Methylated genes composition and preparation diagnosis indication Her-2 overexpression type Bone of Breast Cancer
The purposes of transfering reagent box
Technical field
The invention belongs to biochemical field, is related to diagnosis composition and diagnostic kit, and in particular to one kind methylates
Gene diagnosis composition and the application in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication different molecular hypotype is prepared.
Background technology
Breast cancer is one of malignant tumour for threatening women life and health, there are about the people of 40-45 ten thousand every year and dies from breast cancer (ginseng
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, it is bone tissue (bibliography more than the starting metastasis site of 50% patient:Genes associated with
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,CancerRes Treat,2011).According to ERs
(estrogen receptor, ER), progesterone receptor (progesterone receptor, PR), human epidermal growth factor receptor
Breast cancer, can be divided into by the expression of body -2 (human epidermal growth factor receptor-2, HER-2)
4 hypotypes, it is respectively:Luminal A types, Luminal Type Bs, Her-2 overexpressions type and triple negative breast cancer (bibliography:
Gene expression patterns ofbreast carcinomas distinguish tumor subclasses
with clinical implications,PNAS,2001).Research shows that prognosis and its molecule of Bone of Breast Cancer transfer divide
Closely related (the bibliography such as type, clinical stages, lymph node status:Prevalence and risk factors ofbone
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, determine the difference of its therapeutic modality and prognosis.There is also difference for the gene phenotype of different molecular hypotype Bone of Breast Cancer transfer.
Early diagnosis Bone of Breast Cancer transfer is to save the key of patient vitals.At present, radionuclide bone scan (ECT),
CT scan (CT), nuclear magnetic resonance (MRI), Positron emission computed tomography (PET-CT) and bone tissue
Biopsy is to find and make a definite diagnosis the goldstandard of Bone of Breast Cancer transfer.But there is different deficiencies, such as Laboratory Fee in these methods
With height, intervention diagnosis adds 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, it is primarily involved in the expression regulation of gene.Methylating for 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 abnormal disease such as with tumour of DNA methylation is closely related.Therefore, first
Base gene can be used as the mark of tumor cells diagnosis, while also can be as the target (bibliography of molecular therapy:
Value of the DNA methylation mark in molecule Clinics and Practices, molecule Clinics and Practices magazine in September, 2009 volume 1 the 3rd
Phase).
Research shows 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 the coefficient result of multiple-factor, conclusion prompting, and the transspecific of bone is bone in primary tumo(u)r
The selection of different phenotype tumour cells and the result of bone source sex factor induction, the transfer of different molecular hypotype Bone of Breast Cancer methylate
Gene is expected to turn into the diagnosis marker (bibliography of Bone of Breast Cancer transfer:Kang Y,Siegel PM,Shu W,et
al.Amultigenic program mediatingbreast cancer metastasis to bone.Cancer Cell,
2003)。
Applicant is intended to research and compares the base that methylated in generation Bone tumour and the blood serum of patients with human breast carcinoma that Bone tumour does not occur
The difference of cause, find, checking may be used as diagnosing, indicating the methylated genes mark that different molecular hypotype Bone of Breast Cancer shifts
Thing, with provide it is a kind of can the kit that shifts of quick diagnosis, indication different molecular hypotype Bone of Breast Cancer and method by blood.
The content 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, prepares
Into a kind of testing cost is low, non-invasi, convenient and swift diagnosis indication different molecular hypotype Bone of Breast Cancer transfer diagnostic reagent
Box.
The above-mentioned purpose of the present invention is achieved by following technical scheme:
First,LuminalA types Bone of Breast Cancer shifts
A kind of methylated genes diagnosis composition, by methylating, the PITX1 and AMOT that methylates is formed.
Above-mentioned diagnosis composition is in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication LuminalA types is prepared
Using.
A kind of diagnostic kit for being used to diagnose indication LuminalA types Bone of Breast Cancer transfer, including methylate PITX1 and
Methylate AMOT pcr amplification primer thing.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence NO.1 for the PITX1 that methylates
Shown, PCR expands anti-sense primer as shown in Sequence NO.2.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence NO.4 for the AMOT that methylates
Shown, PCR expands anti-sense primer as shown in Sequence NO.5.
Preferably, the pyrosequencing in described diagnostic kit also including methylate PITX1 and the AMOT that methylates draws
Thing.
Preferably, in described diagnostic kit, the Pyrosequencing primer such as Sequence NO.3 for the PITX1 that methylates
It is shown.
Preferably, in described diagnostic kit, the Pyrosequencing primer such as Sequence NO.6 institutes for the AMOT that methylates
Show.
Preferably, in described diagnostic kit, in addition to PCR amplifications and the enzyme and reagent needed for pyrosequencing.
A kind of method for diagnosing the Bone of Breast Cancer transfer of indication LuminalA types, comprises the following steps:
Step S1, LuminalA type patient with breast cancer's limosis vein bloods are gathered, serum is centrifuged out after natural coagulation;
Step S2, serum STb gene is extracted, expanded through PCR, in the modification of DNA sulphite and pyrosequencing measure STb gene
Methylate PITX1 and the AMOT index that methylates of methylating, and uses X successively1、X2Represent;
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 is less than 0.238 and indicates that Bone tumour occurs for the patient with breast cancer, and bone does not occur more than 0.238 indication turns
Move.
2nd, Luminal Type Bs Bone of Breast Cancer shifts
A kind of methylated genes diagnosis composition, by methylating, the PTPN1 and SLIT2 that methylates is formed.
Above-mentioned diagnosis composition is in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication Luminal Type Bs is prepared
Using.
A kind of diagnostic kit for being used to diagnose indication Luminal Type Bs Bone of Breast Cancer transfer, including methylate PTPN1 and
Methylate SLIT2 pcr amplification primer thing.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence NO.7 for the PTPN1 that methylates
Shown, PCR expands anti-sense primer as shown in Sequence NO.8.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence for the SLIT2 that methylates
Shown in NO.10, PCR expands anti-sense primer as shown in Sequence NO.11.
Preferably, in described diagnostic kit, methylate PTPN1 and the SLIT2 that methylates pyrosequencing are included
Primer.
Preferably, in described diagnostic kit, the Pyrosequencing primer such as Sequence NO.9 for the PTPN1 that methylates
It is shown.
Preferably, in described diagnostic kit, the Pyrosequencing primer such as Sequence NO.12 for the SLIT2 that methylates
It is shown.
Preferably, in described diagnostic kit, in addition to PCR amplifications and the enzyme and reagent needed for pyrosequencing.
A kind of method for diagnosing the Bone of Breast Cancer transfer of indication Luminal Type Bs, comprises the following steps:
Step S1, Luminal Type B patient with breast cancer's limosis vein bloods are gathered, serum is centrifuged out after natural coagulation;
Step S2, serum STb gene is extracted, expanded through PCR, in the modification of DNA sulphite and pyrosequencing measure STb gene
Methylate PTPN1 and the SLIT2 index that methylates of methylating, and uses X successively1、X2Represent;
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 is less than 0.310 and indicates that Bone tumour occurs for the patient with breast cancer, and bone does not occur more than 0.310 indication turns
Move.
3rd,Her-2 overexpression types Bone of Breast Cancer shifts
A kind of methylated genes composition, it is made up of the MYLK2 that methylates, the EFEMP1 and SOSTDC1 that methylates that methylates.
Above-mentioned diagnosis composition is in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication Her-2 overexpression types is prepared
Application.
A kind of diagnostic kit for being used to diagnose the Bone of Breast Cancer transfer of indication Her-2 overexpression types, including methylate
MYLK2, methylate EFEMP1 and the SOSTDC1 that methylates pcr amplification primer thing and Pyrosequencing primer.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence for the MYLK2 that methylates
Shown in NO.13, PCR expands anti-sense primer as shown in Sequence NO.14, Pyrosequencing primer such as Sequence NO.15
It is shown.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence for the EFEMP1 that methylates
Shown in NO.16, PCR expands anti-sense primer as shown in Sequence NO.17, Pyrosequencing primer such as Sequence NO.18
It is shown.
Preferably, in the diagnostic kit, the PCR amplification sense primers such as Sequence for the SOSTDC1 that methylates
Shown in NO.19, PCR expands anti-sense primer as shown in Sequence NO.20, Pyrosequencing primer such as Sequence NO.21
It is shown.
Preferably, in described diagnostic kit, in addition to PCR amplifications and the enzyme and reagent needed for pyrosequencing.
A kind of method for diagnosing the Bone of Breast Cancer transfer of indication Her-2 overexpression types, comprises the following steps:
Step S1, Her-2 overexpression type patient with breast cancer's limosis vein bloods are gathered, bleeding is centrifuged after natural coagulation
Clearly;
Step S2, serum STb gene is extracted, expanded through PCR, in the modification of DNA sulphite and pyrosequencing measure STb gene
Methylate MYLK2, methylate EFEMP1 and the SOSTDC1 that the methylates index that methylates, and is 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 is less than 0.308 and indicates that Bone tumour occurs for the patient with breast cancer, and Bone tumour does not occur more than 0.308 indication.
4th,Triple negative breast cancer Bone tumour
A kind of methylated genes diagnosis composition, by methylating, the MYLK3 and SCARA5 that methylates is formed.
Above-mentioned diagnosis composition answering in terms of the diagnostic kit for diagnosing three negative type breast cancers Bone tumours of indication is prepared
With.
A kind of diagnostic kit for being used to diagnose three negative type breast cancers Bone tumours of indication, including methylate MYLK3 and first
Base SCARA5 pcr amplification primer thing.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence for the MYLK3 that methylates
Shown in NO.22, PCR expands anti-sense primer as shown in Sequence NO.23.
Preferably, in described diagnostic kit, the PCR amplification sense primers such as Sequence for the SCARA5 that methylates
Shown in NO.25, PCR expands anti-sense primer as shown in Sequence NO.26.
Preferably, the pyrosequencing in the diagnostic kit also including methylate MYLK3 and the SCARA5 that methylates draws
Thing.
Preferably, methylate MYLK3 Pyrosequencing primer such as Sequence NO.24 institutes in the diagnostic kit
Show.
Preferably, methylate SCARA5 Pyrosequencing primer such as Sequence NO.27 institutes in the diagnostic kit
Show.
Preferably, in described diagnostic kit, in addition to PCR amplifications and the enzyme and reagent needed for pyrosequencing.
A kind of method for diagnosing three negative type breast cancers Bone tumours of indication, comprises the following steps:
Step S1, three negative type breast cancers patient's limosis vein bloods are gathered, serum is centrifuged out after natural coagulation;
Step S2, serum STb gene is extracted, expanded through PCR, in the modification of DNA sulphite and pyrosequencing measure STb gene
Methylate MYLK3 and the SCARA5 index that methylates of methylating, and uses X successively1、X2Represent;
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 is less than 0.366 and indicates that Bone tumour occurs for the patient with breast cancer, and bone does not occur more than 0.366 indication turns
Move.
It is a discovery of the invention that serum methylates, the PITX1 and AMOT that methylates can combine for diagnosing indication LuminalA types
Breast cancer whether Bone tumour, individual authentication concentrate diagnosis indication rate of accuracy reached more than 90%;Serum methylates PTPN1 and methyl
Change SLIT2 can combine for diagnose indication Luminal Type Bs breast cancer whether Bone tumour, concentrate diagnosis indication in individual authentication
Rate of accuracy reached more than 90%;Methylate MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates of serum can combine for diagnosing
Indicate Her-2 overexpression types breast cancer whether Bone tumour, concentrate diagnosis indication rate of accuracy reached more than 90% in individual authentication;Serum
Methylate MYLK3 and methylate SCARA5 can combine for diagnose indication three negative type breast cancers whether Bone tumour, in independence
Diagnosis indication rate of accuracy reached more than 90% is concentrated in checking.Indication different molecular hypotype breast is diagnosed using above-mentioned serum methylated genes
The degree of accuracy of gland cancer Bone tumour is high, and testing cost is low, non-invasi, convenient and swift, greatly reduces patient suffering and burden.
Brief description of the drawings
Fig. 1 combines for the PITX1 and AMOT that methylates that methylated in test set distinguishes Luminal A type breast cancer for diagnosing
The ROC curve with the transfer of Luminal A types Bone of Breast Cancer is not shifted;
Fig. 2 for checking concentration methylate PITX1 and methylate AMOT combine for diagnose distinguish Luminal A type breast cancer
The accuracy rate with the transfer of Luminal A types Bone of Breast Cancer is not shifted;
Fig. 3 combines for the PTPN1 and SLIT2 that methylates that methylated in test set distinguishes Luminal Type B mammary gland for diagnosing
Cancer does not shift the ROC curve with the transfer of Luminal Type Bs Bone of Breast Cancer;
Fig. 4 for checking concentration methylate PTPN1 and methylate SLIT2 combine for diagnose distinguish Luminal Type B mammary gland
Cancer does not shift the accuracy rate with the transfer of Luminal Type Bs Bone of Breast Cancer;
Fig. 5 combines for methylated in test set MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates to be distinguished for diagnosing
Her-2 overexpression type breast cancer does not shift the ROC curve with the transfer of Her-2 overexpression types Bone of Breast Cancer;
Fig. 6 for checking concentration methylate MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates combine for diagnose distinguish
Her-2 overexpression type breast cancer does not shift the accuracy rate with the transfer of Her-2 overexpression types Bone of Breast Cancer;
Fig. 7 combines for the MYLK3 and SCARA5 that methylates that methylated in test set distinguishes three negative type breast cancers for diagnosing
The ROC curve with three negative type breast cancers Bone tumours is not shifted;
Fig. 8 for checking concentration methylate MYLK3 and methylate SCARA5 Combining diagnosis distinguish three negative type breast cancers do not turn
Move the accuracy rate with three negative type breast cancers Bone tumours.
Embodiment
Essentiality content of the present invention is specifically introduced with reference to the accompanying drawings and examples, but the guarantor of the present invention is not limited with this
Protect scope.
All breast cancer samples of this project are taken from September, 2014 to 2017 Nian9Yue Lai Hospital Attached to Nantong Univ. or south
Tong Shi First People's Hospital 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 types, Luminal Type Bs, Her-2 overexpressions type and three negative types according to SABC 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
It is starting DISTANT METASTASES IN position to belong to bone.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 inspection such as tissue biopsy confirms.The non-transfer group of breast cancer and Bone tumour group patient age compare without bright in each molecular isoform
Significant difference is different, has comparativity.Each group sample is finally half-and-half divided into test set at random and checking collects.
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:Patient limosis vein blood 5.0mL is gathered, 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.
Embodiment 1:LuminalA types Bone of Breast Cancer shifts
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer, the test set of LuminalA type Bone of Breast Cancer transfer groups and checking collection in LuminalA types.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNABlood Midi Kit specifications, and every part of sample uses 0.8mL serum.Extraction
DNA purity UV spectrophotometer measurings, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0.Meter
DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified:
1 μ g DNA are taken, the modification that methylates is carried out to genomic DNA according to DNAMethylation-Goldkit specifications
Afterwards, -70 DEG C save backup.Polymerase chain reaction:Using PCR to PITX1 and AMOT gene promoter methylations area in sample
Domain is expanded.Reaction system includes sulphite processing rear pattern plate 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot star
Taq enzyme, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, the μ l of cumulative volume 50.Blank control is used as using distilled water.
Pyrosequencing detects:
(1) 45 μ l pcr amplification products are taken respectively into PSQ 96Plate Low sample preparation plates A, it is each to add 45 μ l knots
Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR primer is transferred to denaturation
In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes:The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in PITX1 and AMOT promoter regions.
Wherein pcr amplification primer thing and sequencing primer are as follows:
PITX1
- the GGAAGGTATTTAGTATAGGTGAGTTTGA-3 ' of upstream 5 '
- the AAACCTTAATATTCACTACACTTTATC-3 ' of downstream 5 '
5 '-GTGTTTATTTTGGATTGTTTAATT-3 ' are sequenced
AMOT
- the TGAGTTAATATGAAAGAAGATAGTA-3 ' of upstream 5 '
- the TGATCTCTACATCTCAACTAATATAC-3 ' of downstream 5 '
5 '-GTAGGTTTATTTAGGTT-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Equation below calculates the index that methylates of each gene promoter region, and the index can reflect the methyl of the gene promoter region
Change degree:
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2Examine, be that difference is statistically significant with P < 0.05, and establish ROC curve, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, LuminalA types breast cancer does not shift and Bone tumour group methylates PITX1 and the AMOT that methylates methylation
In test set, methylated in each sample PITX1 and the AMOT that the methylates index that methylates are determined respectively.With
The non-transfer group of Luminal A type breast cancer is compared, and methylate PITX1 and methyl in LuminalA type Bone of Breast Cancer transfer group samples
The index that methylates for changing AMOT significantly raises, and Bone tumour group methylates PITX1 and the AMOT index that methylates of methylating is respectively
Non- transfer group methylates (3.5 ± 0.4) times, (3.3 ± 0.5) times of index.
2nd, methylate the PITX1 and AMOT that methylates the index that methylates be individually used for diagnosis distinguish LuminalA type breast cancer
The ROC curve analysis with the transfer of LuminalA types Bone of Breast Cancer is not shifted
The principle of ROC curve evaluation assessment:
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden indexes, 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 (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention
Non- transfer group), the result detected using diagnostic test can be divided into following situation:
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 represented with following table:
Susceptibility=A/ (A+C) of diagnostic test;Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example
Break few for the number of feminine gender, 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 possible diagnosis 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 during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, the curve is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and its TG-AUC AUC size shows
The size of the diagnostic test degree of accuracy.Intrinsic degree of accuracy indexs 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 represents that accuracy rate of diagnosis is relatively low at 0.5~0.7;AUC exists
When 0.7~0.9, represent that diagnostic accuracy is medium;During AUC > 0.9, represent that diagnosis has higher accuracy.
The index that methylates that methylate PITX1 and the AMOT that methylates are drawn in SPSS 19.0 is individually used for diagnosis differentiation
LuminalA types breast cancer does not shift the ROC curve with the transfer of Luminal A types Bone of Breast Cancer, AUC is respectively 0.715,
0.707, there is medium accuracy.
3rd, methylate the PITX1 and AMOT that methylates the index Combining diagnosis model that methylates structure and for diagnose distinguish
LuminalA types breast cancer does not shift the ROC curve analysis with the transfer of LuminalA types Bone of Breast Cancer
Methylate PITX1 using in test set sample and the AMOT index that methylates of methylating (sets X as independent variable1=first
The base PITX1 index that methylates, X2=the AMOT that the methylates index that methylates), with group (i.e. according to the goldstandard sample category
In Bone tumour group still non-transfer group) dependent variable is used as, to methylating PITX1 and AMOT is methylated in LuminalA type breast cancer
The index that methylates shifted with Luminal A types Bone of Breast Cancer in sample is not shifted and carries out dualistic logistic regression, is obtained binary and is patrolled
Collect regression equation:Y=1/ [1+EXP (1.499X1+2.302X2-0.258)];
Methylated in each sample PITX1 and the AMOT that the methylates index that methylates are substituted into the dualistic logistic regression side again
Journey, you can the regressand value Y of each sample is obtained, 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, has higher accuracy.Calculated and tieed up according to the coordinate of ROC curve
Mounting index=specificity+sensitivity -1, corresponding Y value distinguishes LuminalA types breast for that can carry out diagnosis when tieing up mounting index maximum
The optimal cut-off values 0.238 (i.e. diagnostic threshold) of the non-transfer group of gland cancer and Bone tumour group.
4th, checking concentrates the index Combining diagnosis that methylates for verifying the PITX1 and AMOT that methylates that methylates to distinguish Luminal
A types breast cancer does not shift the order of accuarcy with the transfer of LuminalA types Bone of Breast Cancer
Concentrated in checking, the above-mentioned recurrence mould of index substitution that methylates for the PITX1 and AMOT that methylates that each sample is methylated
Type, the regressand value Y, Y for obtaining each sample are predicted as the transfer of LuminalA types Bone of Breast Cancer less than diagnostic threshold 0.238, are higher than
The LuminalA type breast cancer that is predicted as of diagnostic threshold 0.238 does not shift, and the degree of accuracy is 95.5% (105/110), such as Fig. 2 institutes
Show.
Embodiment 2:Luminal Type Bs Bone of Breast Cancer shifts
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer, the test set of Luminal Type B Bone of Breast Cancer transfer groups and checking in Luminal Type Bs
Collection.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNABlood Midi Kit specifications, and every part of sample uses 0.8mL serum.Extraction
DNA purity UV spectrophotometer measurings, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0.Meter
DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified:
1 μ g DNA are taken, the modification that methylates is carried out to genomic DNA according to DNAMethylation-Goldkit specifications
Afterwards, -70 DEG C save backup.Polymerase chain reaction:Using PCR to PTPN1 and SLIT2 gene promoter methylations area in sample
Domain is expanded.Reaction system includes sulphite processing rear pattern plate 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot star
Taq enzyme, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, the μ l of cumulative volume 50.Blank control is used as using distilled water.
Pyrosequencing detects:
(1) 45 μ l pcr amplification products are taken respectively into PSQ 96Plate Low sample preparation plates A, it is each to add 45 μ l knots
Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR primer is transferred to denaturation
In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes:The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in PTPN1 and SLIT2 promoter regions.
Wherein pcr amplification primer thing and sequencing primer are as follows:
PTPN1
- the AGCGGGTTAGAGGGTAGATGT-3 ' of upstream 5 '
- the TAGGTTTCTCCTCTCCCACATAT-3 ' of downstream 5 '
5 '-TTTCCATTCATCCTAA-3 ' are sequenced
SLIT2
- the TGAAGTTTTATTAGGTTGTGGAGGAGTA-3 ' of upstream 5 '
- the ATACCAAATATCCTATCCTTATCTTC-3 ' of downstream 5 '
5 '-GTTTAAGGTTTATGATA-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Equation below calculates the index that methylates of each gene promoter region, and the index can reflect the methyl of the gene promoter region
Change degree:
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2Examine, be that difference is statistically significant with P < 0.05, and establish ROC curve, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, Luminal Type Bs breast cancer does not shift and Bone tumour group methylates PTPN1 and the SLIT2 that the methylates journey that methylates
Degree
In test set, methylated in each sample PTPN1 and the SLIT2 that the methylates index that methylates are determined 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 samples
The base SLIT2 index that methylates significantly raises, and Bone tumour group methylates PTPN1 and the SLIT2 that the methylates index point of methylating
Not Wei non-transfer group methylate (2.9 ± 0.5) times, (3.4 ± 0.5) times of index.
2nd, methylate the PTPN1 and SLIT2 that methylates the index that methylates be individually used for diagnosis distinguish Luminal Type B mammary gland
Cancer does not shift the ROC curve analysis with the transfer of Luminal Type Bs 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 indexes, 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 (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention
Non- transfer group), the result detected using diagnostic test can be divided into following situation:
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 represented with following table:
Susceptibility=A/ (A+C) of diagnostic test;Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example
Break few for the number of feminine gender, 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 possible diagnosis 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 during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, the curve is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and its TG-AUC AUC size shows
The size of the diagnostic test degree of accuracy.Intrinsic degree of accuracy indexs 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 represents that accuracy rate of diagnosis is relatively low at 0.5~0.7;AUC exists
When 0.7~0.9, represent that diagnostic accuracy is medium;During AUC > 0.9, represent that diagnosis has higher accuracy.
The index that methylates that methylate PTPN1 and the SLIT2 that methylates are drawn in SPSS 19.0 is individually used for diagnosis differentiation
Luminal Type Bs breast cancer does not shift the ROC curve with the transfer of Luminal Type Bs Bone of Breast Cancer, AUC is respectively 0.723,
0.741, there is medium accuracy.
3rd, methylate the PTPN1 and SLIT2 that methylates the index Combining diagnosis model that methylates structure and for diagnostic region
Luminal Type Bs breast cancer is divided not shift the ROC curve analysis with the transfer of Luminal Type Bs Bone of Breast Cancer
Methylate PTPN1 using in test set sample and the SLIT2 index that methylates of methylating (sets X as independent variable1=first
The base PTPN1 index that methylates, X2=the SLIT2 that the methylates index that methylates), with group (i.e. according to the goldstandard sample
Belong to Bone tumour group still non-transfer group) dependent variable is used as, to the PTPN1 and SLIT2 that methylates that methylates in Luminal Type Bs breast
Gland cancer does not shift the index that methylates shifted with Luminal Type Bs Bone of Breast Cancer in sample and carries out dualistic logistic regression, obtains two
Metalogic regression equation:Y=1/ [1+EXP (2.016X1+1.898X2-0.455)];
Methylated in each sample PTPN1 and the SLIT2 that the methylates index that methylates are substituted into the dualistic logistic regression side again
Journey, you can the regressand value Y of each sample is obtained, 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, has higher accuracy.Calculated and tieed up according to the coordinate of ROC curve
Mounting index=specificity+sensitivity -1, corresponding Y value distinguishes Luminal Type Bs for that can carry out diagnosis when tieing up mounting index maximum
The optimal cut-off values 0.310 (i.e. diagnostic threshold) of the non-transfer group of breast cancer and Bone tumour group.
4th, checking concentrates the index Combining diagnosis that methylates for verifying the PTPN1 and SLIT2 that methylates that methylates to distinguish Luminal
Type B breast cancer does not shift the order of accuarcy with the transfer of Luminal Type Bs Bone of Breast Cancer
Concentrated in checking, the above-mentioned recurrence mould of index substitution that methylates for the PTPN1 and SLIT2 that methylates that each sample is methylated
Type, the regressand value Y, Y for obtaining each sample are predicted as the transfer of Luminal Type Bs Bone of Breast Cancer less 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 the degree of accuracy is 93.7% (59/63), as shown in Figure 4.
Embodiment 3:Her-2 overexpression types Bone of Breast Cancer shifts
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer in Her-2 overexpression types, Her-2 overexpression type Bone of Breast Cancer transfer groups test set and test
Card collection.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNABlood Midi Kit specifications, and every part of sample uses 0.8mL serum.Extraction
DNA purity UV spectrophotometer measurings, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0.Meter
DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified:
1 μ g DNA are taken, the modification that methylates is carried out to genomic DNA according to DNAMethylation-Goldkit specifications
Afterwards, -70 DEG C save backup.Polymerase chain reaction:Using PCR to MYLK2, EFEMP1 and SOSTDC1 gene promoter in sample
The son region that methylates is expanded.Reaction system includes sulphite processing rear pattern plate 2 μ l, 10 × PCRbuffer, 0.25U/ μ l
Hot star Taq enzymes, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, the μ l of cumulative volume 50.
Pyrosequencing detects:
(1) 45 μ l pcr amplification products are taken respectively into PSQ 96Plate Low sample preparation plates A, it is each to add 45 μ l knots
Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR primer is transferred to denaturation
In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes:The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings 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 thing and sequencing primer are as follows:
MYLK2
- the GAGGGAAAGGATATGGTTGATT-3 ' of upstream 5 '
- the AACTCCACTCCATTCTCCC-3 ' of downstream 5 '
5 '-AGTAAGTTATTTATTTGTTATTTG-3 ' are sequenced
EFEMP1
- the GGTTTAGGTGGGGAGTATGATAG-3 ' of upstream 5 '
- the ACCAACAACCCAACTTTAACATAACC-3 ' of downstream 5 '
5 '-TAATGAGGGGTTGAG-3 ' are sequenced
SOSTDC1
- the GTAAAGGAGAAAGTTTGGTATATGG-3 ' of upstream 5 '
- the CAAAACTATACAAAAGTATCTCTCTCAAT-3 ' of downstream 5 '
5 '-ATAATTTAATTGTTAGAGTTGAATA-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Equation below calculates the index that methylates of each gene promoter region, and the index can reflect the methyl of the gene promoter region
Change degree:
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2Examine, be that difference is statistically significant with P < 0.05, and establish ROC curve, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, Her-2 overexpressions type breast cancer do not shift and Bone tumour group methylate MYLK2, methylating EFEMP1 and methylates
SOSTDC1 methylation
In test set, the MYLK2 that methylated in each sample is determined respectively, methylating EFEMP1 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 samples
In methylate MYLK2, the index that methylates for the EFEMP1 and SOSTDC1 that methylates that methylates significantly raise, respectively non-transfer group
Methylate (3.7 ± 0.6) times, (2.6 ± 0.4), (3.1 ± 0.5) times of index.
2nd, methylate MYLK2, the index that methylates for the EFEMP1 and SOSTDC1 that methylates that methylates be individually used for diagnosis distinguish
Her-2 overexpression type breast cancer does not shift the ROC curve analysis with the transfer of Her-2 overexpression types 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 indexes, 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 (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention
Non- transfer group), the result detected using diagnostic test can be divided into following situation:
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 represented with following table:
Susceptibility=A/ (A+C) of diagnostic test;Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example
Break few for the number of feminine gender, 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 possible diagnosis 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 during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, the curve is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and its TG-AUC AUC size shows
The size of the diagnostic test degree of accuracy.Intrinsic degree of accuracy indexs 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 represents that accuracy rate of diagnosis is relatively low at 0.5~0.7;AUC exists
When 0.7~0.9, represent that diagnostic accuracy is medium;During AUC > 0.9, represent that diagnosis has higher accuracy.
Methylate MYLK2, methylate EFEMP1 and the SOSTDC1 that the methylates index that methylates are drawn in SPSS 19.0
It is individually used for diagnosis differentiation Her-2 overexpression type breast cancer and does not shift the ROC songs shifted with Her-2 overexpression types Bone of Breast Cancer
Line, AUC are respectively 0.794,0.688,0.738, have relatively low or medium accuracy.
3rd, methylate MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates the index Combining diagnosis model that methylates structure
Build and do not shift the ROC curve shifted with Her-2 overexpression types Bone of Breast Cancer for diagnosing differentiation Her-2 overexpression type breast cancer
Analysis
Methylated using in test set sample MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates methylate index as
Independent variable (sets X1=the MYLK2 that the methylates index that methylates, X2=the EFEMP1 that the methylates index that methylates, X3=methylate
The SOSTDC1 index that methylates), using group (i.e. the sample belongs to Bone tumour group still non-transfer group according to goldstandard) as should
Variable, the MYLK2 that methylates, the EFEMP1 and SOSTDC1 that methylates that methylates are not shifted in Her-2 overexpression type breast cancer and
The index that methylates in Her-2 overexpression types Bone of Breast Cancer transfer sample carries out dualistic logistic regression, obtains dualistic logistic regression
Equation:Y=1/ [1+EXP (1.342X1+1.401X2+1.345X3-2.035)];
The MYLK2 that methylated in each sample, the EFEMP1 and SOSTDC1 that methylates that the methylates index that methylates are substituted into again should
Dualistic logistic regression equation, you can obtain the regressand value Y of each sample, using possible regressand value Y as diagnostic points, calculate sensitive
Degree and specificity, draw ROC curve (as shown in Figure 5), AUC 0.950, have higher accuracy accordingly.According to ROC curve
Coordinate calculate dimension mounting index=specificity+sensitivity -1, corresponding Y value is can carry out diagnosis differentiation when tieing up mounting index maximum
The optimal cut-off values 0.308 (diagnostic threshold) of the non-transfer group of Her-2 overexpression type breast cancer and Bone tumour group.
4th, the index joint that methylates for verifying the MYLK2 that methylates, the EFEMP1 and SOSTDC1 that methylates that methylates is concentrated in checking
Diagnosis distinguishes Her-2 overexpression type breast cancer and does not shift the order of accuarcy shifted with Her-2 overexpression types Bone of Breast Cancer
Concentrated in checking, each sample is methylated MYLK2, the EFEMP1 and SOSTDC1 that methylates that the methylates finger that methylates
Number substitutes into above-mentioned regression model, and the regressand value Y, Y for obtaining each sample are predicted as Her-2 overexpressions less than diagnostic threshold 0.308
Type Bone of Breast Cancer shifts, and the Her-2 overexpression type breast cancer that is predicted as higher than diagnostic threshold 0.308 does not shift, and the degree of accuracy is
96.4% (54/56), as shown in Figure 6.
Embodiment 4:Three negative type breast cancers Bone tumours
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer, the test set of three negative type breast cancers Bone tumour groups and checking collection in three negative types.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNABlood Midi Kit specifications, and every part of sample uses 0.8mL serum.Extraction
DNA purity UV spectrophotometer measurings, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0.Meter
DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified:
1 μ g DNA are taken, the modification that methylates is carried out to genomic DNA according to DNAMethylation-Goldkit specifications
Afterwards, -70 DEG C save backup.Polymerase chain reaction:Using PCR to MYLK3 and SCARA5 gene promoter methylations in sample
Region is expanded.Reaction system includes sulphite processing rear pattern plate 2 μ l, 10 × PCRbuffer, 0.25U/ μ l Hot
Star Taq enzymes, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, the μ l of cumulative volume 50.Blank control is used as using distilled water.
Pyrosequencing detects:
(1) 45 μ l pcr amplification products are taken respectively into PSQ 96Plate Low sample preparation plates A, it is each to add 45 μ l knots
Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR primer is transferred to denaturation
In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers
Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes:The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l
Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively
The base frequency of methylation sites in MYLK3 and SCARA5 promoter regions.
Wherein pcr amplification primer thing and sequencing primer are as follows:
MYLK3
- the TAGGGGAGGTTAAGAAAGTGTA-3 ' of upstream 5 '
- the AACTCCTTATCAATTCCTAACATACAAT-3 ' of downstream 5 '
5 '-GGAGTAATGATGTAATGTGTAT-3 ' are sequenced
SCARA5
- the AGGAATTAGGTAAGGTATGTTAGTA-3 ' of upstream 5 '
- the AAAACTCCAACCTATTCCAACCATACCTAC-3 ' of downstream 5 '
5 '-GTTTTAAGTTTTGGTGTTTGATAT-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to
Equation below calculates the index that methylates of each gene promoter region, and the index can reflect the methyl of the gene promoter region
Change degree:
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data
It is expressed as a percentage, using χ2Examine, be that difference is statistically significant with P < 0.05, and establish ROC curve, under calculated curve
Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back
Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, three negative type breast cancers do not shift and Bone tumour group methylates MYLK3 and the SCARA5 that methylates methylation
In test set, methylated in each sample MYLK3 and the SCARA5 that the methylates index that methylates are determined respectively.With three
The non-transfer group of negative type breast cancers is compared, and being methylated in three negative type breast cancers Bone tumour group samples MYLK3 and methylates
The SCARA5 index that methylates significantly raises, Bone tumour group methylate MYLK3 and methylate SCARA5 methylate index difference
Methylate (2.1 ± 0.3) times, (3.6 ± 0.7) times of index for non-transfer group.
2nd, methylate the MYLK3 and SCARA5 that methylates the index that methylates be individually used for diagnosis distinguish three negative type breast cancers
Do not shift and 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 indexes, 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 (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention
Non- transfer group), the result detected using diagnostic test can be divided into following situation:
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 represented with following table:
Susceptibility=A/ (A+C) of diagnostic test;Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example
Break few for the number of feminine gender, 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 possible diagnosis 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 during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, the curve is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and its TG-AUC AUC size shows
The size of the diagnostic test degree of accuracy.Intrinsic degree of accuracy indexs 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 represents that accuracy rate of diagnosis is relatively low at 0.5~0.7;AUC exists
When 0.7~0.9, represent that diagnostic accuracy is medium;During AUC > 0.9, represent that diagnosis has higher accuracy.
The index that methylates that methylate MYLK3 and the SCARA5 that methylates are drawn in SPSS 19.0 is individually used for diagnostic region
Three negative type breast cancers are divided not shift the ROC curve with three negative type breast cancers Bone tumours, AUC is respectively 0.644,0.809, tool
There is relatively low or medium accuracy.
3rd, methylate the MYLK3 and SCARA5 that methylates the index Combining diagnosis model that methylates structure and 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
Methylate MYLK3 using in test set sample and the SCARA5 index that methylates of methylating (sets X as independent variable1=
Methylate the MYLK3 index that methylates, X2=the SCARA5 that the methylates index that methylates), with group (i.e. according to the goldstandard sample
Originally Bone tumour group still non-transfer group is belonged to) dependent variable is used as, to the MYLK3 and SCARA5 that methylates that methylates in three negative types breast
Gland cancer does not shift carries out dualistic logistic regression with the index that methylates 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)];
Methylated in each sample MYLK3 and the SCARA5 that the methylates index that methylates are substituted into the dualistic logistic regression side again
Journey, you can the regressand value Y of each sample is obtained, 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, has higher accuracy.Calculated and tieed up according to the coordinate of ROC curve
Mounting index=specificity+sensitivity -1, corresponding Y value distinguishes three negative type mammary gland for that can carry out diagnosis when tieing up mounting index maximum
The optimal cut-off values 0.366 (i.e. diagnostic threshold) of the non-transfer group of cancer and Bone tumour group.
4th, checking concentrates the index Combining diagnosis that methylates for verifying the MYLK3 and SCARA5 that methylates that methylates to distinguish three feminine genders
Type breast cancer does not shift the order of accuarcy with three negative type breast cancers Bone tumours
Concentrated in checking, the above-mentioned recurrence of index substitution that methylates for the MYLK3 and SCARA5 that methylates that each sample is methylated
Model, the regressand value Y, Y for obtaining each sample are predicted as three negative type breast cancers Bone tumours less 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 the degree of 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
1st, LuminalA types Bone of Breast Cancer transfer diagnosis indication kit
Include methylate PITX1 and the AMOT that methylates pcr amplification primer thing:Methylate PITX1 PCR amplification sense primer
As shown in Sequence NO.1, PCR expands anti-sense primer as shown in Sequence NO.2;Methylate in AMOT PCR amplifications
Primer is swum as shown in Sequence NO.4, PCR expands anti-sense primer as shown in Sequence NO.5;Also include methylating
PITX1 and the AMOT that methylates Pyrosequencing primer, the Pyrosequencing primer such as Sequence NO.3 for the PITX1 that methylates
Shown, the Pyrosequencing primer for the AMOT that methylates is as shown in Sequence NO.6.
Also include PCR amplifications and the enzyme and reagent needed for pyrosequencing.
2nd, Luminal Type Bs Bone of Breast Cancer transfer diagnosis indication kit
Include methylate PTPN1 and the SLIT2 that methylates pcr amplification primer thing;Methylate PTPN1 PCR amplification upstream draw
For thing as shown in Sequence NO.7, PCR expands anti-sense primer as shown in Sequence NO.8;Methylate SLIT2 PCR amplification
For sense primer as shown in Sequence NO.10, PCR expands anti-sense primer as shown in Sequence NO.11;Also include methylating
PTPN1 and the SLIT2 that methylates Pyrosequencing primer, the Pyrosequencing primer such as Sequence NO.9 for the PTPN1 that methylates
Shown, the Pyrosequencing primer for the SLIT2 that methylates is as shown in Sequence NO.12.
Also include PCR amplifications and the enzyme and reagent needed for pyrosequencing.
3rd, Her-2 overexpressions type Bone of Breast Cancer transfer diagnosis indication kit
Pcr amplification primer thing and pyrosequencing including the MYLK2 that methylates, methylate EFEMP1 and the SOSTDC1 that methylates
Primer;Methylate MYLK2 PCR expand sense primer as shown in Sequence NO.13, PCR amplification anti-sense primer such as
Shown in Sequence NO.14, Pyrosequencing primer is as shown in Sequence NO.15;Methylate in EFEMP1 PCR amplifications
Primer is swum as shown in Sequence NO.16, PCR expands anti-sense primer as shown in Sequence NO.17, Pyrosequencing primer
Methylated as shown in Sequence NO.18 SOSTDC1 PCR expand sense primer as shown in Sequence NO.19, PCR expand
Increase anti-sense primer as shown in Sequence NO.20, Pyrosequencing primer is as shown in Sequence NO.21.
Also include PCR amplifications and the enzyme and reagent needed for pyrosequencing.
4th, three negative type breast cancers Bone tumours diagnosis indication kit
Include methylate MYLK3 and the SCARA5 that methylates pcr amplification primer thing;Methylate MYLK3 PCR amplification upstream draw
For thing as shown in Sequence NO.22, PCR expands anti-sense primer as shown in Sequence NO.23;Methylate SCARA5 PCR
Sense primer is expanded as shown in Sequence NO.25, PCR expands anti-sense primer as shown in Sequence NO.26;Also include first
Base MYLK3 and the SCARA5 that methylates Pyrosequencing primer, the Pyrosequencing primer such as Sequence for the MYLK3 that methylates
NO.24, the SCARA5 that methylates Pyrosequencing primer such as Sequence NO.27.
Also include PCR amplifications and the enzyme and reagent needed for pyrosequencing.
In summary, it is a discovery of the invention that serum methylates, PITX1 and the AMOT that methylates can combine for diagnosing indication
LuminalA types breast cancer whether Bone tumour, individual authentication concentrate diagnosis indication rate of accuracy reached more than 90%;Serum methylates
The PTPN1 and SLIT2 that methylates can combine for diagnose indication Luminal Type Bs breast cancer whether Bone tumour, in individual authentication
Concentrate diagnosis indication rate of accuracy reached more than 90%;Serum methylate MYLK2, methylate the EFEMP1 and SOSTDC1 that methylates can be with
Combine for diagnose indication Her-2 overexpression types breast cancer whether Bone tumour, concentrate diagnosis indication rate of accuracy reached in individual authentication
More than 90%;Serum methylate MYLK3 and methylate SCARA5 can combine for diagnose indication three negative type breast cancers whether
Bone tumour, diagnosis indication rate of accuracy reached more than 90% is concentrated in individual authentication.Diagnosed and indicated using above-mentioned serum methylated genes
Not only the degree of accuracy is high for the transfer of different molecular hypotype Bone of Breast Cancer, and testing cost is low, non-invasi, convenient and swift, very big drop
Low patient suffering and burden.
The effect of above-described embodiment is the specific essentiality content for introducing the present invention, but those skilled in the art should know
Road, protection scope of the present invention should not be confined to the specific embodiment.
Sequence table
<110>Xue Shouhai
<120>Methylated genes composition and the purposes for preparing diagnosis indication Her-2 overexpression type Bone of Breast Cancer transfering reagent boxes
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
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<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
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<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
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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