration, the higher the hybridization temperature having specificity, and conversely, the lower the salt concentration, the lower the hybridization temperature having specificity.
  • a probe set corresponding to a partial region containing one or more methylation sites among a plurality of methylation sites included in the above-mentioned region for detecting methylation of the MLH1 gene (methylation-compatible probe and non-methylation-compatible probe).
  • the signal intensities from these probe sets can be used to determine methylation or demethylation for the methylation site.
  • the signal intensity in the methylation-compatible probe and the signal intensity in the non-methylation-compatible probe are measured, respectively, and a judgment value for evaluating the signal intensity derived from the methylation-compatible probe is calculated. do.
  • the judgment value calculated by the above formula is compared with a predetermined threshold value (cutoff value), and if the judgment value exceeds the upper limit threshold value, the region for detecting methylation of the MLH1 gene is used. It is determined that the included CpG island is methylated, and if the determination value is below the lower limit threshold value, it is determined that the included CpG island is not methylated. If the determination value is between the upper and lower thresholds set in advance, it can be determined that about half of the CpG islands contained in the region are methylated (one allele is methylated). can.
  • a single threshold value may be set, and if it exceeds this threshold value, it may be determined that it is methylated, and if it is lower than this threshold value, it may be determined that it is not methylated.
  • the V600E mutation in the BRAF gene can be detected using the genomic DNA after bisulfite treatment (bisulfite treatment) for detecting the methylation of the MLH1 gene. That is, according to the method described above, methylation of the MLH1 gene and V600E mutation in the BRAF gene can be detected at the same time.
  • bisulfite treatment bisulfite treatment
  • methylation of the MLH1 gene and V600E mutation in the BRAF gene can be detected at the same time.
  • Lynch syndrome and sporadic colorectal cancer in colorectal cancer can be discriminated.
  • the MLH1 gene is methylated and the BRAF gene has a V600E mutation, it can be determined to be sporadic MSI-H colorectal cancer. If there is no methylation of the MLH1 gene and there is a V600E mutation in the BRAF gene, it can be determined that there is a high probability of sporadic colorectal cancer that is not MSI-H. Sporadic MSI-H colorectal cancer can also be determined if the MLH1 gene is methylated and there is no V600E mutation in the BRAF gene. Then, in MSI-H colorectal cancer, if there is no methylation of the MLH1 gene and no V600E mutation in the BRAF gene, it can be judged that there is a high possibility of Lynch syndrome in colorectal cancer.
  • FIG. 1 shows the base sequences (SEQ ID NO: 4) of a part of the BRAF gene (SEQ ID NO: 3) and a part of the MLH1 gene (promoter region).
  • FIG. 2 shows the base sequences (SEQ ID NOs: 5 and 6) of the region shown in FIG. 1 after bisulfite treatment (bisulfite treatment). Cytosine becomes uracil by hydrogen sulfite treatment (bisulfite treatment), and in FIG.
  • the uracil is described as thymine for convenience. Further, in FIG. 2, the nucleic acid to be analyzed is surrounded by a square. In FIG. 2, the positions of the primers for amplifying the region containing the nucleic acid to be analyzed are underlined in each base sequence. The base sequence of each primer is shown in Table 1. Table 2 shows the base sequence of the probe that hybridizes with the amplified region.
  • the PCR temperature conditions were set as shown in Table 4.
  • the hybridization reaction was performed using the MLH1 / BRAF analysis chip (MB chip) prepared in advance. Specifically, first, the hybridization oven was set at 60 ° C., a tapper containing 30 mL of water was installed, and the mixture was left for 1 hour or more. Next, the hybridization buffer (2.25 ⁇ SSC, 0.23% SDS) and the PCR product were taken out from the freezer and returned to room temperature. 4 ⁇ l of PCR product and 2 ⁇ l of hybridization buffer were mixed. After adding 3 ⁇ L of the mixed solution to the chip cover, the mixture was set on the chip. The temperature condition in the hybridization reaction was 60 ° C.
  • a cleaning solution (0.1 ⁇ SSC / 0.1% SDS solution) was prepared, the chip after the hybridization reaction was placed in a stainless steel holder, and the inside of the cleaning solution was washed by moving it up and down 10 times, and allowed to stand for 5 minutes. After cleaning, a stainless steel holder holding the chips was placed in 1 ⁇ SSC solution. Then, the water was wiped off, a cover film was covered, and the measurement was performed by the fluorescence intensity estimation method using the gene analyzer BIOSHOT (manufactured by Toyo Kohan Co., Ltd.).
  • Experiment 1 In Experiment 1, we investigated the design of primer concentration for detecting BRAF / MLH1. Specifically, the concentrations of the two types of primer sets corresponding to the BRAF / MLH1 region were carried out under conditions 1 to 3. Each condition is shown in Tables 5 to 7, respectively.
  • the above-mentioned PCR was performed using RKO cell line-derived DNA after bisulfite treatment (bisulfite treatment) as a sample for evaluating the intensity of BRAF / MLH1.
  • the RKO cell line has an MLH1 methylation frequency of about 0% and a BRAF mutation frequency of about 66%.
  • the fluorescence intensity of BRAF / MLH1 is shown in FIG. 3 under conditions 1, 2 and 3.
  • the minimum fluorescence intensity is set to 5000 for stable analysis.
  • the fluorescence intensity of BRAF was less than 5000, and the analysis condition was not satisfied.
  • the fluorescence intensity of BRAF was detected higher than that of condition 1, and under condition 3, the fluorescence intensity of MLH1 and BRAF could be detected to the same extent.
  • the judgment value which is the intensity ratio, was calculated from the fluorescence intensity of each probe obtained by using the control treated with bisulfite (bisulfite treatment) from the following formula.
  • Judgment value strength of mutant probe / (strength of wild-type probe + strength of mutant probe)
  • the results of the judgment values obtained from each control are shown in FIG.
  • the kit was intended to determine the presence or absence of mutation and methylation, and the control with 0% mutation and methylation frequency and the error bar of the determination value were separated in other cases. From this result, it was confirmed that the probe and blocker set in this experiment can detect methylation of the MLH1 gene and V600E mutation in the BRAF gene at the same time.
  • Example 2 PCR conditions were investigated to suppress non-specific amplification of the base sequence in which the conversion from cytosine to uracil did not proceed normally by bisulfite treatment (bisulfite treatment). Specifically, a PCR program in which the annealing temperature and time were changed when the primers designed in Example 1 were used was set (Table 9). In this experiment, a comparative test was carried out between the PCR program shown in Table 4 set in Example 1 and the PCR program shown in Table 9 set in this example. Using genomic DNA derived from normal human tissue (mutation and methylation frequency 0%), treated and untreated samples with bisulfite were prepared, and 20 ng of each sample was added as a template in PCR.
  • the test conditions other than the PCR program were based on Example 1.
  • the fluorescence intensity obtained from the conditions of each PCR program is shown in FIG.
  • the PCR program shown in Table 4 about 4,000 fluorescence intensities were detected from the BRAF wild-type probe and the MLH1 methylation probe even in the sample not treated with bisulfite. It is considered that this is because in the PCR program shown in Table 4, in the annealing reaction, the base sequence untreated with bisulfite and the primer were non-specifically bound to generate an amplicon.

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Abstract

La présente invention détecte précisément la méthylation du gène MLH1 et une mutation spontanée (V600E) dans le gène BRAF. Le procédé comprend une étape de traitement d'un échantillon d'ADN génomique avec du bisulfite, caractérisé en ce qu'une région pour détecter la méthylation du gène MLH1 et une région comportant le site de mutation spontanée V600E dans le gène BRAF sont chacune amplifiées en utilisant une paire d'amorces, et la région pour détecter la méthylation du gène MLH1 et la région comportant le site de mutation spontanée V600E dans le gène BRAF étant contenues dans l'amplicon sont détectées à l'aide d'une sonde.
PCT/JP2022/000095 2021-01-06 2022-01-05 Kit de détection de modification de méthylation mlh1 et de mutation braf WO2022149575A1 (fr)

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