CN107904309B - It is a kind of for diagnose indication three negative type breast cancers Bone tumours gene diagnosis kit - Google Patents
It is a kind of for diagnose indication three negative type breast cancers Bone tumours gene diagnosis kit Download PDFInfo
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Abstract
The invention discloses the purposes of methylated genes composition and preparation diagnosis three negative type breast cancers Bone tumour kits of indication.Present invention discover that, serum methylation PITX1 and methylation AMOT can combine for diagnosing indication LuminalA type Bone of Breast Cancer transfer, serum methylation PTPN1 and methylation SLIT2 can combine for diagnosing indication LuminalB type Bone of Breast Cancer transfer, serum methylation MYLK2, methylation EFEMP1 and methylation SOSTDC1 can combine for diagnosing indication Her-2 overexpression type Bone of Breast Cancer transfer, serum methylation MYLK3 and methylation SCARA5 can combine for diagnosing three negative type breast cancers Bone tumours of indication, accuracy is up to 90% or more, testing cost is low, non-invasi, it is convenient and efficient.
Description
Technical field
The invention belongs to biochemical fields, are related to diagnosis composition and diagnostic kit, and in particular to a kind of methylation
Gene diagnosis composition and the application in terms of the diagnostic kit that preparation diagnoses indication different molecular hypotype Bone of Breast Cancer transfer.
Background technique
Breast cancer is to threaten one of the malignant tumour of women life and health, and there are about ten thousand people of 40-45 to die of breast cancer (ginseng every year
Examine document: different molecular hypotype Bone of Breast Cancer shifts the Clinical symptoms and prognostic analysis of patient, XI AN JIAOTONG UNIVERSITY Subject Index doctor
Learn version, in September, 2017 the 5th phase of volume 38).Breast cancer is very easy to that DISTANT METASTASES IN occurs, and bone is the most common distant place of breast cancer
Metastasis site is bone tissue (bibliography: Genes associated with greater than the starting metastasis site of 50% patient
breast cancer metastatic to bone,J Clin Oncol,2006；Implications of Bone-Only
Metastases in Breast Cancer:Favorable Preference with Excellent Outcomes of
Hormone Receptor Positive Breast Cancer,Cancer Res Treat,2011).According to estrogen receptor
(estrogen receptor, ER), progesterone receptor (progesterone receptor, PR), human epidermal growth factor receptor
Breast cancer, can be divided by the expression of body -2 (human epidermal growth factor receptor-2, HER-2)
4 hypotypes, be respectively as follows: Luminal A type, Luminal Type B, Her-2 overexpression type and triple negative breast cancer (bibliography:
Gene expression patterns of breast carcinomas distinguish tumor subclasses
with clinical implications,PNAS,2001).Studies have shown that prognosis and its molecule point of Bone of Breast Cancer transfer
Closely related (the bibliography: Prevalence and risk factors of bone such as type, clinical stages, lymph node status
metastasis and skeletal related events in patients with primary breast cancer
in Japan,Int J Clin Onco,2014).The specific molecular biology of different molecular hypotype breast cancer and clinical pathology are special
Sign, determines the difference of its therapeutic modality and prognosis.There is also differences for the gene phenotype of different molecular hypotype Bone of Breast Cancer transfer.
Early diagnosis Bone of Breast Cancer transfer is the key that save patient vitals.Currently, radionuclide bone scan (ECT),
CT scan (CT), nuclear magnetic resonance (MRI), Positron emission computed tomography (PET-CT) and bone tissue
Biopsy is discovery and the goldstandard for making a definite diagnosis Bone of Breast Cancer transfer.But there is different deficiencies, such as Laboratory Fee in these methods
With height, intervention diagnosis increases the burden of patient.Which increase the pressure of patient with breast cancer's Bone tumour conventional detection.
DNA methylation refers to one methyl base of covalent bond on No. 5 carbon atoms of cytimidine of genome CpG dinucleotides
Group, is primarily involved in the expression regulation of gene.The methylation of many genes is proved to closely related with various clinical diseases, some
It even can be determined that the pathogenic independent factor of disease.The diseases such as DNA methylation exception and tumour are closely related.Therefore, first
Base gene can be used as tumor cells diagnosis marker, while also can be used as molecular therapy target (bibliography:
Value of the DNA methylation marker in molecular diagnosis and treatment, molecular diagnosis and in September, 2009 volume 1 the 3rd of magazine for the treatment of
Phase).
Studies have shown that the several genes expression of breast cancer primary tumo(u)r is necessary to Bone tumour occurs, it is thus regarded that special
The transfer for determining organ is that multiple-factor is coefficient as a result, the conclusion prompts, and the transspecific of bone is bone in primary tumo(u)r
The selection of different phenotype tumour cells and bone source sex factor induction as a result, different molecular hypotype Bone of Breast Cancer transfer methylation
Gene is expected to become the diagnosis marker (bibliography: Kang Y, Siegel PM, Shu W, et of Bone of Breast Cancer transfer
al.A multigenic program mediating breast cancer metastasis to bone.Cancer
Cell,2003)。
Applicant, which is intended to study to compare, to be occurred Bone tumour and the base that methylates in the blood serum of patients with human breast carcinoma of Bone tumour does not occur
The difference of cause, discovery, verifying may be used as the methylated genes mark of diagnosis, indication different molecular hypotype Bone of Breast Cancer transfer
Object, with provide it is a kind of by blood can quick diagnosis, indication different molecular hypotype Bone of Breast Cancer transfer kit and method.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of methylated genes diagnosis composition, preparations
The diagnostic reagent that low, non-invasi, convenient and efficient diagnosis indication different molecular hypotype Bone of Breast Cancer shift at a kind of testing cost
Box.
Above-mentioned purpose of the invention is achieved by following technical solution:
One,The transfer of Luminal A type Bone of Breast Cancer
A kind of methylated genes diagnosis composition is made of methylation PITX1 and methylation AMOT.
Above-mentioned diagnosis composition is in terms of the diagnostic kit that preparation diagnoses indication Luminal A type Bone of Breast Cancer transfer
Using.
It is a kind of for diagnose indication Luminal A type Bone of Breast Cancer transfer diagnostic kit, including methylation PITX1 and
The PCR amplification primer of methylation AMOT.
Preferably, in the diagnostic kit, the PCR amplification upstream primer for the PITX1 that methylates such as Sequence NO.1
Shown, PCR amplification downstream primer is as shown in Sequence NO.2.
Preferably, in the diagnostic kit, the PCR amplification upstream primer for the AMOT that methylates such as Sequence NO.4
Shown, PCR amplification downstream primer is as shown in Sequence NO.5.
It preferably, further include that methylation PITX1 and the pyrosequencing for the AMOT that methylates draw in the diagnostic kit
Object.
Preferably, in the diagnostic kit, the Pyrosequencing primer for the PITX1 that methylates such as Sequence NO.3
It is shown.
Preferably, in the diagnostic kit, the Pyrosequencing primer for the AMOT that methylates such as Sequence NO.6 institute
Show.
It preferably, further include enzyme needed for PCR amplification and pyrosequencing and reagent in the diagnostic kit.
A method of diagnosis indication Luminal A type Bone of Breast Cancer transfer includes the following steps:
Step S1 acquires Luminal A type patient with breast cancer's limosis vein blood, serum is centrifugated out after natural coagulation；
Step S2 extracts serum total DNA, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measurement total DNA
The methylation index of methylation PITX1 and the AMOT that methylates, successively use X1、X2It indicates；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (1.499X1+2.302X2-
0.258) Y value] is obtained, Y value indicates that Bone tumour occurs for the patient with breast cancer less than 0.238, and bone does not occur greater than 0.238 indication and turns
It moves.
Two, Luminal Type B Bone of Breast Cancer shifts
A kind of methylated genes diagnosis composition is made of methylation PTPN1 and methylation SLIT2.
Above-mentioned diagnosis composition is in terms of the diagnostic kit that preparation diagnoses indication Luminal Type B Bone of Breast Cancer transfer
Using.
It is a kind of for diagnose indication Luminal Type B Bone of Breast Cancer transfer diagnostic kit, including methylation PTPN1 and
The PCR amplification primer of methylation SLIT2.
Preferably, in the diagnostic kit, the PCR amplification upstream primer for the PTPN1 that methylates such as Sequence NO.7
Shown, PCR amplification downstream primer is as shown in Sequence NO.8.
Preferably, in the diagnostic kit, the PCR amplification upstream primer such as Sequence for the SLIT2 that methylates
Shown in NO.10, PCR amplification downstream primer is as shown in Sequence NO.11.
It preferably, further include the pyrosequencing of methylation PTPN1 and the SLIT2 that methylates in the diagnostic kit
Primer.
Preferably, in the diagnostic kit, the Pyrosequencing primer for the PTPN1 that methylates such as Sequence NO.9
It is shown.
Preferably, in the diagnostic kit, the Pyrosequencing primer for the SLIT2 that methylates such as Sequence NO.12
It is shown.
It preferably, further include enzyme needed for PCR amplification and pyrosequencing and reagent in the diagnostic kit.
A method of diagnosis indication Luminal Type B Bone of Breast Cancer transfer includes the following steps:
Step S1 acquires Luminal Type B patient with breast cancer's limosis vein blood, serum is centrifugated out after natural coagulation；
Step S2 extracts serum total DNA, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measurement total DNA
The methylation index of methylation PTPN1 and the SLIT2 that methylates, successively use X1、X2It indicates；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (2.016X1+1.898X2-
0.455) Y value] is obtained, Y value indicates that Bone tumour occurs for the patient with breast cancer less than 0.310, and bone does not occur greater than 0.310 indication and turns
It moves.
Three,The transfer of Her-2 overexpression type Bone of Breast Cancer
A kind of methylated genes composition is made of methylation MYLK2, methylation EFEMP1 and methylation SOSTDC1.
Above-mentioned diagnosis composition is in terms of the diagnostic kit that preparation diagnoses indication Her-2 overexpression type Bone of Breast Cancer transfer
Application.
A kind of diagnostic kit shifted for diagnosing indication Her-2 overexpression type Bone of Breast Cancer, including methylation
The PCR amplification primer and Pyrosequencing primer of MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates.
Preferably, in the diagnostic kit, the PCR amplification upstream primer such as Sequence for the MYLK2 that methylates
Shown in NO.13, PCR amplification downstream primer is as shown in Sequence NO.14, Pyrosequencing primer such as Sequence NO.15
It is shown.
Preferably, in the diagnostic kit, the PCR amplification upstream primer such as Sequence for the EFEMP1 that methylates
Shown in NO.16, PCR amplification downstream primer is as shown in Sequence NO.17, Pyrosequencing primer such as Sequence NO.18
It is shown.
Preferably, in the diagnostic kit, the PCR amplification upstream primer such as Sequence for the SOSTDC1 that methylates
Shown in NO.19, PCR amplification downstream primer is as shown in Sequence NO.20, Pyrosequencing primer such as Sequence NO.21
It is shown.
It preferably, further include enzyme needed for PCR amplification and pyrosequencing and reagent in the diagnostic kit.
A method of diagnosis indication Her-2 overexpression type Bone of Breast Cancer transfer includes the following steps:
Step S1 acquires Her-2 overexpression type patient with breast cancer's limosis vein blood, is centrifugated bleeding after natural coagulation
Clearly；
Step S2 extracts serum total DNA, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measurement total DNA
The methylation index of methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates, are followed successively by X1、X2、X3；
Step S3, by X1、X2、X3Substitute into equation Y=1/ [1+EXP (1.342X1+1.401X2+1.345X3- 2.035)]
To Y value, Y value indicates that Bone tumour occurs for the patient with breast cancer less than 0.308, and Bone tumour does not occur greater than 0.308 indication.
Four,Triple negative breast cancer Bone tumour
A kind of methylated genes diagnosis composition is made of methylation MYLK3 and methylation SCARA5.
Above-mentioned diagnosis composition answering in terms of the diagnostic kit that preparation diagnoses three negative type breast cancers Bone tumours of indication
With.
It is a kind of for diagnosing the diagnostic kit of three negative type breast cancers Bone tumours of indication, including methylation MYLK3 and first
The PCR amplification primer of base SCARA5.
Preferably, in the diagnostic kit, the PCR amplification upstream primer such as Sequence for the MYLK3 that methylates
Shown in NO.22, PCR amplification downstream primer is as shown in Sequence NO.23.
Preferably, in the diagnostic kit, the PCR amplification upstream primer such as Sequence for the SCARA5 that methylates
Shown in NO.25, PCR amplification downstream primer is as shown in Sequence NO.26.
It preferably, further include that methylation MYLK3 and the pyrosequencing for the SCARA5 that methylates draw in the diagnostic kit
Object.
Preferably, in the diagnostic kit methylate MYLK3 Pyrosequencing primer such as Sequence NO.24 institute
Show.
Preferably, in the diagnostic kit methylate SCARA5 Pyrosequencing primer such as Sequence NO.27 institute
Show.
It preferably, further include enzyme needed for PCR amplification and pyrosequencing and reagent in the diagnostic kit.
A method of diagnosis three negative type breast cancers Bone tumours of indication include the following steps:
Step S1 acquires three negative type breast cancers patient's limosis vein bloods, serum is centrifugated out after natural coagulation；
Step S2 extracts serum total DNA, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measurement total DNA
The methylation index of methylation MYLK3 and the SCARA5 that methylates, successively use X1、X2It indicates；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (1.775X1+1.236X2-
0.398) Y value] is obtained, Y value indicates that Bone tumour occurs for the patient with breast cancer less than 0.366, and bone does not occur greater than 0.366 indication and turns
It moves.
It is a discovery of the invention that serum methylation PITX1 and methylation AMOT can combine for diagnosing indication Luminal A type
Breast cancer whether Bone tumour, concentrate diagnosis indication accuracy rate up to 90% or more in individual authentication；Serum methylation PTPN1 and methyl
Change SLIT2 can combine for diagnose indication Luminal Type B breast cancer whether Bone tumour, individual authentication concentrate diagnosis indication
Accuracy rate is up to 90% or more；Serum methylation MYLK2, methylation EFEMP1 and methylation SOSTDC1 can combine for diagnosing
Indicate Her-2 overexpression type breast cancer whether Bone tumour, concentrate diagnosis indication accuracy rate up to 90% or more in individual authentication；Serum
Methylation MYLK3 and methylation SCARA5 can combine for diagnose indication three negative type breast cancers whether Bone tumour, in independence
Verifying concentrates diagnosis indication accuracy rate up to 90% or more.Indication different molecular hypotype cream is diagnosed using above-mentioned serum methylated genes
Gland cancer Bone tumour accuracy is high, and testing cost is low, non-invasi, convenient and efficient, greatly reduces patient suffering and burden.
Detailed description of the invention
Fig. 1 is that methylation PITX1 and methylation AMOT combines for diagnosing differentiation Luminal A type breast cancer in test set
The ROC curve with the transfer of Luminal A type Bone of Breast Cancer is not shifted；
Fig. 2 is that verifying concentrates methylation PITX1 and methylation AMOT joint to distinguish Luminal A type breast cancer for diagnosing
The accuracy rate with the transfer of Luminal A type Bone of Breast Cancer is not shifted；
Fig. 3 is that methylation PTPN1 and methylation SLIT2 combines for diagnosing differentiation Luminal Type B mammary gland in test set
Cancer does not shift the ROC curve with the transfer of Luminal Type B Bone of Breast Cancer；
Fig. 4 is that verifying concentrates methylation PTPN1 and methylation SLIT2 joint to distinguish Luminal Type B mammary gland for diagnosing
Cancer does not shift the accuracy rate with the transfer of Luminal Type B Bone of Breast Cancer；
Fig. 5 is that methylation MYLK2, methylation EFEMP1 and methylation SOSTDC1 joint are distinguished for diagnosis in test set
Her-2 overexpression type breast cancer does not shift the ROC curve with the transfer of Her-2 overexpression type Bone of Breast Cancer；
Fig. 6 is that verifying concentrates methylation MYLK2, methylation EFEMP1 and methylation SOSTDC1 joint to distinguish for diagnosing
Her-2 overexpression type breast cancer does not shift the accuracy rate with the transfer of Her-2 overexpression type Bone of Breast Cancer；
Fig. 7 is that methylation MYLK3 and methylation SCARA5 combines for diagnosing three negative type breast cancers of differentiation in test set
The ROC curve with three negative type breast cancers Bone tumours is not shifted；
Fig. 8 is verifying concentration methylation MYLK3 and methylation SCARA5 Combining diagnosis is distinguished three negative type breast cancers and do not turned
Move the accuracy rate with three negative type breast cancers Bone tumours.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
All breast cancer samples of this project are taken from September, 2014 to 2017 Nian9Yue Lai Hospital Attached to Nantong Univ. or south
The First People's Hospital Tong Shi or Nanjing drum tower hospital inspection are diagnosed as the patient of other malignant tumours of breast cancer and nonjoinder.It is all
Sample is divided into Luminal A type, Luminal Type B, Her-2 overexpression type and three negative types according to immunohistochemistry detection, various
Molecular isoform is according to whether transfer is divided into the non-transfer group of breast cancer and Bone of Breast Cancer transfer group, and each case of Bone of Breast Cancer transfer group
Belonging to bone is starting DISTANT METASTASES IN position.The non-transfer group of breast cancer and Bone of Breast Cancer transfer group pass through radionuclide bone scan
(ECT), CT scan (CT), nuclear magnetic resonance (MRI), Positron emission computed tomography (PET-CT)
And/or the inspections such as tissue biopsy confirm.The non-transfer group of breast cancer and Bone tumour group patient age compare without bright in each molecular isoform
Significant difference is different, is comparable.Each group sample is finally half-and-half divided into test set and verifying collection at random.
All sample packet information and sample number are as shown in the table after the diagnosis of above-mentioned goldstandard:
The collection of serum specimen: acquisition patient's limosis vein blood 5.0mL, be centrifuged after natural coagulation (4000r/min, 2860
× g) serum is isolated after 7min, -80 DEG C of preservations are placed in, for detecting target methylated genes in serum.
The transfer of embodiment 1:Luminal A type Bone of Breast Cancer
One, experiment sample and experimental method
1, experiment sample
The non-transfer group of breast cancer, the test set of Luminal A type Bone of Breast Cancer transfer group and verifying in Luminal A type
Collection.
2, serum Genome DNA extraction
Serum CRP is extracted to carry out according to DNA Blood Midi Kit specification, and every part of sample uses 0.8mL serum.It mentions
The DNA purity UV spectrophotometer measuring taken, absorbance A 260/A280 ratio carry out subsequent operation between 1.7-2.0.
DNA content is calculated, -70 DEG C save backup.
3, the modification of DNA sulphite and pyrosequencing detection
The modification of DNA sulphite:
1 μ g DNA is taken, methylation modification is carried out to genomic DNA according to DNA Methylation-Gold kit specification
Afterwards, it saves backup for -70 DEG C.Polymerase chain reaction: using PCR to PITX1 and AMOT gene promoter methylation area in sample
Domain is expanded.Reaction system includes sulphite processing 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot star of rear pattern plate
Taq enzyme, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of total volume.Using distilled water as blank control.
Pyrosequencing detection:
(1) take 45 μ l pcr amplification products into 96 Plate Low sample preparation plate A of PSQ respectively, it is each that 45 μ l knot is added
It closes buffer and 8 μ l is coated with the magnetic bead of streptavidin, 43 DEG C of oscillation 25min.The magnetic bead of PCR product will be combined to be transferred to denaturation
In the plate B of buffer, it is denaturalized double-stranded DNA sufficiently.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillation wash 3min.
(2) sequencing primer hybridizes: the magnetic bead of single stranded PCR products will be combined to be transferred in 50 μ l renaturation buffers, 10 μ l are added
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencing instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in PITX1 and AMOT promoter region.
Wherein PCR amplification primer and sequencing primer are as follows:
PITX1
Upstream 5 '-GGAAGGTATTTAGTATAGGTGAGTTTGA-3 '
Downstream 5 '-AAACCTTAATATTCACTACACTTTATC-3 '
5 '-GTGTTTATTTTGGATTGTTTAATT-3 ' are sequenced
AMOT
Upstream 5 '-TGAGTTAATATGAAAGAAGATAGTA-3 '
Downstream 5 '-TGATCTCTACATCTCAACTAATATAC-3 '
5 '-GTAGGTTTATTTAGGTT-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Following formula calculates the methylation index of each gene promoter region, which can reflect the methyl of the gene promoter region
Change degree:
4, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is indicated with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2It examines, is that difference is statistically significant, and establishes ROC curve with P < 0.05, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, generates one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
Two, experimental result
1, Luminal A type breast cancer do not shift and Bone tumour group methylation PITX1 and methylate AMOT methylation journey
Degree
In test set, the methylation index of methylate in each sample PITX1 and the AMOT that methylates are measured respectively.With
The non-transfer group of Luminal A type breast cancer is compared, and methylate PITX1 and first in Luminal A type Bone of Breast Cancer transfer group sample
The methylation index of base AMOT significantly increases, the methylation index difference of Bone tumour group methylation PITX1 and the AMOT that methylates
It methylates (3.5 ± 0.4) times, (3.3 ± 0.5) times of index for non-transfer group.
2, the methylation index of methylation PITX1 and the AMOT that methylates are individually used for diagnosis and distinguish Luminal A type mammary gland
Cancer does not shift the ROC curve analysis with the transfer of Luminal A type Bone of Breast Cancer
The principle of ROC curve evaluation assessment:
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden index, ROC,
AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark
Accurately fixed disease group (the Bone of Breast Cancer transfer group being equivalent in the present invention) and healthy group (are equivalent to the breast cancer in the present invention
Non- transfer group), following situation can be divided into using the result that diagnostic test detects:
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP): diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN): diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be indicated with following table:
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property is it can be concluded that diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.Disease example is examined in the representative of susceptibility height
Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With diagnosis possible in diagnostic test
Dividing value calculates corresponding susceptibility and specificity as diagnostic points, according to above table.Then, using susceptibility as ordinate,
1- specificity is abscissa, the susceptibility of each point when each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, which is ROC curve.Diagnostic points are arranged much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and the size of area under the curve AUC shows
The size of diagnostic test accuracy.Intrinsic accuracy index of the area AUC as diagnostic test Authentic Assessment under ROC curve
It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC indicates that accuracy rate of diagnosis is lower at 0.5~0.7；AUC exists
When 0.7~0.9, indicate that diagnostic accuracy is medium；When AUC > 0.9, indicate that diagnosis has higher accuracy.
The methylation index that the methylation PITX1 and AMOT that methylates is drawn in SPSS 19.0 is individually used for diagnosis and distinguishes
Luminal A type breast cancer does not shift the ROC curve with the transfer of Luminal A type Bone of Breast Cancer, AUC is respectively 0.715,
0.707, there is medium accuracy.
3, methylate PITX1 and methylate AMOT methylation index Combining diagnosis model building and for diagnose distinguish
Luminal A type breast cancer does not shift the ROC curve analysis with the transfer of Luminal A type Bone of Breast Cancer
The methylation index of the methylation PITX1 using in test set sample and the AMOT that methylates (set X as independent variable1=first
The methylation index of base PITX1, X2The methylation index of=methylation AMOT), with group (i.e. according to the goldstandard sample category
In Bone tumour group still non-transfer group) it is used as dependent variable, to methylation PITX1 and methylation AMOT in Luminal A type mammary gland
Cancer, which does not shift, carries out dualistic logistic regression with the methylation index in Luminal A type Bone of Breast Cancer transfer sample, obtains binary
Logistic regression equation: Y=1/ [1+EXP (1.499X1+2.302X2-0.258)]；
The methylation index of methylate in each sample PITX1 and the AMOT that methylates are substituted into the dualistic logistic regression side again
The regressand value Y of each sample can be obtained in journey, using possible regressand value Y as diagnostic points, meter sensitivity and specificity, according to
This draws ROC curve (as shown in Figure 1), AUC 0.935, accuracy with higher.Coordinate according to ROC curve calculates dimension
Mounting index=specificity+sensitivity -1, corresponding Y value is that can be carried out diagnosis to distinguish Luminal A type when tieing up mounting index maximum value
The best cut-off value 0.238 (i.e. diagnostic threshold) of the non-transfer group of breast cancer and Bone tumour group.
4, verifying concentrates verifying methylation PITX1 and the methylation index Combining diagnosis for the AMOT that methylates to distinguish Luminal
A type breast cancer does not shift the order of accuarcy with the transfer of Luminal A type Bone of Breast Cancer
It is concentrated in verifying, the methylation index of each sample methylation PITX1 and the AMOT that methylates is substituted into above-mentioned recurrence mould
Type, the regressand value Y, Y for obtaining each sample are predicted as the transfer of Luminal A type Bone of Breast Cancer lower than diagnostic threshold 0.238, are higher than
The Luminal A type breast cancer that is predicted as of diagnostic threshold 0.238 does not shift, and accuracy is 95.5% (105/110), such as Fig. 2 institute
Show.
The transfer of embodiment 2:Luminal Type B Bone of Breast Cancer
One, experiment sample and experimental method
1, experiment sample
The non-transfer group of breast cancer, the test set of Luminal Type B Bone of Breast Cancer transfer group and verifying in Luminal Type B
Collection.
2, serum Genome DNA extraction
Serum CRP is extracted to carry out according to DNA Blood Midi Kit specification, and every part of sample uses 0.8mL serum.It mentions
The DNA purity UV spectrophotometer measuring taken, absorbance A 260/A280 ratio carry out subsequent operation between 1.7-2.0.
DNA content is calculated, -70 DEG C save backup.
3, the modification of DNA sulphite and pyrosequencing detection
The modification of DNA sulphite:
1 μ g DNA is taken, methylation modification is carried out to genomic DNA according to DNA Methylation-Gold kit specification
Afterwards, it saves backup for -70 DEG C.Polymerase chain reaction: using PCR to PTPN1 and SLIT2 gene promoter methylation area in sample
Domain is expanded.Reaction system includes sulphite processing 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot star of rear pattern plate
Taq enzyme, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of total volume.Using distilled water as blank control.
Pyrosequencing detection:
(1) take 45 μ l pcr amplification products into 96 Plate Low sample preparation plate A of PSQ respectively, it is each that 45 μ l knot is added
It closes buffer and 8 μ l is coated with the magnetic bead of streptavidin, 43 DEG C of oscillation 25min.The magnetic bead of PCR product will be combined to be transferred to denaturation
In the plate B of buffer, it is denaturalized double-stranded DNA sufficiently.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillation wash 3min.
(2) sequencing primer hybridizes: the magnetic bead of single stranded PCR products will be combined to be transferred in 50 μ l renaturation buffers, 10 μ l are added
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencing instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in PTPN1 and SLIT2 promoter region.
Wherein PCR amplification primer and sequencing primer are as follows:
PTPN1
Upstream 5 '-AGCGGGTTAGAGGGTAGATGT-3 '
Downstream 5 '-TAGGTTTCTCCTCTCCCACATAT-3 '
5 '-TTTCCATTCATCCTAA-3 ' are sequenced
SLIT2
Upstream 5 '-TGAAGTTTTATTAGGTTGTGGAGGAGTA-3 '
Downstream 5 '-ATACCAAATATCCTATCCTTATCTTC-3 '
5 '-GTTTAAGGTTTATGATA-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Following formula calculates the methylation index of each gene promoter region, which can reflect the methyl of the gene promoter region
Change degree:
4, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is indicated with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2It examines, is that difference is statistically significant, and establishes ROC curve with P < 0.05, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, generates one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
Two, experimental result
1, Luminal Type B breast cancer do not shift and Bone tumour group methylation PTPN1 and methylate SLIT2 methylation journey
Degree
In test set, the methylation index of methylate in each sample PTPN1 and the SLIT2 that methylates are measured respectively.With
The non-transfer group of Luminal Type B breast cancer is compared, and methylate PTPN1 and first in Luminal Type B Bone of Breast Cancer transfer group sample
The methylation index of base SLIT2 significantly increases, the methylation index point of Bone tumour group methylation PTPN1 and the SLIT2 that methylates
Not Wei non-transfer group methylate (2.9 ± 0.5) times, (3.4 ± 0.5) times of index.
2, the methylation index of methylation PTPN1 and the SLIT2 that methylates are individually used for diagnosis and distinguish Luminal Type B mammary gland
Cancer does not shift the ROC curve analysis with the transfer of Luminal Type B Bone of Breast Cancer
The principle of ROC curve evaluation assessment:
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden index, ROC,
AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark
Accurately fixed disease group (the Bone of Breast Cancer transfer group being equivalent in the present invention) and healthy group (are equivalent to the breast cancer in the present invention
Non- transfer group), following situation can be divided into using the result that diagnostic test detects:
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP): diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN): diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be indicated with following table:
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property is it can be concluded that diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.Disease example is examined in the representative of susceptibility height
Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With diagnosis possible in diagnostic test
Dividing value calculates corresponding susceptibility and specificity as diagnostic points, according to above table.Then, using susceptibility as ordinate,
1- specificity is abscissa, the susceptibility of each point when each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, which is ROC curve.Diagnostic points are arranged much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and the size of area under the curve AUC shows
The size of diagnostic test accuracy.Intrinsic accuracy index of the area AUC as diagnostic test Authentic Assessment under ROC curve
It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC indicates that accuracy rate of diagnosis is lower at 0.5~0.7；AUC exists
When 0.7~0.9, indicate that diagnostic accuracy is medium；When AUC > 0.9, indicate that diagnosis has higher accuracy.
The methylation index that the methylation PTPN1 and SLIT2 that methylates is drawn in SPSS 19.0 is individually used for diagnosis and distinguishes
Luminal Type B breast cancer does not shift the ROC curve with the transfer of Luminal Type B Bone of Breast Cancer, AUC is respectively 0.723,
0.741, there is medium accuracy.
3, methylate PTPN1 and methylate SLIT2 methylation index Combining diagnosis model building and be used for diagnostic region
Luminal Type B breast cancer is divided not shift the ROC curve analysis with the transfer of Luminal Type B Bone of Breast Cancer
The methylation index of the methylation PTPN1 using in test set sample and the SLIT2 that methylates (set X as independent variable1=first
The methylation index of base PTPN1, X2The methylation index of=methylation SLIT2), with group (i.e. according to the goldstandard sample
Belong to Bone tumour group still non-transfer group) as dependent variable, to methylation PTPN1 and methylation SLIT2 in Luminal Type B cream
Gland cancer, which does not shift, carries out dualistic logistic regression with the methylation index in Luminal Type B Bone of Breast Cancer transfer sample, obtains two
Metalogic regression equation: Y=1/ [1+EXP (2.016X1+1.898X2-0.455)]；
The methylation index of methylate in each sample PTPN1 and the SLIT2 that methylates are substituted into the dualistic logistic regression side again
The regressand value Y of each sample can be obtained in journey, using possible regressand value Y as diagnostic points, meter sensitivity and specificity, according to
This draws ROC curve (as shown in Figure 3), AUC 0.942, accuracy with higher.Coordinate according to ROC curve calculates dimension
Mounting index=specificity+sensitivity -1, corresponding Y value is that can be carried out diagnosis to distinguish Luminal Type B when tieing up mounting index maximum value
The best cut-off value 0.310 (i.e. diagnostic threshold) of the non-transfer group of breast cancer and Bone tumour group.
4, verifying concentrates verifying methylation PTPN1 and the methylation index Combining diagnosis for the SLIT2 that methylates to distinguish Luminal
Type B breast cancer does not shift the order of accuarcy with the transfer of Luminal Type B Bone of Breast Cancer
It is concentrated in verifying, the methylation index of each sample methylation PTPN1 and the SLIT2 that methylates is substituted into above-mentioned recurrence mould
Type, the regressand value Y, Y for obtaining each sample are predicted as the transfer of Luminal Type B Bone of Breast Cancer lower than diagnostic threshold 0.310, are higher than
The Luminal Type B breast cancer that is predicted as of diagnostic threshold 0.310 does not shift, and accuracy is 93.7% (59/63), as shown in Figure 4.
The transfer of embodiment 3:Her-2 overexpression type Bone of Breast Cancer
One, experiment sample and experimental method
1, experiment sample
The non-transfer group of breast cancer in Her-2 overexpression type, Her-2 overexpression type Bone of Breast Cancer transfer group test set and test
Card collection.
2, serum Genome DNA extraction
Serum CRP is extracted to carry out according to DNA Blood Midi Kit specification, and every part of sample uses 0.8mL serum.It mentions
The DNA purity UV spectrophotometer measuring taken, absorbance A 260/A280 ratio carry out subsequent operation between 1.7-2.0.
DNA content is calculated, -70 DEG C save backup.
3, the modification of DNA sulphite and pyrosequencing detection
The modification of DNA sulphite:
1 μ g DNA is taken, methylation modification is carried out to genomic DNA according to DNA Methylation-Gold kit specification
Afterwards, it saves backup for -70 DEG C.Polymerase chain reaction: using PCR to MYLK2, EFEMP1 and SOSTDC1 gene promoter in sample
Son methylation region is expanded.Reaction system includes sulphite processing 2 μ l, 10 × PCR buffer, 0.25U/ μ of rear pattern plate
L Hot star Taq enzyme, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of total volume.
Pyrosequencing detection:
(1) take 45 μ l pcr amplification products into 96 Plate Low sample preparation plate A of PSQ respectively, it is each that 45 μ l knot is added
It closes buffer and 8 μ l is coated with the magnetic bead of streptavidin, 43 DEG C of oscillation 25min.The magnetic bead of PCR product will be combined to be transferred to denaturation
In the plate B of buffer, it is denaturalized double-stranded DNA sufficiently.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillation wash 3min.
(2) sequencing primer hybridizes: the magnetic bead of single stranded PCR products will be combined to be transferred in 50 μ l renaturation buffers, 10 μ l are added
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencing instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in MYLK2, EFEMP1 and SOSTDC1 promoter region.
Wherein PCR amplification primer and sequencing primer are as follows:
MYLK2
Upstream 5 '-GAGGGAAAGGATATGGTTGATT-3 '
Downstream 5 '-AACTCCACTCCATTCTCCC-3 '
5 '-AGTAAGTTATTTATTTGTTATTTG-3 ' are sequenced
EFEMP1
Upstream 5 '-GGTTTAGGTGGGGAGTATGATAG-3 '
Downstream 5 '-ACCAACAACCCAACTTTAACATAACC-3 '
5 '-TAATGAGGGGTTGAG-3 ' are sequenced
SOSTDC1
Upstream 5 '-GTAAAGGAGAAAGTTTGGTATATGG-3 '
Downstream 5 '-CAAAACTATACAAAAGTATCTCTCTCAAT-3 '
5 '-ATAATTTAATTGTTAGAGTTGAATA-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Following formula calculates the methylation index of each gene promoter region, which can reflect the methyl of the gene promoter region
Change degree:
4, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is indicated with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2It examines, is that difference is statistically significant, and establishes ROC curve with P < 0.05, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, generates one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
Two, experimental result
1, Her-2 overexpression type breast cancer does not shift and Bone tumour group methylation MYLK2, methylation EFEMP1 and methylation
The methylation of SOSTDC1
In test set, the MYLK2 that methylates in each sample, methylation EFEMP1 are measured respectively and methylates SOSTDC1's
Methylate index.Compared with the non-transfer group of Her-2 overexpression type breast cancer, Her-2 overexpression type Bone of Breast Cancer transfer group sample
The methylation index of middle methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates significantly increase, respectively non-transfer group
Methylate (3.7 ± 0.6) times, (2.6 ± 0.4), (3.1 ± 0.5) times of index.
2, the methylation index of methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates are individually used for diagnosis differentiation
Her-2 overexpression type breast cancer does not shift the ROC curve analysis with the transfer of Her-2 overexpression type Bone of Breast Cancer
The principle of ROC curve evaluation assessment:
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden index, ROC,
AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark
Accurately fixed disease group (the Bone of Breast Cancer transfer group being equivalent in the present invention) and healthy group (are equivalent to the breast cancer in the present invention
Non- transfer group), following situation can be divided into using the result that diagnostic test detects:
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP): diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN): diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be indicated with following table:
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property is it can be concluded that diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.Disease example is examined in the representative of susceptibility height
Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With diagnosis possible in diagnostic test
Dividing value calculates corresponding susceptibility and specificity as diagnostic points, according to above table.Then, using susceptibility as ordinate,
1- specificity is abscissa, the susceptibility of each point when each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, which is ROC curve.Diagnostic points are arranged much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and the size of area under the curve AUC shows
The size of diagnostic test accuracy.Intrinsic accuracy index of the area AUC as diagnostic test Authentic Assessment under ROC curve
It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC indicates that accuracy rate of diagnosis is lower at 0.5~0.7；AUC exists
When 0.7~0.9, indicate that diagnostic accuracy is medium；When AUC > 0.9, indicate that diagnosis has higher accuracy.
The methylation index list of methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates are drawn in SPSS19.0
It is solely used in diagnosis differentiation Her-2 overexpression type breast cancer and does not shift the ROC curve shifted with Her-2 overexpression type Bone of Breast Cancer,
AUC is respectively 0.794,0.688,0.738, has lower or medium accuracy.
3, the structure of the methylation index Combining diagnosis model of methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates
It builds and does not shift the ROC curve shifted with Her-2 overexpression type Bone of Breast Cancer for diagnosing differentiation Her-2 overexpression type breast cancer
Analysis
Using in test set sample methylate MYLK2, methylation EFEMP1 and methylate SOSTDC1 methylation index as
Independent variable (sets X1The methylation index of=methylation MYLK2, X2The methylation index of=methylation EFEMP1, X3=methylation
The methylation index of SOSTDC1), using group (i.e. the sample belongs to Bone tumour group still non-transfer group according to goldstandard) as answering
Variable, methylation MYLK2, methylation EFEMP1 and methylation SOSTDC1 are not shifted in Her-2 overexpression type breast cancer and
Her-2 overexpression type Bone of Breast Cancer shifts the methylation index in sample and carries out dualistic logistic regression, obtains dualistic logistic regression
Equation: Y=1/ [1+EXP (1.342X1+1.401X2+1.345X3-2.035)]；
The methylation index of the MYLK2 that methylates in each sample, methylation EFEMP1 and the SOSTDC1 that methylates are substituted into again should
The regressand value Y of each sample can be obtained in dualistic logistic regression equation, using possible regressand value Y as diagnostic points, calculates sensitive
Degree and specificity, draw ROC curve (as shown in Figure 5) accordingly, AUC 0.950, accuracy with higher.According to ROC curve
Coordinate calculate dimension mounting index=specificity+sensitivity -1, corresponding Y value is that can be carried out diagnosis to distinguish when tieing up mounting index maximum value
The best cut-off value 0.308 (diagnostic threshold) of the non-transfer group of Her-2 overexpression type breast cancer and Bone tumour group.
4, the methylation index joint of verifying methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates are concentrated in verifying
Diagnosis distinguishes Her-2 overexpression type breast cancer and does not shift the order of accuarcy shifted with Her-2 overexpression type Bone of Breast Cancer
It is concentrated in verifying, the methylation of the methylation of each sample MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates is referred to
Number substitutes into above-mentioned regression model, and the regressand value Y, Y for obtaining each sample are predicted as Her-2 overexpression lower than diagnostic threshold 0.308
The transfer of type Bone of Breast Cancer, the Her-2 overexpression type breast cancer that is predicted as higher than diagnostic threshold 0.308 do not shift, and accuracy is
96.4% (54/56), as shown in Figure 6.
4: three negative type breast cancers Bone tumour of embodiment
One, experiment sample and experimental method
1, experiment sample
The non-transfer group of breast cancer, the test set of three negative type breast cancers Bone tumour groups and verifying collection in three negative types.
2, serum Genome DNA extraction
Serum CRP is extracted to carry out according to DNA Blood Midi Kit specification, and every part of sample uses 0.8mL serum.It mentions
The DNA purity UV spectrophotometer measuring taken, absorbance A 260/A280 ratio carry out subsequent operation between 1.7-2.0.
DNA content is calculated, -70 DEG C save backup.
3, the modification of DNA sulphite and pyrosequencing detection
The modification of DNA sulphite:
1 μ g DNA is taken, methylation modification is carried out to genomic DNA according to DNA Methylation-Gold kit specification
Afterwards, it saves backup for -70 DEG C.Polymerase chain reaction: using PCR to MYLK3 and SCARA5 gene promoter methylation in sample
Region is expanded.Reaction system includes sulphite processing 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot of rear pattern plate
Star Taq enzyme, 0.5mmol/LdNTP, each 1 μ l of upstream and downstream primer, 50 μ l of total volume.Using distilled water as blank control.
Pyrosequencing detection:
(1) take 45 μ l pcr amplification products into 96 Plate Low sample preparation plate A of PSQ respectively, it is each that 45 μ l knot is added
It closes buffer and 8 μ l is coated with the magnetic bead of streptavidin, 43 DEG C of oscillation 25min.The magnetic bead of PCR product will be combined to be transferred to denaturation
In the plate B of buffer, it is denaturalized double-stranded DNA sufficiently.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillation wash 3min.
(2) sequencing primer hybridizes: the magnetic bead of single stranded PCR products will be combined to be transferred in 50 μ l renaturation buffers, 10 μ l are added
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencing instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in MYLK3 and SCARA5 promoter region.
Wherein PCR amplification primer and sequencing primer are as follows:
MYLK3
Upstream 5 '-TAGGGGAGGTTAAGAAAGTGTA-3 '
Downstream 5 '-AACTCCTTATCAATTCCTAACATACAAT-3 '
5 '-GGAGTAATGATGTAATGTGTAT-3 ' are sequenced
SCARA5
Upstream 5 '-AGGAATTAGGTAAGGTATGTTAGTA-3 '
Downstream 5 '-AAAACTCCAACCTATTCCAACCATACCTAC-3 '
5 '-GTTTTAAGTTTTGGTGTTTGATAT-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Following formula calculates the methylation index of each gene promoter region, which can reflect the methyl of the gene promoter region
Change degree:
4, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is indicated with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2It examines, is that difference is statistically significant, and establishes ROC curve with P < 0.05, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, generates one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
Two, experimental result
1, three negative type breast cancers do not shift and Bone tumour group methylation MYLK3 and methylate SCARA5 methylation
In test set, the methylation index of methylate in each sample MYLK3 and the SCARA5 that methylates are measured respectively.With three
The non-transfer group of negative type breast cancers is compared, and methylate MYLK3 and methylation in three negative type breast cancers Bone tumour group samples
The methylation index of SCARA5 significantly increases, the methylation index difference of Bone tumour group methylation MYLK3 and the SCARA5 that methylates
It methylates (2.1 ± 0.3) times, (3.6 ± 0.7) times of index for non-transfer group.
2, the methylation index of methylation MYLK3 and the SCARA5 that methylates are individually used for diagnosis and distinguish three negative type breast cancers
It does not shift and is analyzed with the ROC curve of three negative type breast cancers Bone tumours
The principle of ROC curve evaluation assessment:
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden index, ROC,
AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark
Accurately fixed disease group (the Bone of Breast Cancer transfer group being equivalent in the present invention) and healthy group (are equivalent to the breast cancer in the present invention
Non- transfer group), following situation can be divided into using the result that diagnostic test detects:
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP): diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN): diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be indicated with following table:
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property is it can be concluded that diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.Disease example is examined in the representative of susceptibility height
Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With diagnosis possible in diagnostic test
Dividing value calculates corresponding susceptibility and specificity as diagnostic points, according to above table.Then, using susceptibility as ordinate,
1- specificity is abscissa, the susceptibility of each point when each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, which is ROC curve.Diagnostic points are arranged much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and the size of area under the curve AUC shows
The size of diagnostic test accuracy.Intrinsic accuracy index of the area AUC as diagnostic test Authentic Assessment under ROC curve
It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC indicates that accuracy rate of diagnosis is lower at 0.5~0.7；AUC exists
When 0.7~0.9, indicate that diagnostic accuracy is medium；When AUC > 0.9, indicate that diagnosis has higher accuracy.
The methylation index that the methylation MYLK3 and SCARA5 that methylates is drawn in SPSS19.0 is individually used for diagnosis and distinguishes
Three negative type breast cancers do not shift the ROC curve with three negative type breast cancers Bone tumours, and AUC is respectively 0.644,0.809, have
Lower or medium accuracy.
3, methylate MYLK3 and methylate SCARA5 methylation index Combining diagnosis model building and be used for diagnostic region
Point three negative type breast cancers do not shift and the analysis of the ROC curve of three negative type breast cancers Bone tumours
The methylation index of the methylation MYLK3 using in test set sample and the SCARA5 that methylates (set X as independent variable1=
The methylation index of methylation MYLK3, X2The methylation index of=methylation SCARA5), with group (i.e. according to the goldstandard sample
Originally belong to Bone tumour group still non-transfer group) as dependent variable, to methylation MYLK3 and methylation SCARA5 in three negative types cream
Gland cancer, which does not shift, carries out dualistic logistic regression with the methylation index in three negative type breast cancers Bone tumour samples, obtains binary and patrols
Collect regression equation: Y=1/ [1+EXP (1.775X1+1.236X2-0.398)]；
The methylation index of methylate in each sample MYLK3 and the SCARA5 that methylates are substituted into the dualistic logistic regression side again
The regressand value Y of each sample can be obtained in journey, using possible regressand value Y as diagnostic points, meter sensitivity and specificity, according to
This draws ROC curve (as shown in Figure 7), AUC 0.954, accuracy with higher.Coordinate according to ROC curve calculates dimension
Mounting index=specificity+sensitivity -1, corresponding Y value is that can be carried out diagnosis to distinguish three negative type mammary gland when tieing up mounting index maximum value
The best cut-off value 0.366 (i.e. diagnostic threshold) of the non-transfer group of cancer and Bone tumour group.
4, verifying concentrates the methylation index Combining diagnosis of verifying methylation MYLK3 and the SCARA5 that methylates to distinguish three negative
Type breast cancer does not shift the order of accuarcy with three negative type breast cancers Bone tumours
It is concentrated in verifying, the methylation index of each sample methylation MYLK3 and the SCARA5 that methylates is substituted into above-mentioned recurrence
Model, the regressand value Y, Y for obtaining each sample are predicted as three negative type breast cancers Bone tumours lower than diagnostic threshold 0.366, are higher than
Three negative type breast cancers that are predicted as of diagnostic threshold 0.366 do not shift, and accuracy is 94.6% (53/56), as shown in Figure 8.
Embodiment 5: the diagnostic kit of diagnosis indication different subtype Bone of Breast Cancer transfer
1, Luminal A type Bone of Breast Cancer transfer diagnosis indication kit
PCR amplification primer including methylation PITX1 and the AMOT that methylates: the PCR amplification upstream primer for the PITX1 that methylates
As shown in Sequence NO.1, PCR amplification downstream primer is as shown in Sequence NO.2；It methylates in the PCR amplification of AMOT
Primer is swum as shown in Sequence NO.4, PCR amplification downstream primer is as shown in Sequence NO.5；It further include methylation
The Pyrosequencing primer of PITX1 and methylation AMOT, the Pyrosequencing primer for the PITX1 that methylates such as Sequence NO.3
Shown, the Pyrosequencing primer for the AMOT that methylates is as shown in Sequence NO.6.
It further include enzyme needed for PCR amplification and pyrosequencing and reagent.
2, Luminal Type B Bone of Breast Cancer transfer diagnosis indication kit
PCR amplification primer including methylation PTPN1 and the SLIT2 that methylates；Draw the PCR amplification upstream of methylation PTPN1
Object is as shown in Sequence NO.7, and PCR amplification downstream primer is as shown in Sequence NO.8；The PCR amplification of methylation SLIT2
Upstream primer is as shown in Sequence NO.10, and PCR amplification downstream primer is as shown in Sequence NO.11；It further include methylation
The Pyrosequencing primer of PTPN1 and methylation SLIT2, the Pyrosequencing primer for the PTPN1 that methylates such as Sequence NO.9
Shown, the Pyrosequencing primer for the SLIT2 that methylates is as shown in Sequence NO.12.
It further include enzyme needed for PCR amplification and pyrosequencing and reagent.
3, Her-2 overexpression type Bone of Breast Cancer transfer diagnosis indication kit
PCR amplification primer and pyrosequencing including methylation MYLK2, methylation EFEMP1 and the SOSTDC1 that methylates
Primer；The PCR amplification upstream primer of methylation MYLK2 is as shown in Sequence NO.13, and PCR amplification downstream primer is such as
Shown in Sequence NO.14, Pyrosequencing primer is as shown in Sequence NO.15；It methylates in the PCR amplification of EFEMP1
Primer is swum as shown in Sequence NO.16, PCR amplification downstream primer is as shown in Sequence NO.17, Pyrosequencing primer
As shown in Sequence NO.19, PCR expands the PCR amplification upstream primer of methylation SOSTDC1 as shown in Sequence NO.18
Increase downstream primer as shown in Sequence NO.20, Pyrosequencing primer is as shown in Sequence NO.21.
It further include enzyme needed for PCR amplification and pyrosequencing and reagent.
4, three negative type breast cancers Bone tumours diagnosis indication kit
PCR amplification primer including methylation MYLK3 and the SCARA5 that methylates；Draw the PCR amplification upstream of methylation MYLK3
Object is as shown in Sequence NO.22, and PCR amplification downstream primer is as shown in Sequence NO.23；The PCR of methylation SCARA5
Upstream primer is expanded as shown in Sequence NO.25, PCR amplification downstream primer is as shown in Sequence NO.26；It further include first
The Pyrosequencing primer of base MYLK3 and methylation SCARA5, the Pyrosequencing primer for the MYLK3 that methylates such as Sequence
NO.24, the Pyrosequencing primer for the SCARA5 that methylates such as Sequence NO.27.
It further include enzyme needed for PCR amplification and pyrosequencing and reagent.
In summary, it is a discovery of the invention that serum methylation PITX1 and methylation AMOT can combine for diagnosing indication
Luminal A type breast cancer whether Bone tumour, concentrate diagnosis indication accuracy rate up to 90% or more in individual authentication；Serum methylation
PTPN1 and methylation SLIT2 can combine for diagnose indication Luminal Type B breast cancer whether Bone tumour, in individual authentication
Concentrate diagnosis indication accuracy rate up to 90% or more；Serum methylates MYLK2, methylation EFEMP1 and methylation SOSTDC1 can be with
Joint for diagnose indication Her-2 overexpression type breast cancer whether Bone tumour, individual authentication concentrate diagnosis indication accuracy rate reach
90% or more；Serum methylation MYLK3 and methylation SCARA5 can combine for whether diagnosing three negative type breast cancers of indication
Bone tumour concentrates diagnosis indication accuracy rate up to 90% or more in individual authentication.It is diagnosed and is indicated using above-mentioned serum methylated genes
Not only accuracy is high for the transfer of different molecular hypotype Bone of Breast Cancer, but also testing cost is low, non-invasi, convenient and efficient, very big drop
Low patient suffering and burden.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Sequence table
<110>Xue Shouhai
<120>purposes of methylated genes composition and preparation diagnosis three negative type breast cancers Bone tumour kits of indication
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggaaggtatt tagtataggt gagtttga 28
<210> 2
<211> 27
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aaaccttaat attcactaca ctttatc 27
<210> 3
<211> 24
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtgtttattt tggattgttt aatt 24
<210> 4
<211> 25
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgagttaata tgaaagaaga tagta 25
<210> 5
<211> 26
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgatctctac atctcaacta atatac 26
<210> 6
<211> 17
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gtaggtttat ttaggtt 17
<210> 7
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 7
agcgggttag agggtagatg t 21
<210> 8
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 8
taggtttctc ctctcccaca tat 23
<210> 9
<211> 16
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 9
tttccattca tcctaa 16
<210> 10
<211> 28
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tgaagtttta ttaggttgtg gaggagta 28
<210> 11
<211> 26
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ataccaaata tcctatcctt atcttc 26
<210> 12
<211> 17
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gtttaaggtt tatgata 17
<210> 13
<211> 22
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gagggaaagg atatggttga tt 22
<210> 14
<211> 19
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aactccactc cattctccc 19
<210> 15
<211> 24
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 15
agtaagttat ttatttgtta tttg 24
<210> 16
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ggtttaggtg gggagtatga tag 23
<210> 17
<211> 26
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 17
accaacaacc caactttaac ataacc 26
<210> 18
<211> 15
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 18
taatgagggg ttgag 15
<210> 19
<211> 25
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gtaaaggaga aagtttggta tatgg 25
<210> 20
<211> 29
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 20
caaaactata caaaagtatc tctctcaat 29
<210> 21
<211> 25
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ataatttaat tgttagagtt gaata 25
<210> 22
<211> 3
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 22
<210> 23
<211> 22
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 23
taggggaggt taagaaagtg ta 22
<210> 24
<211> 28
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 24
aactccttat caattcctaa catacaat 28
<210> 25
<211> 22
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ggagtaatga tgtaatgtgt at 22
<210> 26
<211> 3
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 26
<210> 27
<211> 25
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 27
aggaattagg taaggtatgt tagta 25
<210> 28
<211> 30
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 28
aaaactccaa cctattccaa ccatacctac 30
<210> 29
<211> 24
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gttttaagtt ttggtgtttg atat 24
Claims (2)
1. a kind of for diagnosing the gene diagnosis kit of three negative type breast cancers Bone tumours of indication, it is characterised in that: including first
PCR amplification primer, the Pyrosequencing primer of base MYLK3 and methylation SCARA5；The PCR amplification upstream of methylation MYLK3
Primer is as shown in Sequence NO. 23, and PCR amplification downstream primer is as shown in Sequence NO. 24, pyrosequencing
Primer is as shown in Sequence NO. 25；Methylate SCARA5 PCR amplification upstream primer as shown in Sequence NO. 27,
Its PCR amplification downstream primer is as shown in Sequence NO. 28, and Pyrosequencing primer is as shown in Sequence NO. 29.
2. gene diagnosis kit according to claim 1, it is characterised in that: further include PCR amplification and pyrosequencing
Required enzyme and reagent.
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| CN201711200315.0A CN107904309B (en) | 2017-11-20 | 2017-11-20 | It is a kind of for diagnose indication three negative type breast cancers Bone tumours gene diagnosis kit |
| PCT/CN2018/072269 WO2019095541A1 (en) | 2017-11-17 | 2018-01-11 | Composition and method for diagnosing and predicting breast cancer bone metastases |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711200315.0A CN107904309B (en) | 2017-11-20 | 2017-11-20 | It is a kind of for diagnose indication three negative type breast cancers Bone tumours gene diagnosis kit |
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