WO2022181496A1 - バイサルファイト試薬の評価方法及び遺伝子検査方法 - Google Patents
バイサルファイト試薬の評価方法及び遺伝子検査方法 Download PDFInfo
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- methylation
- bisulfite reagent
- error rate
- bisulfite
- dna
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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
- 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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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
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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
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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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Definitions
- the present disclosure relates to a method for evaluating a bisulfite reagent and a method for genetic testing.
- DNA methylation is known to be correlated with the onset or progression of disease, and is drawing attention as useful information for disease diagnosis.
- Patent Document 1 describes adjusting the hydrogen sulfite concentration for the purpose of reducing incomplete conversion.
- Patent Document 2 describes adjusting the hydrogen sulfite concentration and the reaction time for incomplete conversion.
- the incomplete conversion and inappropriate conversion by the bisulfite reagent are in a trade-off relationship, and the decrease of the incomplete conversion increases the inappropriate conversion, and the decrease of the inappropriate conversion increases the incomplete conversion. could be.
- An object of the present disclosure is to provide a method for evaluating a bisulfite reagent for proper use according to the target of genetic testing, and a genetic testing method for selectively using the bisulfite reagent according to the target of genetic testing.
- a method for evaluating a bisulfite reagent including the following (a) to (c). (a) Prepare a DNA sample 1 in which the CpG site to be measured is unmethylated, treat the DNA sample 1 with the bisulfite reagent to be evaluated, measure the degree of methylation of the CpG site to be measured, and measure the error Calculate the rate 1.
- An error rate of 1 is the average degree of methylation at the CpG site to be measured.
- (b) Prepare a DNA sample 2 that has the same sequence as the DNA sample 1 and has a methylated CpG site to be measured, treat the DNA sample 2 with the bisulfite reagent to be evaluated, and then treat the CpG site to be measured. Measure the degree of methylation of and calculate the error rate 2.
- the error rate 2 is 100-(average degree of methylation of the CpG site to be measured).
- a genetic testing method for detecting a disease whose onset or progression correlates with gene methylation or non-methylation and analyzing the degree of gene methylation The subject has no history of disease, treating the subject's DNA with a bisulfite reagent;
- the bisulfite reagent is a bisulfite reagent that is evaluated as having an inappropriate conversion tendency by the method for evaluating a bisulfite reagent according to ⁇ 1> when a disease whose onset or progression is correlated with gene methylation is to be detected.
- a genetic testing method for detecting a disease whose onset or progression correlates with methylation or non-methylation of a gene and analyzing the degree of methylation of the gene The subject has a history of disease, treating the subject's DNA with a bisulfite reagent;
- the bisulfite reagent is a bisulfite reagent that is evaluated as having a tendency to incomplete conversion by the method for evaluating a bisulfite reagent according to ⁇ 1> when a disease whose onset or progression is correlated with gene methylation is to be detected.
- Method. ⁇ 4> The genetic testing method according to claim 2 or 3, wherein a false positive rate and a false negative rate in the test are estimated from the values of the error rate 1 and the error rate 2 in the evaluation of the bisulfite reagent.
- ⁇ 5> The genetic testing method according to any one of ⁇ 2> to ⁇ 4>, wherein the disease is cancer.
- a method for evaluating a bisulfite reagent for proper use according to the target of genetic testing and a method for genetic testing that selectively uses the bisulfite reagent according to the target of genetic testing are provided.
- process includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.
- a numerical range indicated using “to” indicates a range including the numerical values before and after "to” as the minimum and maximum values, respectively.
- the upper limit or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described step by step.
- the upper or lower limits of the numerical ranges may be replaced with the values shown in the examples.
- sequencer is a term that includes first-generation sequencers (capillary sequencers), second-generation sequencers (next-generation sequencers), third-generation sequencers, fourth-generation sequencers, and sequencers to be developed in the future.
- the sequencer may be a capillary sequencer, a next-generation sequencer, or any other sequencer.
- a next-generation sequencer is preferable from the viewpoints of speed of analysis, a large number of samples that can be processed at one time, and the like.
- a next generation sequencer refers to a sequencer classified as opposed to a capillary sequencer (called a first generation sequencer) using the Sanger method.
- next-generation sequencers are sequencers based on the principle of determining base sequences by capturing fluorescence or luminescence associated with complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase.
- Specific examples include MiSeq (Illumina), HiSeq2000 (Illumina, HiSeq is a registered trademark), Roche454 (Roche), and the like.
- methylation of cytosine refers to addition of a methyl group to the 5-position carbon of the pyrimidine ring of cytosine.
- Bisulfite reagents can, albeit infrequently, undergo two types of mistransformation.
- One is “failed conversion” in which unmethylated cytosine is not converted to uracil and remains as cytosine.
- the other is “inappropriate conversion", in which methylated cytosine is converted to uracil.
- incomplete conversion occurs, unmethylated cytosines are erroneously identified as being methylated and the degree of DNA methylation is measured higher than it should be.
- inappropriate conversion occurs, methylated cytosines are erroneously identified as unmethylated and the degree of DNA methylation is measured lower than it should be.
- the present inventors have found that the above two types of conversion errors can be used as means for reducing methylation signals or amplifying methylation signals when using the degree of DNA methylation as a biomarker.
- the inventors of the present invention have classified bisulfite reagents into reagents that are likely to cause incomplete conversion (referred to as "reagents that tend to be incompletely converted") and reagents that are likely to cause inappropriate conversions (“reagents that tend to be improperly converted”). I found out that there is. Then, the present inventor found that the bisulfite reagent is used properly according to the nature of genetic testing (for example, genetic testing that prioritizes sensitivity and genetic testing that prioritizes specificity).
- Methods for evaluating the bisulfite reagent of the present disclosure include the following (a) to (c).
- (a) Prepare a DNA sample 1 in which the CpG site to be measured is unmethylated, treat the DNA sample 1 with the bisulfite reagent to be evaluated, measure the degree of methylation of the CpG site to be measured, and measure the error Calculate the rate 1.
- An error rate of 1 is the average degree of methylation at the CpG site to be measured.
- (b) Prepare a DNA sample 2 that has the same sequence as the DNA sample 1 and has a methylated CpG site to be measured, treat the DNA sample 2 with the bisulfite reagent to be evaluated, and then treat the CpG site to be measured.
- the error rate 2 is 100-(average degree of methylation of the CpG site to be measured). (c) Comparing error rate 1 and error rate 2, when error rate 1 ⁇ error rate 2, the bisulfite reagent to be evaluated is evaluated as having an inappropriate conversion tendency, and when error rate 1 > error rate 2, Evaluate the bisulfite reagent to be evaluated as having a tendency to incomplete conversion.
- a bisulfite reagent is a reagent that converts unmethylated cytosine in a base sequence to uracil by a bisulfite reaction, and contains hydrogen sulfite as a main component.
- a bisulfite reagent is used in the bisulfite sequencing method.
- the outline of the bisulfite sequencing method is as follows. Treatment of DNA with a bisulfite reagent converts unmethylated cytosines to uracil, while methylated cytosines remain as cytosines. That is, bisulfite treatment converts the modification state of cytosine (unmethylated or methylated) into sequence information (uracil or cytosine) at that position. Next, DNA is amplified by PCR (polymerase chain reaction). Uracil is converted to thymine in this process. The sequence of the amplified product is then analyzed using a sequencer. Knowing the modification state (unmethylated or methylated) of cytosine at the target position in DNA by determining whether the base at the position to be analyzed is thymine or cytosine can be done.
- the bisulfite reagent to be evaluated may be a commercially available product or a newly prepared reagent.
- Commercially available bisulfite reagents include EpiTect Plus Bisulfite Conversion Kit (manufactured by Qiagen), EpiTect Fast DNA Bisulfite Kit (manufactured by Qiagen), EZ DNA Methylation Direct Kit (manufactured by Zymo research), EZ DNA Methylation Gold Kit (manufactured by Zymo research), EZ DNA Methylation Lightning Kit (Zymo research), innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena), innuCONVERT Bisulfite Basic Kit (Analytik Jena), Premium Bisulfite (Diagenode), etc. be done.
- the bisulfite reagent is a commercially available product, it is preferable that the treatment of DNA sample 1 and DNA sample 2 with the bisulfite reagent follow the recommended protocol for that bisulfite reagent.
- reaction time of less than 8 hours corresponds to the former, and a reaction time of 8 hours or more corresponds to the latter.
- a CpG site is a two-base sequence in which cytosine is followed by guanine.
- the degree of methylation at a CpG site is a value calculated from a set of DNA fragments, and is calculated for each CpG site.
- the degree of methylation at a certain CpG site is ⁇ the number of DNA fragments whose CpG site is methylated ⁇ (the number of DNA fragments whose CpG site is methylated + the number of DNA fragments whose CpG site is not methylated). ⁇ , expressed in percentage (%).
- the degree of methylation of the CpG sites to be measured is the average value of the degree of methylation of each CpG site.
- DNA sample 1 and DNA sample 2 have the same base sequence, but differ in the modification state (unmethylated or methylated) of the CpG site to be measured.
- DNA sample 1 and DNA sample 2 may be synthetic DNA or standard DNA.
- DNA sample 1 the cytosine at the CpG site to be measured is unmethylated.
- DNA sample 1 is defined as DNA with a methylation degree of 0% to 5% measured by a common methylation measurement technique.
- DNA sample 2 the cytosine at the CpG site to be measured is in a methylated state.
- DNA sample 2 is defined as DNA with a methylation degree of 95% to 100% measured by a common methylation measurement technique.
- the base lengths of DNA sample 1 and DNA sample 2 are preferably 50 to 150 bases, more preferably 50 to 125 bases, and still more preferably 50 to 100 bases.
- the number of CpG sites to be measured in DNA sample 1 and DNA sample 2 is preferably 1 to 10, more preferably 2 to 8, and still more preferably 3 to 6.
- An error rate of 1 is, in short, the rate at which unmethylated CpG sites are discriminated as being methylated.
- An error rate of 1 is determined by treating DNA sample 1 with the bisulfite reagent to be evaluated, followed by PCR and sequencing. Bisulfite treatment, PCR, and sequence analysis of DNA sample 1 were performed three times, and the average value (%) of the degree of methylation for the three times was defined as an error rate of 1 (%).
- the error rate of 2 is, in short, the rate at which methylated CpG sites are discriminated as unmethylated.
- An error rate of 2 is determined by treating DNA sample 2 with the bisulfite reagent to be evaluated, followed by PCR and sequencing. Bisulfite treatment, PCR, and sequence analysis of DNA sample 2 are performed three times, and the average value (%) of the degree of methylation of the three times is subtracted from 100, and the resulting value is defined as error rate 2 (%).
- Error rate 1 and error rate 2 are compared, and when error rate 1 ⁇ error rate 2, the bisulfite reagent to be evaluated is evaluated as having an inappropriate conversion tendency.
- error rate 1 ⁇ error rate 2 When inappropriate conversion occurs, methylated cytosines are erroneously identified as unmethylated and the degree of DNA methylation is measured lower than it should be.
- a bisulfite reagent with a tendency to convert inappropriately is more likely to measure the degree of DNA methylation lower than the original value, rather than to measure the degree of DNA methylation higher than the original value. It is a high bisulfite reagent.
- a bisulfite reagent that tends to be inappropriately converted can be said to be a means for reducing methylation signals.
- Error rate 1 and error rate 2 are compared, and when error rate 1 > error rate 2, the bisulfite reagent to be evaluated is evaluated as having an incomplete conversion tendency.
- error rate 1 When incomplete conversion occurs, unmethylated cytosines are erroneously identified as being methylated and the degree of DNA methylation is measured higher than it should be.
- a bisulfite reagent that tends to be incompletely converted is more likely to measure the degree of DNA methylation higher than the original value, rather than to measure the degree of DNA methylation lower than the original value. It is a high bisulfite reagent.
- a bisulfite reagent that tends to be incompletely converted can be said to be a methylation signal amplification means.
- the bisulfite reagent is a commercial product, the following can generally be said.
- a bisulfite reagent having a recommended protocol with a reaction time of 8 hours or longer is likely to be evaluated as a bisulfite reagent with a tendency to be inappropriately converted.
- a bisulfite reagent with a recommended protocol in which the reaction time is less than 8 hours is likely to be evaluated as a bisulfite reagent prone to incomplete conversion. Therefore, with the recommended protocol, it is possible to predict whether a bisulfite reagent tends to be improperly converted or to be incompletely converted before performing the bisulfite reagent evaluation method of the present disclosure.
- a subject for the genetic testing method of the present disclosure is a human. Examinees are, for example, those who voluntarily undergo health checkups, those suspected of having a disease at a medical institution, patients undergoing treatment, and former patients who have recovered from a disease.
- the information obtained by the genetic testing method of the present disclosure is information that assists the doctor's diagnosis, the basis for the doctor or the subject to determine the necessity of a detailed examination (e.g., imaging test), and the doctor's treatment method or therapeutic drug. It is useful as a basis for selection, motivation for subjects to improve their lifestyle habits, and the like.
- the genetic testing method of the present disclosure is a genetic testing method for analyzing the degree of methylation of genes.
- Diseases to be tested by the genetic testing method of the present disclosure are not particularly limited as long as they are diseases associated with methylation or non-methylation of cytosine in genes.
- Diseases include cancer, autoimmune disease, neurological disease, heart disease, cardiovascular disease, cerebrovascular disease, metabolic disease, endocrine disease, and the like.
- Cancers include lung cancer, esophageal cancer, stomach cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, kidney cancer, bladder cancer, urinary tract cancer, breast cancer, and ovarian cancer.
- Diseases other than cancer include rheumatoid arthritis and schizophrenia.
- the genetic testing method of the present disclosure targets diseases whose onset or progression correlates with gene methylation or non-methylation, and analyzes the degree of gene methylation.
- the genetic testing method of the present disclosure uses different bisulfite reagents according to the disease history of the subject and the correlation between the disease and gene methylation or non-methylation. This disclosure discloses two embodiments of the genetic testing method.
- the subject is a person who has no history of the disease to be detected.
- a first embodiment includes treating a subject's DNA with a bisulfite reagent, and the bisulfite reagent of the present disclosure is evaluated when a disease whose onset or progression is correlated with gene methylation is targeted for detection.
- the bisulfite reagent that has been evaluated as having an inappropriate conversion tendency by the method is used to detect a disease whose onset or progression is correlated with gene demethylation, the incomplete conversion tendency is detected by the method for evaluating a bisulfite reagent of the present disclosure.
- the subject is a person who has a history of the disease to be detected.
- a second embodiment includes treating a subject's DNA with a bisulfite reagent, and the bisulfite reagent of the present disclosure is evaluated when a disease whose onset or progression is correlated with gene methylation is targeted for detection.
- the bisulfite reagent evaluated for incomplete conversion tendency by the method is used to detect a disease whose onset or progression is correlated with gene demethylation, the inappropriate conversion tendency is detected by the bisulfite reagent evaluation method of the present disclosure.
- the properties of genetic testing e.g., genetic testing that prioritizes specificity, sensitivity that prioritizes gene inspection
- genetic testing that prioritizes specificity, sensitivity that prioritizes gene inspection
- a disease whose onset or progression is correlated with gene methylation refers to a disease in which the CpG site of a gene is unmethylated under normal conditions and becomes methylated as the disease develops or progresses.
- esophageal cancer correlates with methylation of the MT1M (metallothionin 1M) gene.
- the MT1M gene is unmethylated in health and methylated in cancer tissues of many esophageal cancer patients.
- a disease whose onset or progression is correlated with gene unmethylation refers to a disease in which the CpG site of a gene is methylated in a healthy state and becomes unmethylated as the disease develops or progresses.
- hepatocellular carcinoma correlated with unmethylation of the LINE-1 (long interspersed nucleotide element 1) gene.
- the LINE-1 gene is methylated in a healthy state and unmethylated in cancer tissues of many hepatocellular carcinoma patients.
- false positive means detecting unmethylated cytosines as being methylated in diseases whose onset or progression is correlated with gene methylation.
- false negative means detecting methylated cytosines as unmethylated in diseases whose onset or progression is correlated with gene methylation.
- false positive means detecting methylated cytosine as unmethylated in a disease whose onset or progression is correlated with unmethylation of the gene.
- false negative means detecting unmethylated cytosine as being methylated in a disease whose onset or progression is correlated with unmethylation of the gene. do.
- a “person with no history of disease” is, for example, a subject who undergoes a health checkup voluntarily. Medical examinations should avoid misdiagnosing healthy individuals as affected. In addition, since health examinations are generally conducted on a relatively large scale (for example, primary screening for cancer in Japan is expected to involve hundreds of thousands of patients annually), secondary screening (for example, imaging examinations) ), misdiagnosis is undesirable from the viewpoint of medical economy. Therefore, when the subject has no history of disease, the genetic test preferably has a high specificity, ie a high true negative rate, in other words a low false positive rate. Therefore, when the subject has no history of disease, the following (1) and (2) can be used properly as a preferred form.
- the detection target is a disease whose onset or progression is correlated with gene methylation
- the possibility that the degree of DNA methylation is measured higher than the original value is more likely than the original degree of DNA methylation.
- a bisulfite reagent evaluated to have an inappropriate conversion tendency is used.
- the conversion error of the bisulfite reagent is used as a means of reducing the methylation signal.
- the detection target is a disease whose onset or progression is correlated with gene unmethylation
- the degree of DNA methylation is lower than the possibility that the degree of DNA methylation is measured lower than the original value.
- the conversion error of the bisulfite reagent is a means of amplifying the methylation signal.
- a "person with a history of disease” is, for example, a patient under treatment or a former patient who has recovered from a disease. Oversight of patients should be avoided in monitoring or follow-up examinations of patients or former patients. Therefore, when the subject has a history of disease, the genetic test preferably has high sensitivity, ie, a high true positive rate, in other words a low false negative rate. Accordingly, when the subject has a history of disease, the following (3) and (4) can be used properly as a preferred form.
- the detection target is a disease whose onset or progression is correlated with gene methylation
- the possibility that the degree of DNA methylation is measured lower than the original value is more likely.
- the detection target is a disease whose onset or progression is correlated with gene unmethylation
- the degree of DNA methylation is higher than the possibility that the degree of DNA methylation is measured higher than the original value.
- a bisulfite reagent that is more likely to be measured lower than the value of .
- a bisulfite reagent evaluated to have an inappropriate conversion tendency is used.
- the conversion error of the bisulfite reagent is used as a means of reducing the methylation signal.
- DNA which is a sample for analyzing the degree of methylation of genes
- Biological samples are, for example, tissue, blood cells, blood, lymph, urine, feces, saliva, tears, cerebrospinal fluid, pericardial fluid, pleural fluid, ascites fluid.
- the biological sample is preferably selected according to the type of disease to be tested.
- the biological sample is preferably blood, urine, stool, saliva, cerebrospinal fluid, pericardial effusion, pleural effusion, or ascites.
- Blood, urine, feces, saliva, cerebrospinal fluid, pericardial effusion, pleural effusion and ascites are known to contain ctDNA (circulating tumor DNA) released from cancer cells or tumor cells. It is a versatile biological sample. As the biological sample, blood, urine, stool, or saliva is preferable from the viewpoint of low invasiveness to the subject. is preferred.
- blood refers to blood itself and blood diluted with physiological saline; stored blood obtained by adding additives such as glucose and anticoagulants to blood; fractions thereof (e.g., plasma and serum); and so on.
- Extracting DNA from a biological sample may be extracting DNA from cells contained in the biological sample, or extracting cell-free DNA contained in the biological sample.
- the degree of methylation of genes is analyzed using the bisulfite sequencing method.
- a bisulfite reagent with a tendency to inappropriate conversion or a bisulfite reagent with a tendency to incomplete conversion which is selected according to the target of genetic testing, is used.
- DNA sample 1-1 DNA having the sequence of SEQ ID NO: 1 was synthesized. This DNA has CpG sites at the 25th, 28th, 38th, 57th, 69th and 74th cytosines from the 5' end, but all CpG sites are unmethylated. This DNA is hereinafter referred to as DNA sample 1-1.
- DNA sample 1-1 (SEQ ID NO: 1) TTGATGGTATTGCACAGAATATGGCGGCGGATGCTGACCGGCAGTGAGCAGAACTGGCGCAGCTTCACCCGTTCCGTGCTGTCCATGATGACAGAAATTC
- DNA sample 1-1 was treated with EZ DNA Methylation Gold Kit, a bisulfite reagent. (manufactured by Zymo research). Bisulfite treatment was performed according to the recommended protocol for this product.
- PCR Dispense 25 ⁇ L of 2X PCR Buffer for KOD -Multi & Epi-, 1 ⁇ L of KOD -Multi & Epi-, 15 ⁇ L of 1 ⁇ M primer mix, 8.5 ⁇ L of bisulfite-treated DNA, and 0.5 ⁇ L of water into a PCR tube. did. PCR was carried out for 40 cycles of 3 steps of 94° C./2 minutes for 1 cycle, 98° C./10 seconds, 58° C./30 seconds and 68° C./15 seconds. The amplification reaction solution was purified using AMPure XP (manufactured by BECMAN COULTER), and the purified DNA was collected in 40 ⁇ L of Tris-EDTA buffer.
- AMPure XP manufactured by BECMAN COULTER
- a primer having the sequence of SEQ ID NO: 4 as a forward primer (manufactured by FASMAC Co., Ltd.) and a primer having the sequence of SEQ ID NO: 5 as a reverse primer (manufactured by FASMAC Co., Ltd.)
- indexes were added.
- DNA was amplified by PCR.
- Index-added PCR was performed using Multiplex PCR Assay Kit (manufactured by Takara Bio Inc.).
- a reaction solution was prepared with 1 ⁇ L each of 1.25 ⁇ M primers, 0.125 ⁇ L of Multiplex PCR Mix1, 12.5 ⁇ L of Multiplex PCR Mix2, and water to a final volume of 25 ⁇ L.
- PCR 1 cycle of 94°C/3 minutes, 5 cycles of 3 steps of 94°C/45 seconds, 50°C/60 seconds, 72°C/30 seconds, 94°C/45 seconds, 55°C/60 seconds, 72°C 11 cycles of 3 steps of /30 seconds were performed.
- the resulting PCR product was purified using AMPure XP Kit (manufactured by BECMAN COULTER).
- the DNA concentration after purification was quantified using BioAnalyzer (manufactured by Agilent Technologies), and more accurately quantified using KAPA Library Quantification Kit (manufactured by KAPA Biosystems).
- the purified DNA was used as a sample and sequenced using Miseq Reagent Kit v2 300 Cycle (manufactured by Illumina). Information on the degree of methylation was obtained by mapping the resulting FastQ file to the human genome sequence using Bismark. In this way, the degree of methylation was obtained for each of the 6 CpG sites of DNA sample 1-1.
- DNA sample 2-1 DNA having the sequence of SEQ ID NO:6 was synthesized. This DNA has the same sequence as DNA sample 1-1, except that all CpG sites are methylated (i.e., 25th, 28th, 38th, 57th from the 5' end, The 69th and 74th cytosines are methylated. ). This DNA is hereinafter referred to as DNA sample 2-1.
- DNA sample 2-1 (SEQ ID NO: 6) TTGATGGTATTGCACAGAATATGG[5MedC]GG[5MedC]GATGCTGAC[5MedC]GGCAGTGAGCAGAACTGG[5MedC]GCAGCTTCACC[5MedC]GTTC[5MedC]GTGCTGTCCATGATGACAGAAATTC[5MedC] represents methylated cytosine.
- DNA sample 2-1 was treated with EZ DNA Methylation Gold Kit (manufactured by Zymo Research) in the same manner as in [Bisulfite treatment of DNA sample 1-1].
- Example 2 Genetic test for cancer> The prevalence of cancer among people in their 40s in Japan is about 0.05% (about 50 people per 100,000 population). From this, among 100,000 people in their 40s who have no history of cancer, it is estimated that there are 50 potential cancer patients, and 99,950 people are estimated not to have cancer.
- the cancer to be tested is cancer whose onset or progression is correlated with gene methylation.
- a relatively large-scale primary screening of healthy subjects should preferably have high specificity, that is, should have a low false positive rate.
- the bisulfite reagent that treats the subject's DNA is more likely to be inappropriately converted. It is preferred over the bisulfite reagent, which is rated for complete conversion tendency.
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Abstract
Description
特許文献1には、亜硫酸水素塩で処理した核酸サンプルを、サイズ排除技術及びサイズ排除デバイスを使用して精製する方法が開示されている。
特許文献2には、複数の核酸フラグメントにおけるシトシンの部分的な及び不完全なバイサルファイト変換を含む、核酸サンプルのシーケンス決定方法が開示されている。
本開示は、遺伝子検査の対象に応じた使い分けのためのバイサルファイト試薬の評価方法と、遺伝子検査の対象に応じてバイサルファイト試薬を使い分ける遺伝子検査方法とを提供することを課題とする。
<1> 下記の(a)~(c)を含む、バイサルファイト試薬の評価方法。
(a)計測対象のCpGサイトがメチル化されていないDNAサンプル1を用意し、DNAサンプル1を評価対象のバイサルファイト試薬で処理した後、計測対象のCpGサイトのメチル化度を計測し、エラー率1を算出すること。エラー率1は、計測対象のCpGサイトのメチル化度の平均値である。
(b)DNAサンプル1と同一配列であり且つ計測対象のCpGサイトがメチル化されているDNAサンプル2を用意し、DNAサンプル2を評価対象のバイサルファイト試薬で処理した後、計測対象のCpGサイトのメチル化度を計測し、エラー率2を算出すること。エラー率2は、100-(計測対象のCpGサイトのメチル化度の平均値)である。
(c)エラー率1とエラー率2とを比較し、エラー率1≦エラー率2のとき、評価対象のバイサルファイト試薬を不適切変換傾向と評価し、エラー率1>エラー率2のとき、評価対象のバイサルファイト試薬を不完全変換傾向と評価すること。
<2> 発症又は進行が遺伝子のメチル化又は非メチル化と相関する疾患を検出対象とし、遺伝子のメチル化度を解析する遺伝子検査方法であって、
被検者が疾患の罹患歴がない者であり、
被検者のDNAをバイサルファイト試薬で処理することを含み、
バイサルファイト試薬が、発症又は進行が遺伝子のメチル化と相関する疾患を検出対象とするとき、<1>に記載のバイサルファイト試薬の評価方法によって不適切変換傾向と評価されたバイサルファイト試薬であり、発症又は進行が遺伝子の非メチル化と相関する疾患を検出対象とするとき、<1>に記載のバイサルファイト試薬の評価方法によって不完全変換傾向と評価されたバイサルファイト試薬である、遺伝子検査方法。
<3> 発症又は進行が遺伝子のメチル化又は非メチル化と相関する疾患を検出対象とし、遺伝子のメチル化度を解析する遺伝子検査方法であって、
被検者が疾患の罹患歴がある者であり、
被検者のDNAをバイサルファイト試薬で処理することを含み、
バイサルファイト試薬が、発症又は進行が遺伝子のメチル化と相関する疾患を検出対象とするとき、<1>に記載のバイサルファイト試薬の評価方法によって不完全変換傾向と評価されたバイサルファイト試薬であり、発症又は進行が遺伝子の非メチル化と相関する疾患を検出対象とするとき、<1>に記載のバイサルファイト試薬の評価方法によって不適切変換傾向と評価されたバイサルファイト試薬である、遺伝子検査方法。
<4> 前記バイサルファイト試薬の評価における前記エラー率1および前記エラー率2の値から、検査における偽陽性率および偽陰性率を推定する、請求項2又は請求項3に記載の遺伝子検査方法。
<5> 疾患ががんである、<2>から<4>のいずれかに記載の遺伝子検査方法。
本開示中に段階的に記載されている数値範囲において、一つの数値範囲で記載された上限値又は下限値は、他の段階的な記載の数値範囲の上限値又は下限値に置き換えてもよい。また、本開示中に記載されている数値範囲において、その数値範囲の上限値又は下限値は、実施例に示されている値に置き換えてもよい。
バイサルファイト試薬は、低頻度ではあるが、2種類の誤変換を起こし得る。一つは、非メチル化シトシンがウラシルに変換されずシトシンとして残存する「不完全変換(failed conversion)」である。もう一つは、メチル化シトシンがウラシルに変換される「不適切変換(inappropriate conversion)」である。
不完全変換が起きると、非メチル化シトシンが誤ってメチル化状態であると判別され、DNAのメチル化度が本来の値よりも高く計測される。
不適切変換が起きると、メチル化シトシンが誤って非メチル化状態であると判別され、DNAのメチル化度が本来の値よりも低く計測される。
また、本発明者は、バイサルファイト試薬には、不完全変換を起こしやすい試薬(「不完全変換傾向の試薬」という。)と、不適切変換を起こしやすい試薬(「不適切変換傾向の試薬」という。)とがあることを見出した。
そして、本発明者は、遺伝子検査の性質(例えば、感度を優先する遺伝子検査、特異度を優先する遺伝子検査)に応じてバイサルファイト試薬を使い分けることを見出した。
(a)計測対象のCpGサイトがメチル化されていないDNAサンプル1を用意し、DNAサンプル1を評価対象のバイサルファイト試薬で処理した後、計測対象のCpGサイトのメチル化度を計測し、エラー率1を算出すること。エラー率1は、計測対象のCpGサイトのメチル化度の平均値である。
(b)DNAサンプル1と同一配列であり且つ計測対象のCpGサイトがメチル化されているDNAサンプル2を用意し、DNAサンプル2を評価対象のバイサルファイト試薬で処理した後、計測対象のCpGサイトのメチル化度を計測し、エラー率2を算出すること。エラー率2は、100-(計測対象のCpGサイトのメチル化度の平均値)である。
(c)エラー率1とエラー率2とを比較し、エラー率1≦エラー率2のとき、評価対象のバイサルファイト試薬を不適切変換傾向と評価し、エラー率1>エラー率2のとき、評価対象のバイサルファイト試薬を不完全変換傾向と評価すること。
バイサルファイト試薬とは、バイサルファイト反応によって塩基配列中のメチル化されていないシトシンをウラシルに変換する試薬であって、亜硫酸水素塩を主要成分として含む試薬である。
DNAをバイサルファイト試薬で処理すると、非メチル化シトシンがウラシルへと変換される一方、メチル化シトシンはシトシンとして残存する。つまり、バイサルファイト処理により、シトシンの修飾状態(メチル化されていない、又は、メチル化されている)は、その位置の配列情報(ウラシル又はシトシン)に変換される。次いで、PCR(polymerase chain reaction)によってDNAの増幅を行う。この過程でウラシルはチミンへと変換される。次いで、増幅産物の配列をシーケンサーを用いて解析する。解析対象の位置の塩基がチミン又はシトシンのいずれであるかを決定することにより、DNA中の目的の位置のシトシンの修飾状態(メチル化されていない、又は、メチル化されている)を知ることができる。
CpGサイトとは、シトシンの次にグアニンが現れる2塩基配列である。
DNAサンプル1とDNAサンプル2とは、塩基配列が同一であるが、計測対象のCpGサイトの修飾状態(メチル化されていない、又は、メチル化されている)が相違する。
エラー率1は、要するに、メチル化されていないCpGサイトをメチル化状態と判別する率である。エラー率1は、DNAサンプル1を評価対象のバイサルファイト試薬で処理し、次いでPCR及び配列解析をして求める。DNAサンプル1のバイサルファイト処理、PCR及び配列解析を3回行い、3回のメチル化度を平均した値(%)をエラー率1(%)とする。
エラー率1とエラー率2とを比較し、エラー率1≦エラー率2のとき、評価対象のバイサルファイト試薬を不適切変換傾向であると評価する。不適切変換が起きると、メチル化シトシンが誤って非メチル化状態であると判別され、DNAのメチル化度が本来の値よりも低く計測される。不適切変換傾向のバイサルファイト試薬は、DNAのメチル化度が本来の値よりも高く計測される可能性よりも、DNAのメチル化度が本来の値よりも低く計測される可能性の方が高いバイサルファイト試薬である。不適切変換傾向のバイサルファイト試薬は、メチル化シグナル低減手段といえる。
本開示の遺伝子検査方法の被検者は、ヒトである。被検者は、例えば、自らの意思で行う健康診断の受診者、医療機関において疾患を疑われた者、治療中の患者、疾患から快復した元患者である。
DNAのメチル化度を疾患のバイオマーカーとして利用するとき、発症又は進行が遺伝子のメチル化と相関する疾患において偽陰性とは、メチル化シトシンを非メチル化状態であると検出することを意味する。
DNAのメチル化度を疾患のバイオマーカーとして利用するとき、発症又は進行が遺伝子の非メチル化と相関する疾患において偽陰性とは、非メチル化シトシンをメチル化状態であると検出することを意味する。
したがって、被検者が疾患の罹患歴がない者であるとき、遺伝子検査は特異度が高いことが好ましく、即ち真陰性率が高いことが好ましく、言い換えると偽陽性率が低いことが好ましい。そうすると、被検者が疾患の罹患歴がない者であるとき、下記の(1)と(2)の使い分けが好ましい形態であると導かれる。
(1)検出対象が発症又は進行が遺伝子のメチル化と相関する疾患であるとき、DNAのメチル化度が本来の値よりも高く計測される可能性よりも、DNAのメチル化度が本来の値よりも低く計測される可能性の方が高いバイサルファイト試薬を用いる。つまり、不適切変換傾向と評価されたバイサルファイト試薬を用いる。言い換えると、バイサルファイト試薬の変換エラーをメチル化シグナル低減手段とする。
(2)検出対象が発症又は進行が遺伝子の非メチル化と相関する疾患であるとき、DNAのメチル化度が本来の値よりも低く計測される可能性よりも、DNAのメチル化度が本来の値よりも高く計測される可能性の方が高いバイサルファイト試薬を用いる。つまり、不完全変換傾向と評価されたバイサルファイト試薬を用いる。言い換えると、バイサルファイト試薬の変換エラーをメチル化シグナル増幅手段とする。
したがって、被検者が疾患の罹患歴がある者であるとき、遺伝子検査は感度が高いことが好ましく、即ち真陽性率が高いことが好ましく、言い換えると偽陰性率が低いことが好ましい。そうすると、被検者が疾患の罹患歴がある者であるとき、下記の(3)と(4)の使い分けが好ましい形態であると導かれる。
(3)検出対象が発症又は進行が遺伝子のメチル化と相関する疾患であるとき、DNAのメチル化度が本来の値よりも低く計測される可能性よりも、DNAのメチル化度が本来の値よりも高く計測される可能性の方が高いバイサルファイト試薬を用いる。つまり、不完全変換傾向と評価されたバイサルファイト試薬を用いる。言い換えると、バイサルファイト試薬の変換エラーをメチル化シグナル増幅手段とする。
(4)検出対象が発症又は進行が遺伝子の非メチル化と相関する疾患であるとき、DNAのメチル化度が本来の値よりも高く計測される可能性よりも、DNAのメチル化度が本来の値よりも低く計測される可能性の方が高いバイサルファイト試薬を用いる。つまり、不適切変換傾向と評価されたバイサルファイト試薬を用いる。言い換えると、バイサルファイト試薬の変換エラーをメチル化シグナル低減手段とする。
生体試料は、検査対象である疾患の種類に応じて選択することが好ましい。検査対象ががんである場合、生体試料としては、血液、尿、便、唾液、脳脊髄液、心嚢水、胸水又は腹水が好ましい。血液、尿、便、唾液、脳脊髄液、心嚢水、胸水及び腹水にはがん細胞又は腫瘍細胞から放出されたctDNA(circulating tumor DNA)が存在することが知られており、複数種のがんに汎用性のある生体試料である。生体試料としては、被検者への侵襲性が低い観点から、血液、尿、便又は唾液が好ましく、ctDNAの濃度が比較的高い観点及び多種のがんのctDNAを含み得るという観点から、血液が好ましい。
[DNAサンプル1-1の用意]
DNAサンプル1の実施形態例として、配列番号1の配列を有するDNAを合成した。このDNAは、5’末端から25番目、28番目、38番目、57番目、69番目及び74番目のシトシンがCpGサイトであるが、すべてのCpGサイトがメチル化されていない。以下、このDNAを、DNAサンプル1-1という。
DNAサンプル1-1を、バイサルファイト試薬であるEZ DNA Methylation Gold Kit
(Zymo research社製)で処理した。バイサルファイト処理は、この製品の推奨プロトコルに従って行った。
フォワードプライマーとして配列番号2の配列を有するプライマー及びリバースプライマーとして配列番号3の配列を有するプライマーを用いて、マルチプレックスPCRにて、バイサルファイト処理後のDNAサンプル1-1を増幅した。マルチプレックスPCRは、KOD -Multi & Epi-(東洋紡株式会社製)を用いて、この製品の説明書に従い行った。PCRチューブに、2X PCR Buffer for KOD -Multi & Epi-を25μL、KOD -Multi & Epi-を1μL、1μMプライマーミックスを15μL、バイサルファイト処理後のDNAを8.5μL、水を0.5μL分注した。PCRは、94℃/2分を1サイクル、98℃/10秒、58℃/30秒、68℃/15秒の3ステップを40サイクル行った。増幅反応液をAMPure XP(BECMAN COULTER社製)を用いて精製し、精製したDNAを40μLのTris-EDTA緩衝液に回収した。
各回それぞれ6箇所のCpGサイトのメチル化度を平均し、さらに3回のメチル化度を平均したところ、メチル化度の平均値は0.5%であった。この値をエラー率1とした。
DNAサンプル2の実施形態例として、配列番号6の配列を有するDNAを合成した。
このDNAは、DNAサンプル1-1と同一配列を有し、ただしすべてのCpGサイトがメチル化されている(つまり、5’末端から25番目、28番目、38番目、57番目、
69番目及び74番目のシトシンがメチル化されている。)。以下、このDNAを、DNAサンプル2-1という。
DNAサンプル2-1を、[DNAサンプル1-1のバイサルファイト処理]における処理と同様に、EZ DNA Methylation Gold Kit(Zymo research社製)で処理した。
[DNAサンプル1-1のメチル化度の計測]と同じ工程を行い、DNAサンプル2-
1の6箇所のCpGサイトについてそれぞれのメチル化度を得た。
各回それぞれ6箇所のCpGサイトのメチル化度を平均し、さらに3回のメチル化度を平均したところ、メチル化度の平均値は97.6%であった。この値からエラー率2を2.4%と算出した。
バイサルファイト試薬であるEZ DNA Methylation Gold Kit(Zymo research社製)は、
エラー率1=0.5%よりもエラー率2=2.4%が大きい値であった。したがって、このバイサルファイト試薬を、不適切変換傾向と評価した。
バイサルファイト試薬であるEpiTect Plus Bisulfite Conversion Kit(Qiagen社製)についても、DNAサンプル1-1及びDNAサンプル2-1を用いて上記と同じ工程を行ったところ、エラー率1=1.3%且つエラー率2=1.2%という結果が得られた。
エラー率1よりもエラー率2が小さい値であった。したがって、このバイサルファイト試薬を不完全変換傾向と評価した。
日本における40代のがん罹患率は0.05%程度である(人口10万人に対して50人程度)。このことから、がんの罹患歴がない40代10万人において、潜在的ながん罹患者は50人と推定され、99950人はがんに罹患していないと推定される。
がんの罹患歴がない40代10万人を被検者とし、がんスクリーニング検査を実施する。検査対象のがんは、発症又は進行が遺伝子のメチル化と相関するがんとする。
不適切変換傾向のバイサルファイト試薬であるEZ DNA Methylation Gold Kit(Zymo research社製)を用いて、検査対象の遺伝子について、被検者のDNAメチル化度を解析する。
EZ DNA Methylation Gold Kitは、エラー率1が0.5%であり、エラー率2が2.4%である。したがって、EZ DNA Methylation Gold Kitを用いてバイサルファイトシーケンスすると、偽陽性499人(99950人×0.5%)且つ偽陰性1人(50人×2.4%)となり得る。
不完全変換傾向のバイサルファイト試薬であるEpiTect Plus Bisulfite Conversion Kit(Qiagen社製)を用いて、検査対象の遺伝子について、被検者のDNAメチル化度を解析する。
EpiTect Plus Bisulfite Conversion Kitは、エラー率1が1.3%であり、エラー率2が1.2%である。したがって、EpiTect Plus Bisulfite Conversion Kitを用いてバイサルファイトシーケンスすると、偽陽性1299人(99950人×1.3%)且つ偽陰性0人(50人×1.2%)となり得る。
Claims (5)
- 下記の(a)~(c)を含む、バイサルファイト試薬の評価方法。
(a)計測対象のCpGサイトがメチル化されていないDNAサンプル1を用意し、前記DNAサンプル1を評価対象のバイサルファイト試薬で処理した後、前記計測対象のCpGサイトのメチル化度を計測し、エラー率1を算出すること。前記エラー率1は、前記計測対象のCpGサイトのメチル化度の平均値である。
(b)前記DNAサンプル1と同一配列であり且つ前記計測対象のCpGサイトがメチル化されているDNAサンプル2を用意し、前記DNAサンプル2を前記評価対象のバイサルファイト試薬で処理した後、前記計測対象のCpGサイトのメチル化度を計測し、エラー率2を算出すること。前記エラー率2は、100-(前記計測対象のCpGサイトのメチル化度の平均値)である。
(c)前記エラー率1と前記エラー率2とを比較し、前記エラー率1≦前記エラー率2のとき、前記評価対象のバイサルファイト試薬を不適切変換傾向と評価し、前記エラー率1>前記エラー率2のとき、前記評価対象のバイサルファイト試薬を不完全変換傾向と評価すること。 - 発症又は進行が遺伝子のメチル化又は非メチル化と相関する疾患を検出対象とし、前記遺伝子のメチル化度を解析する遺伝子検査方法であって、
被検者が前記疾患の罹患歴がない者であり、
前記被検者のDNAをバイサルファイト試薬で処理することを含み、
前記バイサルファイト試薬が、発症又は進行が遺伝子のメチル化と相関する疾患を検出対象とするとき、請求項1に記載のバイサルファイト試薬の評価方法によって不適切変換傾向と評価されたバイサルファイト試薬であり、発症又は進行が遺伝子の非メチル化と相関する疾患を検出対象とするとき、請求項1に記載のバイサルファイト試薬の評価方法によって不完全変換傾向と評価されたバイサルファイト試薬である、遺伝子検査方法。 - 発症又は進行が遺伝子のメチル化又は非メチル化と相関する疾患を検出対象とし、前記遺伝子のメチル化度を解析する遺伝子検査方法であって、
被検者が前記疾患の罹患歴がある者であり、
前記被検者のDNAをバイサルファイト試薬で処理することを含み、
前記バイサルファイト試薬が、発症又は進行が遺伝子のメチル化と相関する疾患を検出対象とするとき、請求項1に記載のバイサルファイト試薬の評価方法によって不完全変換傾向と評価されたバイサルファイト試薬であり、発症又は進行が遺伝子の非メチル化と相関する疾患を検出対象とするとき、請求項1に記載のバイサルファイト試薬の評価方法によって不適切変換傾向と評価されたバイサルファイト試薬である、遺伝子検査方法。 - 前記バイサルファイト試薬の評価における前記エラー率1および前記エラー率2の値から、検査における偽陽性率および偽陰性率を推定する、請求項2又は請求項3に記載の遺伝子検査方法。
- 前記疾患ががんである、請求項2から4のいずれか1項に記載の遺伝子検査方法。
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