CN108300787A - Special application of the methylation sites as early diagnosing mammary cancer marker - Google Patents
Special application of the methylation sites as early diagnosing mammary cancer marker Download PDFInfo
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- CN108300787A CN108300787A CN201810343233.XA CN201810343233A CN108300787A CN 108300787 A CN108300787 A CN 108300787A CN 201810343233 A CN201810343233 A CN 201810343233A CN 108300787 A CN108300787 A CN 108300787A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
A kind of application the invention discloses special methylation sites as early diagnosing mammary cancer marker.Wherein, special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One or more of 92883861.The present invention establishes the method and standard of the screening relevant special methylation sites of disease, and screened by taking breast cancer as an example with relevant ten special methylation sites of the early diagnosis of breast cancer, the special methylation sites screened can be used for the quick diagnosis of disease as the early diagnosis marker of breast cancer.
Description
Technical field
The present invention relates to biotechnologies, in particular to a kind of special methylation sites as breast cancer early stage
The application of diagnosis marker.
Background technology
Liquid Biopsy is chosen as 20,150 big Progress ＆ New Products by MIT technology, is tumor drug resistance biomarker
Research provides new thinking.Liquid biopsy is numerous compared to tissue biopsy advantage, as non-invasi, it is repeatable obtain it is swollen
Tumor sample, Small side effects, easy to operate, cost is relatively low, detection speed is fast.
Circulating tumor DNA (cfDNA) carries mutation and the methylation information of Tumour DNA, using cfDNA as tumour mark
The false positive rate of will object, testing result is low, high sensitivity, and accuracy is high.Most protein tumor markers can be in blood
There are several weeks, the half-life period of cfDNA can utilize the development of cfDNA real time monitoring tumours only less than two hours.
CfDNA is increasingly becoming one of classical detection object of tumour liquid biopsy.But due to cfDNA contents extremely low (nanogram rank), drop
Solution is serious, and liquid biopsy development meets with bottleneck, is faced with huge challenge.
Cancer, be otherwise known as malignant tumour, be as control growth and proliferation of cell mechanism it is not normal caused by disease.Normally
In the case of, the growing multiplication of cell is tightly adjusted by the gene of some regulation and control growth and development including oncogene and tumor suppressor gene
Control, keeps normal physiological status；Radiated, damaged, chemical contamination, virus infection and endocrine imbalance etc. it is a variety of because
Under the influence of element, lead to the activation of internal oncogene and the inactivation of tumor suppressor gene, so as to cause cancer generation.In addition to base
Except mutation, epigenetic such as histone modification, DNA methylation etc. is adjusted also in the activation of oncogene, tumor suppressor gene extremely
Inactivation and follow-up tumour generation hair evolution in play an important role, apparent repair is found in kinds cancer
The exception of decorations.In short, one extremely complex process when the generation of tumour, is due to internal oncogene, tumor suppressor gene and table
See the abnormal result for adjusting and accumulating such as modification.
DNA methylation is a kind of common epigenetic (epigenetic) modification, under dnmt rna catalysis,
The methyl provided using S-adenosylmethionine, the 5th carbon atom of cytimidine is methylated, to make Cytosines be
5-methylcytosine has vital effect to the expression regulation of gene.
DNA methylation and cancer have close relationship, and it is different to have been found that there are DNA methylations in many cancers
Normal phenomenon.DNA methylation has certain stability, it is the earliest events during cancer occurs.Many research cards in recent years
Bright, the aberrant methylation of DNA can be as a kind of biomarker of cancer diagnosis.
The Main Diagnosis method of breast cancer is to use serum antigen CA153, CA125, CEA, iconography, histogenic immunity at present
Slice etc., wherein histogenic immunity slice are the gold diagnostic methods of breast cancer, but since it will obtain the mammary gland of patient first
Tissue, larger pain is brought to patient.CA153 is a kind of marker currently used for breast cancer diagnosis, but its detection is only
Suitable for advanced breast cancer, it is not exactly accurate to be used for early diagnosing mammary cancer, and there are certain gray areas.So finding one
Kind facilitates accurate diagnosis marker very urgent for the early diagnosis of breast cancer.
In the prior art, there are the use that methylates of the various methods for DNA methylation monitoring, such as Septin9 genes
It is a kind of quantitative analysis method of disclosed DNA methylation monitoring in the detection of the carcinoma of the rectum.But at this stage in detection methyl
Change whether it is abnormal when lack a unified, accurate standard, can not by the variation of specific site methylation level with
Risk of the prediction with cancer as early as possible.
Invention content
The present invention is intended to provide a kind of application of special methylation sites as early diagnosing mammary cancer marker, to solve
Marker in the prior art can not easily and accurately diagnose the technical issues of early-stage breast cancer.
To achieve the goals above, according to an aspect of the invention, there is provided in a kind of detection subject's sample to be tested
Purposes of the reagent of the methylation level of the special methylation sites of cfDNA in reagent preparation box.Wherein, detection subject is to be measured
In sample the reagent of the methylation level of special methylation sites reagent preparation box be used for selected from use below on the way one or
It is multinomial：The early diagnosis of breast cancer, the assessment of breast cancer risk, the assessment of breast cancer treatment effect and breast cancer treatment medicine
The screening of object；Special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:
106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:One or more of 92883861.
Further, sample to be tested is Healthy People and/or the blood sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, sample to be tested is Healthy People and/or the plasma sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
Further, detecting method used by the methylation level of special methylation sites in subject's sample to be tested is
Pyrosequencing, bisulfite sequencing, methylation-specific PCR quantitatively and/or qualitatively, it is quantitative and/or
Qualitative bisulfites specific polymerase chain reaction, digital polymerase chain reaction, the targeting for combining bisulfites
Sequencing, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the islands CpG microarray, mononucleotide primer
Extend (SNUPE), joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to another aspect of the present invention, it provides a kind of for detecting the special methyl of cfDNA in subject's sample to be tested
Change the kit of the methylation level in site.Include special methylation sites in detection subject's sample to be tested in the kit
The reagent of methylation level, kit are used for selected from using on the way one or more below：The early diagnosis of breast cancer, breast cancer
The assessment of risk, the assessment and breast cancer treatment drug of breast cancer cancer therapeutic effect screening；Special methylation sites choosing
From chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869,
chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:In 92883861
It is one or more.
Further, sample to be tested is Healthy People and/or the blood sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, sample to be tested is Healthy People and/or the plasma sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
Further, the reagent for detecting the methylation level of special methylation sites in subject's sample to be tested is for coke
Phosphoric acid sequencing, methylation-specific PCR quantitatively and/or qualitatively, quantifies and/or determines bisulfite sequencing
Property the reaction of bisulfites specific polymerase chain, digital polymerase chain reaction, combine bisulfites targeting survey
Sequence, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the islands CpG microarray, mononucleotide primer prolong
Stretch the reagent of (SNUPE), joint sodium hydrogensulfite restriction endonuclease analysis or Mass Spectrometer Method.
In accordance with a further aspect of the present invention, a kind of method for treating the drug screening of breast cancer is provided.This method
It include the steps that the methylation level for detecting the special methylation sites of cfDNA in subject's sample to be tested；Special methylation sites
Selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869,
chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:In 92883861
It is one or more.
Further, compared with using before screening drug, it is selected from chr10:24489173, chr10:38795361,
chr2:89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:
93251945, chr9:32183334 and chrX:The methylation level liter of one or more of 92883861 methylation sites
Height, show subject for screening drug it is effective.
Further, sample to be tested is Healthy People and/or the blood sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, sample to be tested is Healthy People and/or the plasma sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
Further, detecting method used by the methylation level of special methylation sites in subject's sample to be tested is
Pyrosequencing, bisulfite sequencing, methylation-specific PCR quantitatively and/or qualitatively, it is quantitative and/or
Qualitative bisulfites specific polymerase chain reaction, digital polymerase chain reaction, the targeting for combining bisulfites
Sequencing, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the islands CpG microarray, mononucleotide primer
Extend (SNUPE), joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to another aspect of the invention, a kind of early diagnosis, risk assessment, treatment for breast cancer is provided
Recruitment evaluation or the device of drug screening.The device includes the special methylation sites of cfDNA in detection subject's sample to be tested
The module of methylation level；Special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:
89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:
93251945, chr9:32183334 and chrX:One or more of 92883861.
Further, device further includes evaluation module, and evaluation module is used to detect subject's detected value and normal specimens
Value or normal reference value are compared, when selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:
106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:When the methylation level of one or more of 92883861 methylation sites reduces, preliminary judgement is
Subject is early-stage breast cancer patient.
Further, evaluation module will be additionally operable to before treating or uses subject's detected value before screening drug and treatment
It compares afterwards or using subject's detected value after screening drug, when selected from chr10:24489173, chr10:38795361,
chr2:89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:
93251945, chr9:32183334 and chrX:The methylation level of one or more of 92883861 methylation sites increases
When, it is determined as that drug of the treatment of subject effectively or for screening is effective.
Further, sample to be tested is Healthy People and/or the blood sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, sample to be tested is Healthy People and/or the plasma sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
Further, detecting method used by the methylation level of special methylation sites in subject's sample to be tested is
Pyrosequencing, bisulfite sequencing, methylation-specific PCR quantitatively and/or qualitatively, it is quantitative and/or
Qualitative bisulfites specific polymerase chain reaction, digital polymerase chain reaction, the targeting for combining bisulfites
Sequencing, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the islands CpG microarray, mononucleotide primer
Extend (SNUPE), joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
In accordance with a further aspect of the present invention, a kind of early diagnosis, risk assessment, treatment for breast cancer is provided
The biomarker of recruitment evaluation and drug screening.The biomarker is the special methylation sites of cfDNA, and specifically methylate position
Point is selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869,
chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:In 92883861
It is one or more.
According to another aspect of the invention, a kind of screen and the relevant specific chromosomal methylation sites of disease is provided
Method.This approach includes the following steps：A) Healthy People and blood samples of patients sample are obtained；B) it is detached from the sample that a) step obtains
Obtain blood plasma；C) cfDNA is extracted from the blood plasma of b) step；D) cfDNA of c) step extraction methylate building library；E) it determines
The full-length genome methylation level of sample；F) cfDNA methylation informations are analyzed；And it g) screens in Healthy People and patient with bright
The different conserved positions that methylate of significant difference, as with the relevant specific chromosomal methylation sites of disease.
Further, the conserved positions that methylate refer to that the standard variance SD values of the methylation level in Healthy People sample are less than
Methylation sites equal to 0.1.
Further, it refers to that Healthy People sample and clinical samples compare to have notable difference, and methylation level changes 0.2
More than, and p value is less than or equal to 0.05 site；Variation is to be raised and lowered.
Further, patient is early-stage breast cancer patient.
According to another aspect of the invention, a kind of early diagnosis of breast cancer, risk assessment or treatment effect are provided
The method of fruit assessment.This method includes detecting the methylation level of the special methylation sites of cfDNA in subject's sample to be tested
Step；Special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:
106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:One or more of 92883861.
Further, by subject's detected value compared with normal specimens detected value or normal reference value, when selected from chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
When the methylation level of multiple methylation sites reduces, preliminary judgement is that subject is early-stage breast cancer patient.
Further, sample to be tested is Healthy People and/or the blood sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, sample to be tested is Healthy People and/or the plasma sample of patient；Preferably, patient is early-stage breast cancer
Patient.
Further, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
Further, detecting method used by the methylation level of special methylation sites in subject's sample to be tested is
Pyrosequencing, bisulfite sequencing, methylation-specific PCR quantitatively and/or qualitatively, it is quantitative and/or
Qualitative bisulfites specific polymerase chain reaction, digital polymerase chain reaction, the targeting for combining bisulfites
Sequencing, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the islands CpG microarray, mononucleotide primer
Extend (SNUPE), joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to another aspect of the invention, a kind of special methylation sites of cfDNA are provided to be used for as biomarker
The purposes of early diagnosis, the risk assessment, therapeutic effect assessment and drug screening of breast cancer, special methylation sites are selected from
chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
It is multiple.
The present invention establishes the method and standard of the screening relevant special methylation sites of disease, and is sieved by taking breast cancer as an example
It chooses and relevant ten special methylation sites of the early diagnosis of breast cancer.The special methylation sites screened are as breast
The early diagnosis marker of gland cancer can be used for the diagnosis of disease, quick diagnosis.
Description of the drawings
The accompanying drawings which form a part of this application are used to provide further understanding of the present invention, and of the invention shows
Meaning property embodiment and its explanation are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 shows the methylation level thermal map of 54984 conserved positions filtered out in normal sample in embodiment 2；
Fig. 2 shows 534 that normal sample in embodiment 3 and the comparison of early-stage breast cancer sample filter out to have apparent become
The methylation sites of change；
Fig. 3 shows the site guarded in 54984 normal samples in embodiment 4, and 165 in early-stage breast cancer sample
In apparent variation has occurred；And
Fig. 4 shows the methylation level in the 10 final sites filtered out in embodiment 5 and according to this 10 site
Methylation level carry out sample clustering result.
Specific implementation mode
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
The present inventor establish unification, standard the variation feelings with the methylation level of disease detection specific site
The method that condition carrys out auxiliary diagnosis early-stage breast cancer, and screen with breast cancer illness and early stage relevant special methylation sites,
It has thus completed the present invention.
In the present invention, " diagnosis ", " risk assessment " and " therapeutic effect assessment " has meaning well known in the art,
For example, whether " diagnosis " is to suffering from the disease and judging, " risk assessment " is the size to risk and controls
The size of risk of recurrence is assessed after treatment, and whether " therapeutic effect assessment " is to there is therapeutic effect to assess, for example, such as
Fruit symptom mitigates, disappears, and lesion reduction or disappearance or disease are no longer in progress, then treatment is effective.
In the present invention, " methylate " refers to methylating for the sites CpG.
In the present invention, representation method well known in the art may be used in methylation level, such as can be examined with when being sequenced
The methylated cytosine C measured accounts for the ratio of the total C detected to indicate, specially β value (beta value), and numberical range is
0~1 or 0~100%.In some embodiments, the representation method of methylation level is β value (beta value), numerical value
Ranging from 0~1.
In the present invention, the judgment method of methylation level being raised and lowered is that the difference of β value is more than or equal to 0.2.
In the present invention, " SD values " i.e. standard deviation value has meaning well known in the art and computational methods, such as can join
It examines《The attached experimental design of biometrics》.
In the present invention, " p value " and " q values " has meaning well known in the art and computational methods, such as can refer to《Biology
Count attached experimental design》, such as p value=hypothesis is probability=feminine gender number/total number that is correct but being rejected, is pair and sample
One inspection probability of notebook data.
According to a kind of typical embodiment of the present invention, the special first of cfDNA in a kind of detection subject's sample to be tested is provided
Purposes of the reagent of the methylation level in base site in reagent preparation box, kit are used for selected from one used below on the way
Or it is multinomial：The early diagnosis of breast cancer, the assessment of breast cancer risk, the assessment of breast cancer treatment effect and breast cancer treatment
The screening of drug；Special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:One or more of 92883861.
The present invention establishes the method and standard of the screening relevant special methylation sites of disease, and is sieved by taking breast cancer as an example
It chooses and relevant ten special methylation sites of the early diagnosis of breast cancer.The special methylation sites screened are as breast
The early diagnosis marker of gland cancer can be used for the diagnosis of disease, quick diagnosis.
Preferably, sample to be tested is that Healthy People and/or the blood sample of patient, the sample facilitate acquisition, will not give patient
Excessive pain is brought, in the present embodiment, it is preferred that patient is early-stage breast cancer patient.
Preferably, sample to be tested is that Healthy People and/or the plasma sample of patient, the sample facilitate acquisition, will not give patient
Excessive pain is brought, in the present embodiment, patient is early-stage breast cancer patient.
Preferably, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861, while ten special methylation sites are detected, accuracy rate higher.
According to a kind of typical embodiment of the present invention, the methyl of special methylation sites in subject's sample to be tested is detected
It is pyrosequencing, bisulfite sequencing, methylation-specific polymerization quantitatively and/or qualitatively to change method used by level
Enzyme chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase chain reaction,
The targeting sequencing of joint bisulfites, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, CpG
Island microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to a kind of typical embodiment of the present invention, reagent includes Oligonucleolide primers, which is used for
Nucleotide sequence of the amplification containing special methylation sites.
In the present invention, one or more reagent selected from the following can also be contained in the kit：DNTP delays
Fliud flushing, archaeal dna polymerase, restriction endonuclease (including Methylation specific endonuclease) and marker (including fluorescence mark
Remember object, chemiluminescent labels and radio-labeled object etc.).
According to a kind of typical embodiment of the present invention, provide a kind of special for detecting cfDNA in subject's sample to be tested
The kit of the methylation level of different methylation sites.Include specifically to methylate in detection subject's sample to be tested in the kit
The reagent of the methylation level in site, kit are used for selected from using on the way one or more below：The early diagnosis of breast cancer,
The assessment of breast cancer risk, the assessment and breast cancer treatment drug of breast cancer cancer therapeutic effect screening；Specifically methylate
Site is selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:
189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:
One or more of 92883861.
Preferably, sample to be tested is Healthy People and/or the blood sample of patient, and patient is early-stage breast cancer patient.
Preferably, sample to be tested is Healthy People and/or the plasma sample of patient, and patient is early-stage breast cancer patient.
Preferably, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
According to a kind of typical embodiment of the present invention, the methyl of special methylation sites in subject's sample to be tested is detected
It is for pyrosequencing, bisulfite sequencing, methylation-specific polymerase quantitatively and/or qualitatively to change horizontal reagent
Chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase chain reaction, connection
The targeting sequencing of conjunction bisulfites, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the islands CpG
Microarray, mononucleotide primer extend the examination of SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or Mass Spectrometer Method
Agent.
According to a kind of typical embodiment of the present invention, reagent includes Oligonucleolide primers, which is used for
Nucleotide sequence of the amplification containing special methylation sites.
In the present invention, one or more reagent selected from the following can also be contained in the kit：DNTP delays
Fliud flushing, archaeal dna polymerase, restriction endonuclease (including Methylation specific endonuclease) and marker (including fluorescence mark
Remember object, chemiluminescent labels and radio-labeled object etc.).
According to a kind of typical embodiment of the present invention, a kind of method for treating the drug screening of breast cancer is provided.
This includes the steps that the methylation level for detecting the special methylation sites of cfDNA in subject's sample to be tested；The special position that methylates
Point is selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869,
chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:In 92883861
It is one or more.
According to a kind of typical embodiment of the present invention chr10 is selected from compared with using before screening drug:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
The methylation levels of multiple methylation sites increases, show subject for screening drug it is effective.
Preferably, sample to be tested is Healthy People and/or the blood sample of patient, and patient is early-stage breast cancer patient.
Preferably, sample to be tested is Healthy People and/or the plasma sample of patient, and patient is early-stage breast cancer patient.
Preferably, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
According to a kind of typical embodiment of the present invention, the methyl of special methylation sites in subject's sample to be tested is detected
It is pyrosequencing, bisulfite sequencing, methylation-specific polymerization quantitatively and/or qualitatively to change method used by level
Enzyme chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase chain reaction,
The targeting sequencing of joint bisulfites, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, CpG
Island microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to a kind of typical embodiment of the present invention, the methyl of special methylation sites in subject's sample to be tested is detected
The method of change level includes the steps that using nucleotide sequence of the Oligonucleolide primers amplification containing special methylation sites.
According to a kind of typical embodiment of the present invention, provides and a kind of commented for the early diagnosis of breast cancer, risk
Estimate, the device of therapeutic effect assessment or drug screening.The device includes detecting cfDNA in subject's sample to be tested specifically to methylate
The module of the methylation level in site；Special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:
89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:
93251945, chr9:32183334 and chrX:One or more of 92883861.
According to a kind of typical embodiment of the present invention, device further includes evaluation module, and evaluation module is used for subject
Detected value is compared with normal specimens detected value or normal reference value, when selected from chr10:24489173, chr10:38795361,
chr2:89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:
93251945, chr9:32183334 and chrX:The methylation level of one or more of 92883861 methylation sites reduces
When, preliminary judgement is that subject is early-stage breast cancer patient.
According to a kind of typical embodiment of the present invention, before evaluation module will be additionally operable to before treating or uses screening drug
Subject's detected value with after treatment or use screening drug after subject's detected value compared with, when selected from chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
When the methylation level of multiple methylation sites increases, it is determined as that drug of the treatment of subject effectively or for screening is effective.
Preferably, sample to be tested is Healthy People and/or the blood sample of patient, and patient is early-stage breast cancer patient.
Preferably, sample to be tested is Healthy People and/or the plasma sample of patient, and patient is early-stage breast cancer patient.
Preferably, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
According to a kind of typical embodiment of the present invention, the methyl of special methylation sites in subject's sample to be tested is detected
It is pyrosequencing, bisulfite sequencing, methylation-specific polymerization quantitatively and/or qualitatively to change method used by level
Enzyme chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase chain reaction,
The targeting sequencing of joint bisulfites, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, CpG
Island microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to a kind of typical embodiment of the present invention, provides and a kind of commented for the early diagnosis of breast cancer, risk
Estimate, the biomarker of therapeutic effect assessment and drug screening.The biomarker is the special methylation sites of cfDNA, specifically
Methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637,
chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:
One or more of 92883861.
According to a kind of typical embodiment of the present invention, a kind of screen is provided and is methylated with the relevant specific chromosomal of disease
The method in site.This approach includes the following steps：A) Healthy People and blood samples of patients sample are obtained；B) sample obtained from a) step
In isolated blood plasma；C) cfDNA is extracted from the blood plasma of b) step；D) cfDNA of c) step extraction methylate building library；
E) the full-length genome methylation level of sample is determined；F) cfDNA methylation informations are analyzed；And it g) screens in Healthy People and patient
The conserved positions that methylate with notable difference, as with the relevant specific chromosomal methylation sites of disease.
According to a kind of typical embodiment of the present invention, the conserved positions that methylate refer to the water that methylates in Healthy People sample
Flat standard variance SD values are less than or equal to 0.1 methylation sites.Detailed screening process is as follows：Measure the full-length genome first of sample
Baseization is horizontal, is generated according to illumina officials Methylation Analysis Algorithms each containing each sample
The methylation level in site, that is, Raw data.By Quality Control and standardization, ambient noise and missing values are removed, by remaining whole
Methylation information carries out analysis comparison.The conservative position that methylates is screened according to the SD values of the methylation level (β value) between healthy sample
Point, i.e. sd (betas of non relapse samples)≤0.1.
According to a kind of typical embodiment of the present invention, it refers to Healthy People sample and clinical samples ratio to have notable difference
Compared with methylation level variation is 0.2 or more, and p value is less than or equal to 0.05 site, changes to be raised and lowered.Screening in detail
Process is as follows：After trial by the different difference criterias such as 0.1,0.15,0.2,0.25 and 0.3, it is apparent poor finally to determine
The absolute value of the difference of the different methylation level mean value between Healthy People sample and early stage patient cancer sample is more than or equal to 0.2, i.e., |
mean(betas of non relapse samples)–mean(betas of relapse samples)|≥0.2.This
The establishment of standard, ensure that can have apparent classifying quality and retain effective site as much as possible for sieving in next step
Choosing.Using limma software packages, compared according to the cfDNA methylation levels of Healthy People and early carcinoma patient, by 0.1,0.5,
After the trial of the different difference criterias such as 0.01 and 0.001, finally it is determined that notable difference is that p value is less than or equal to 0.05, i.e.,
T.test (betas of non relapse samples, betas of relapse samples)≤0.05, this standard
Establishment, ensure that can have apparent classifying quality and retain effective site as much as possible for screening in next step.
According to a kind of typical embodiment of the present invention, the special methylation sites as early-stage breast cancer diagnosis marker
Refer to being guarded in healthy sample and being abnormal and methylate in early stage sample, and to classification percentage contribution maximum site.
Detailed screening process is as follows：The big site of percentage contribution is filtered out using Random Froest algorithms.By different loci quantity
Trial after, finally determined maximum 10 sites of classification percentage contribution, i.e. CpG sites with top
10MeanDecreaseAccuracy calculated by randomForest R package.It both ensure that so bright
Aobvious classifying quality, and effective site as few as possible can be retained to facilitate clinical rapid checking.Later according only to this 10 positions
The methylation level of point clusters sample, and cluster result meets clinical expection.This 10 special methylation sites can be with
Marker as early diagnosing mammary cancer.
According to a kind of typical embodiment of the present invention, patient is early-stage breast cancer patient.
According to a kind of typical embodiment of the present invention, provide a kind of early diagnosis of breast cancer, risk assessment or
The method of therapeutic effect assessment.This method includes detecting cfDNA special methylation sites in subject's sample to be tested to methylate
Horizontal step；Special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:One or more of 92883861.
Preferably, by subject's detected value compared with normal specimens detected value or normal reference value, when selected from chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
When the methylation level of multiple methylation sites reduces, preliminary judgement is that subject is early-stage breast cancer patient.
Preferably, sample to be tested is Healthy People and/or the blood sample of patient, and patient is early-stage breast cancer patient.
Preferably, sample to be tested is Healthy People and/or the plasma sample of patient, and patient is early-stage breast cancer patient.
Preferably, special methylation sites are chr10:24489173, chr10:38795361, chr2:89864021,
chr3:106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:92883861.
According to a kind of typical embodiment of the present invention, the methyl of special methylation sites in subject's sample to be tested is detected
It is pyrosequencing, bisulfite sequencing, methylation-specific polymerization quantitatively and/or qualitatively to change method used by level
Enzyme chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase chain reaction,
The targeting sequencing of joint bisulfites, southern blotting technique method, restricted boundary mark genome scanning, the extension of mononucleotide primer, CpG
Island microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
According to a kind of typical embodiment of the present invention, a kind of special methylation sites of cfDNA are provided as biological marker
Purposes of the object for early diagnosis, the risk assessment, therapeutic effect assessment and drug screening of breast cancer, specifically methylate position
Point is selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869,
chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:In 92883861
It is one or more.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
The collection of Healthy People and early-stage breast cancer disease blood samples of patients sample is identified
The blood sample of Healthy People and early-stage breast cancer patient is collected, and is received through the doctor with rich experiences by verifying
Collect (sample is provided by Tianjin tumour hospital).
Embodiment 2
The screening of conservative methylation sites
The blood sample for obtaining Healthy People and early-stage breast cancer patient is collected, and utilizes QIAamp Circulating
Nucleic Acid Kit kits extract cfDNA from blood plasma.
Concrete operation step is as follows：1) density-gradient centrifugation method is utilized, is centrifuged with the rotating speed of 1900g, by red blood cell, white thin
Born of the same parents and blood plasma separation, it is limpid for blood plasma in top layer, blood plasma is taken out, the rotating speed of 14000rpm is recycled, is carried out high again
Speed centrifugation, upper layer is blood plasma；2) 1) it in the 50ml centrifuge tubes that the blood plasma of extraction has been added to protease k, adds and splits
Liquid ACL (having contained CarrierRNA) is solved, the first mixed liquor is obtained；3) it by the first mixed liquor mixing of last gained in 2), is put in
60 DEG C of water-baths 30 minutes；4) it will complete 3) that the first mixed liquor takes out from water-bath afterwards, and ACB be added, by 1：1.8 ratio row, i.e. 1ml
Blood plasma, 1.8mlACB obtain the second mixed liquor；5) vacuum pump is utilized to filter the second mixed liquor obtained in 4), it is sharp after having filtered
It is washed with ACW1, ACW2 and absolute ethyl alcohol；6) 14000rpm skies were evaporated removal ethyl alcohol from 3 minutes, 56 DEG C；7) 30ul is utilized
Water elution obtains cfDNA；8) 7) cfDNA of middle gained methylate building library.
It is methylated two generation sequenator X-ten using complete genome DNA, measures the full-length genome methylation level of sample, root
Methylating containing each site of each sample is generated according to illumina officials Methylation Analysis Algorithms
Level is that whole methylation informations are carried out analysis comparison by Raw data, according to the SD of the methylation level (β value) between sample
Value filters out 54984 conserved positions that methylate (value≤0.1 SD), as a result referring to Fig. 1.
Embodiment 3
The screening of the methylation sites of notable difference
After trial by the different difference criterias such as 0.1,0.15,0.2,0.25 and 0.3, notable difference is finally determined
The absolute value of the difference of methylation level mean value is more than or equal to 0.2 this mark between Healthy People sample and early stage patient cancer sample
Accurate establishment, ensure that can have apparent classifying quality and retain effective site as much as possible for screening in next step.
Using limma software packages, compared according to the cfDNA methylation levels of Healthy People and early-stage breast cancer patient, by 0.1,0.5,
After the trial of the different difference criterias such as 0.01 and 0.001, finally it is determined that notable difference is that p value is less than or equal to 0.05 this mark
Accurate establishment, ensure that can have apparent classifying quality and retain effective site as much as possible for screening in next step.
Figure is depicted according to the standard established after screening, referring to Fig. 2.
Embodiment 4
Filter out the conserved sites that significant change occurs
According to it is in embodiment 2 and embodiment 3 as a result, the intersection both filtered out is guarded i.e. in Healthy People and in early days
The methylation sites that significant change occurs in patient with breast cancer, as a result referring to Fig. 3.
Embodiment 5
Further filter out the special methylation sites as early-stage breast cancer diagnosis marker
According to embodiment 4 as a result, the big site of percentage contribution is filtered out using Random Froest algorithms, by difference
After the trial of bit number of points, finally maximum 10 sites of classification percentage contribution have been determined.It both ensure that so apparent
Classifying quality, and effective site as few as possible can be retained to facilitate clinical rapid checking.Later according only to this 10 sites
Methylation level sample is clustered (see Fig. 4), this 10 special methylation sites can be used as breast cancer early stage examine
Disconnected marker.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (28)
1. the reagent of the methylation level of the special methylation sites of cfDNA is in reagent preparation in a kind of detection subject's sample to be tested
Purposes in box, which is characterized in that the kit is used for selected from using on the way one or more below：The early stage of breast cancer examines
The disconnected, assessment of breast cancer risk, the assessment of breast cancer treatment effect and breast cancer treatment drug screening；The special first
Base site is selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:
189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:
One or more of 92883861.
2. purposes according to claim 1, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Sample；
Preferably, the patient is early-stage breast cancer patient.
3. purposes according to claim 1, which is characterized in that the sample to be tested is Healthy People and/or the blood plasma of patient
Sample；
Preferably, the patient is early-stage breast cancer patient.
4. purposes according to claim 2 or 3, which is characterized in that the special methylation sites are chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:92883861.
5. purposes according to claim 1, which is characterized in that specifically methylate position in detection subject's sample to be tested
Method used by the methylation level of point is pyrosequencing, bisulfite sequencing, the spy that methylates quantitatively and/or qualitatively
Specific polymerase chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase chain
Formula reaction, the targeting sequencing of joint bisulfites, southern blotting technique method, restricted boundary mark genome scanning, mononucleotide primer prolong
It stretches, the islands CpG microarray, mononucleotide primer extend SNUPE, combine sodium hydrogensulfite restriction endonuclease analysis or mass spectrum.
6. a kind of kit for detecting the methylation level of the special methylation sites of cfDNA in subject's sample to be tested,
It is characterized in that, the examination of the methylation level of special methylation sites in detection subject's sample to be tested is included in the kit
Agent, the kit are used for selected from using on the way one or more below：The early diagnosis of breast cancer, breast cancer risk
It assesses, the screening of the assessment and breast cancer treatment drug of breast cancer cancer therapeutic effect；The special methylation sites are selected from
chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
It is multiple.
7. kit according to claim 6, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Liquid sample；Preferably, the patient is early-stage breast cancer patient.
8. kit according to claim 6, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Starch sample；Preferably, the patient is early-stage breast cancer patient.
9. kit according to claim 7 or 8, which is characterized in that the special methylation sites are chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:92883861.
10. kit according to claim 6, which is characterized in that special methyl in detection subject's sample to be tested
The reagent for changing the methylation level in site is for pyrosequencing, bisulfite sequencing, methylating quantitatively and/or qualitatively
Specific polymerase chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase
Chain reaction, the targeting sequencing for combining bisulfites, southern blotting technique method, restricted boundary mark genome scanning, mononucleotide primer
Extension, the islands CpG microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or mass spectrum
The reagent of detection.
11. a kind of method for treating the drug screening of breast cancer, which is characterized in that including in detection subject's sample to be tested
The step of methylation level of the special methylation sites of cfDNA；The special methylation sites are selected from chr10:24489173,
chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:
133579577, chr6:93251945, chr9:32183334 and chrX:One or more of 92883861.
12. according to the method for claim 11, which is characterized in that compared with using before screening drug, be selected from chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
The methylation levels of multiple methylation sites increases, show subject for screening drug it is effective.
13. according to the method for claim 11, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Liquid sample；Preferably, the patient is early-stage breast cancer patient.
14. according to the method for claim 11, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Starch sample；Preferably, the patient is early-stage breast cancer patient.
15. the method according to claim 13 or 14, which is characterized in that the special methylation sites are chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:92883861.
16. according to the method for claim 11, which is characterized in that specifically methylate in detection subject's sample to be tested
Method used by the methylation level in site is pyrosequencing, bisulfite sequencing, methylating quantitatively and/or qualitatively
Specific polymerase chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase
Chain reaction, the targeting sequencing for combining bisulfites, southern blotting technique method, restricted boundary mark genome scanning, mononucleotide primer
Extension, the islands CpG microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or matter
Spectrum.
17. a kind of device of early diagnosis, risk assessment, therapeutic effect assessment or drug screening for breast cancer,
It is characterized in that, described device includes detecting the mould of the methylation level of the special methylation sites of cfDNA in subject's sample to be tested
Block；The special methylation sites are selected from chr10:24489173, chr10:38795361, chr2:89864021, chr3:
106632637, chr4:189869, chr5:151552535, chr6:133579577, chr6:93251945, chr9:
32183334 and chrX:One or more of 92883861.
18. device according to claim 17, which is characterized in that described device further includes evaluation module, the assessment mould
Block is used for by subject's detected value compared with normal specimens detected value or normal reference value, when selected from chr10:24489173,
chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:151552535, chr6:
133579577, chr6:93251945, chr9:32183334 and chrX:One or more of 92883861 methylation sites
Methylation level reduce when, preliminary judgement is that subject is early-stage breast cancer patient.
19. device according to claim 17, which is characterized in that before the evaluation module is additionally operable to will to treat or
Using subject's detected value before screening drug with after treatment or compared with using subject's detected value after screening drug, it is elected to
From chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869,
chr5:151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:In 92883861
When the methylation level of one or more methylation sites increases, it is determined as that subject's treats drug effective or for screening
Effectively.
20. device according to claim 17, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Liquid sample；
Preferably, the patient is early-stage breast cancer patient.
21. device according to claim 17, which is characterized in that the sample to be tested is Healthy People and/or the blood of patient
Starch sample；
Preferably, the patient is early-stage breast cancer patient.
22. the device according to claim 20 or 21, which is characterized in that the special methylation sites are chr10:
24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:92883861.
23. device according to claim 17, which is characterized in that specifically methylate in detection subject's sample to be tested
Method used by the methylation level in site is pyrosequencing, bisulfite sequencing, methylating quantitatively and/or qualitatively
Specific polymerase chain reaction, bisulfites specific polymerase chain reaction quantitatively and/or qualitatively, digital polymerase
Chain reaction, the targeting sequencing for combining bisulfites, southern blotting technique method, restricted boundary mark genome scanning, mononucleotide primer
Extension, the islands CpG microarray, mononucleotide primer extend SNUPE, joint sodium hydrogensulfite restriction endonuclease analysis or matter
Spectrum.
24. a kind of biological marker of early diagnosis, risk assessment, therapeutic effect assessment and drug screening for breast cancer
Object, which is characterized in that the biomarker is the special methylation sites of cfDNA, and the special methylation sites are selected from
chr10:24489173, chr10:38795361, chr2:89864021, chr3:106632637, chr4:189869, chr5:
151552535, chr6:133579577, chr6:93251945, chr9:32183334 and chrX:One in 92883861 or
It is multiple.
25. a kind of method of screening and the relevant specific chromosomal methylation sites of disease, which is characterized in that including following step
Suddenly：
A) Healthy People and blood samples of patients sample are obtained；
B) the isolated blood plasma from the sample that a) step obtains；
C) cfDNA is extracted from the blood plasma of b) step；
D) cfDNA of c) step extraction methylate building library；
E) the full-length genome methylation level of sample is determined；
F) cfDNA methylation informations are analyzed；And
G) conserved positions that methylate with notable difference in Healthy People and patient are screened, as with the relevant specific staining of disease
Body methylation sites.
26. according to the method for claim 25, which is characterized in that the conserved positions that methylate refer in Healthy People sample
The standard variance SD values of middle methylation level are less than or equal to 0.1 methylation sites.
27. according to the method for claim 26, which is characterized in that it is described have notable difference refer to Healthy People sample and trouble
Person's sample compares, and methylation level variation is 0.2 or more, and p value is less than or equal to 0.05 site；The variation is raising or drop
It is low.
28. according to the method for claim 25, which is characterized in that the patient is early-stage breast cancer patient.
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