CN106399304A

CN106399304A - Breast cancer related SNP marker

Info

Publication number: CN106399304A
Application number: CN201611027831.3A
Authority: CN
Inventors: 何劲松; 陈伟财; 欧阳依雯; 毛有胜; 罗雪莹; 潘悦; 刘宝儿; 李锋
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen bike Biotechnology Co., Ltd.
Priority date: 2016-11-18
Filing date: 2016-11-18
Publication date: 2017-02-15
Anticipated expiration: 2036-11-18
Also published as: CN106399304B

Abstract

The invention discloses a breast cancer related biomarker. The biomarker comprises an SNP site of which base in the position 8187 from the 5' end of the nucleotide sequence of gene C12orf45 is G, wherein the site is C12orf45:NM_152318:exon4:c.C368G. In the invention, the application prospect of SNP in breast cancer assisted diagnosis is studied, the influence of SNP on the breast cancer progress is elaborated, and the diagnosis value thereof is revealed. Therefore, through the development and application of the SNP biomarker and diagnosis kit, the diagnosis of breast cancer is more convenient and easier to implement, a foundation is laid for the quick and accurate control of the patient condition by clinical doctors and for the evaluation of clinical treatment effect, and the discovery of a novel small-molecule drug target with potential treatment value is facilitated.

Description

A kind of SNP marker related to breast cancer

Technical field

The present invention relates to field of biomedicine technology is and in particular to a kind of SNP marker related to breast cancer.

Background technology

Breast cancer is that a kind of systemic disease, its occurrence and development are one and are related to multifactor, too many levels complex process, Inactivation of activation including oncogene and tumor suppressor gene etc..Therefore, gene mutation rises in the generation, evolution of breast cancer Very important effect.

Breast cancer is a multifactor hereditary variability disease, only less than 10% because single-gene defect causes. With the development of high flux gene technology, get more and more and be found with breast cancer related gene, potentially heredity on these genes Variation (mononucleotide polymorphic and copy number variation) may cause the difference of breast cancer medicines therapeutic effect.Due to hereditary variation Exist so that the metabolic pathway of antineoplastic and pharmaceutically-active target gene may be affected, and then affect the treatment and Prognosis.

SNP (singlenucleotidepolymorphism, SNP, i.e. SNP) is 1996 by the U.S. The molecule genetic marker that the human genome research center scholar Lander of the Massachusetts Institute of Technology proposes, is primarily referred to as gene By the DNA sequence polymorphism that the variation of single nucleotide acid is caused in group level.The polymorphism that SNP shows relates only to single The variation of base, performance is that have conversion, transversion, insertion and disappearance etc..SNP is third generation genetic marker, human body Many phenotypic differences, all may be relevant with SNP to neurological susceptibility of medicine or disease etc..Pre- for different parting breast cancer at present Afterwards, the predictive research of curative effect is concentrated mainly on SNP level.

SNP gives the individual differential responses to environmental exposure, drug therapy etc., thus producing different phenotypes, therefore SNP Possibly lead to the important hereditary basis of individual disease development difference.SNP spectrum using disease-susceptible humans diagnoses the illness, and has Quickly, sensitive, accurate the features such as, thus have a extensive future.In recent years, become using the generation development that SNP diagnoses the illness The clinical study hotspot with researcher.

However, there is presently no the report that SNP is applied to breast cancer diagnosis, if the SNP of breast cancer susceptibility can be filtered out As biomarker, and develop corresponding diagnostic kit, will effectively promote the present situation of China's early diagnosing mammary cancer, And open up new approach for its drug screening, evaluating drug effect and targeted therapy.

Content of the invention

The purpose of the present invention is for above-mentioned technical problem, proposes a kind of biological marker related to Computer-aided Diagnosis of Breast Cancer Thing.

Second object of the present invention is the application providing described biomarker in prediction breast cancer diagnosis reagent.

Third object of the present invention is to provide Computer-aided Diagnosis of Breast Cancer kit.

Inventor is passed through to separate and studies patient with breast cancer and compareed in peripheral blood DNA with the healthy women of its age-matched SNP, find the SNP of one group of high specific with breast cancer height correlation and sensitiveness, and develop can be just In the Computer-aided Diagnosis of Breast Cancer kit of clinical practice, the examination for breast cancer and diagnosis provide data to support.

The purpose of the present invention is realized by following technical proposal：

A kind of SNP marker related to breast cancer, described biomarker is included positioned at gene C 12orf45 nucleotides sequence Row the 8187th bit base from 5 ' ends there occurs that, by the SNP site mutation of C to G, described SNP site sports C12orf45:NM_ 152318:exon4:c.C368G:p.S123W.

Wherein, gene C 12orf45 (chromosome 12open reading frame 45), is in the mankind the 12nd An ORFs on number chromosome, its genome sequence is classified as NC_000012.12, common 8408bp；NM_152318 is One transcript of C12orf45, described SNP site mutation occurs on the 4th extron of this transcript, and the 368th There occurs by the missense mutation of C to G, this mutation result in the transformation by serine S to tryptophan W for the encoding proteins.

Further, the invention provides described biomarker prediction breast cancer diagnosis reagent in application.

Preferably, described reagent includes the primer pair for expanding described SNP site, or includes described for expanding The primer pair of SNP site and restriction enzyme.

Preferably, the primer pair expanding described SNP site has SEQIDNO：Nucleotide sequence shown in 3-4.

Preferably, the nucleotide sequence SEQIDNO of described primer pair amplifies：Shown in 1.

Closer, the invention provides a kind of kit of Computer-aided Diagnosis of Breast Cancer, it includes detection and is located at gene The reagent of C12orf45 nucleotide sequence the 8187th SNP site genotype from 5 ' ends.

Preferably, described kit also includes the conventional enzyme of PCR reaction and reagent, such as dNTPs, Taq enzyme, Mg²⁺, PCR anti- Answer buffer solution etc.；Standard items and/or reference substance can also be contained.

Beneficial effect of the present invention：

Present invention research SNP, in the application prospect of Computer-aided Diagnosis of Breast Cancer, illustrates the impact for breast cancer progression for the SNP, Disclose its diagnostic value.Therefore, the present invention passes through development and the application of SNP biomarker and diagnostic kit, can make breast The diagnosis of gland cancer is more convenient and easy, is that clinician quick and precisely grasps conditions of patients, is that clinical therapeutic efficacy evaluation is established Basis, and the new small molecule drug targets for finding to have potential therapeutic value provide help.

Specific embodiment

Following examples are used for the present invention is described, but are not limited to the scope of the present invention.If not specializing, embodiment In the conventional meanses that are well known to those skilled in the art of technological means used.

Technical scheme specifically includes：Gather standard compliant blood sample, the complete demography of systematic collection Data and clinical data；Genotype detection：Breast cancer case is selected to compare with the healthy women of breast cancer case age-matched, Using extron sequencing, find out the SNP related to breast cancer；To the positive association SNP filtering out, adopt Genotyping further Detected, verified that it is applied to the repeatability of clinical diagnosis；The development of Computer-aided Diagnosis of Breast Cancer kit：According to breast cancer The genotype distribution frequency SNP that there were significant differences exploitation SNP auxiliary diagnostic box in case and healthy women comparison.

In data analysis, each numerical value is expressed as follows：

1、ljb23_sift：SIFT score value (version 2.3), represents the impact to protein sequence for this variation, comprises three Individual value, one is SIFT initial value, and two is the value (1-SIFT) after conversion, and three is T or D.When this variation affect simultaneously multiple During protein sequence, there is a SIFT value to every protein sequence, take minimum of a value.SIFT score value is more little more " being harmful to ", shows this SNP Lead to the possibility that protein structure or function change big；D:Deleterious(sift<=0.05)；T:tolerated(sift> 0.05))；

2、ljb23_pp2hvar：Predict this variation to protein sequence using PolyPhen2 based on HumanVar database Impact, for single gene inheritance disease.This row comprises two values, and first is PolyPhen2 score value, and numerical value is more big more " being harmful to ", Show that this SNP leads to the possibility that protein structure or function change big；Second is D or P or B (D:Probably damaging (>=0.909), P:possibly damaging(0.447<=pp2_hvar<=0.909)；B:benign(pp2_hvar<= 0.446))；

3、ljb23_pp2hdiv：Predict this variation to protein sequence using PolyPhen2 based on HumanDiv database Impact, for complex disease.This row comprises two values, and first is PolyPhen 2 score value, and numerical value is more big more " being harmful to ", shows This SNP leads to the possibility that protein structure or function change big；Second is D or P or B (D:Probably damaging(>= 0.957),P:possibly damaging(0.453<=pp2_hdiv<=0.956)；B:benign(pp2_hdiv<= 0.452))；

4、ljb23_mt：TionTaster score value (version 2.3), represents the impact to protein sequence for this variation, bag It is worth containing three, one is Mutation Taster initial value, two is the value after conversion, and three is A, D, N or P.Second value is got over Greatly more " be harmful to ", show this SNP leads to protein structure or function changes possibility greatly, wherein " A " (" disease_ causing_automatic")；"D"("disease_causing")；"N"("polymorphism")；"P"(" polymorphism_automatic").

The experimental technique specifically studied mainly includes following components：

1. study the selection of sample

(1) breast cancer case 25 clarified a diagnosis through pathology and the healthy women 10 with breast cancer case age-matched Example, as comparison, wherein has 3 patients to have cancer family history in breast cancer case；

(2) radiotherapy or chemotherapy, no the past tumour medical history were not accepted before taking a blood sample；

(3) compare with the healthy women of case age-matched

2. phenol-chloroform method extracts peripheral blood genomic DNA, operates according to a conventional method.Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D：2800D) in 1.6-2.0.

3. full Exon chip detection

(1) take experimenter's complete genome DNA sample；

(2) it is scanned on full Exon chip (Beijing Nuo Hezhi source Science and Technology Co., Ltd., similarly hereinafter)；

(3) detect and compare difference difference in breast cancer case is compareed with healthy women for each genotype.

4. the Genotyping of single SNP

(1) take experimenter's DNA sample；

(2) design the specificity amplification primer of single SNP；

(3) enter performing PCR reaction, recovery product is sequenced；

(4) compare the distributional difference of different genotype during breast cancer case is compareed with healthy women.

5. diagnostic reagent box preparation method

Gene during determination breast cancer case is compareed with healthy women after Exon chip is scanned detecting with single SNP entirely The type distribution frequency SNP that there were significant differences, as the index of breast cancer diagnosis.The SNP relevant with pathogenesis of breast carcinoma filtering out Auxiliary diagnostic box, it includes detection and is located at gene C 12orf45 nucleotide sequence the 8187th SNP site base from 5 ' ends Because of the reagent of type, diagnostic kit can also include the specificity amplification primer of these SNP, and the reagent such as Taq enzyme, dNTPs.

6. clinical practice example

Using the present inventor preparation Computer-aided Diagnosis of Breast Cancer kit detection treat examination patient with breast cancer and with reality Clinical detection compares to determine the validity of Computer-aided Diagnosis of Breast Cancer kit.Specifically include mensure experimenter's blood specimen In cDNA, the specificity amplification primer of above-mentioned SNP and other detection reagents, are the disease that clinician quick and precisely grasps patient State and coincident with severity degree of condition, take the control prece of more personalized to provide in time and support.

The collection of embodiment 1 sample and the arrangement of sample data

Inventor have collected substantial amounts of new hair-cream gland in January, 2010 in December, 2015 in Shenzhen City Second People's Hospital Cancer patient's blood specimen, by the arrangement to sample data, inventor therefrom have selected 25 samples meeting following standard, simultaneously Select 10 ages 25-55 year healthy women compare and carry out full Exon chip detection, sample selection criteria is as follows：

1st, the breast cancer case clarified a diagnosis through pathology, wherein has 3 patients to have cancer family history and mark respectively For X1, X2, X3；

2nd, radiotherapy or chemotherapy, no the past tumour medical history were not accepted before taking a blood sample；

3 are compareed with the healthy women of case age-matched

And situations such as the system acquisition demographic data of these samples and clinical data.

The extraction of embodiment 2 peripheral blood DNA and purifying

In above-mentioned qualified 25 patient with breast cancers and 10 healthy women comparisons, two groups of age equilibriums are comparable.

Concretely comprise the following steps：

1st, (i.e. lysate, 40 deal collocation methods are such as to add hemolyzing reagent to the peripheral blood being stored in 2mL cryopreservation tube Under：After sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20mL mixing, use TrisHcl solution It is settled to 2000mL, similarly hereinafter), proceed to completely after reverse mixing.

2nd, remove red blood cell：With hemolyzing reagent, 5mL centrifuge tube is mended to 4mL, overturns and mix, 4000rpm is centrifuged 10 minutes, Abandon supernatant.Add 4mL hemolyzing reagent in precipitation, overturn again and mix cleaning once, 4000rpm is centrifuged 10 minutes, abandons supernatant.

3rd, extract DNA：Add 1mL extract and (in every 300mL, contain 122.5mL0.2M sodium chloride, 14.4mL in precipitation 0.5M ethylenediamine tetra-acetic acid, 15mL10% lauryl sodium sulfate, 148.1mL distilled water, similarly hereinafter) and 8 μ L Proteinase Ks, vibration On device, fully vibration mixes, and 37 DEG C of water-baths are overnight.

4th, remove isolating protein：Plus 1mL saturated phenol fully mixes (hand jog 15 minutes), 4000rpm is centrifuged 10 minutes, takes Proceed to clearly in new 5mL centrifuge tube.Equal-volume chloroform and isoamyl alcohol mixed liquor (chloroform is added in supernatant：Isoamyl alcohol=24: 1, v/v, similarly hereinafter), after fully mixing (hand 15 minutes), 4000rpm is centrifuged 10 minutes, take supernatant (be divided into two 1.5mL from Heart pipe).

5th, DNA precipitation：Add the sodium acetate 60 μ L of 3M in supernatant, add the anhydrous second with the isopyknic ice of supernatant Alcohol, upper and lower jog is centrifuged 10min it is seen that white flock precipitate thing, then with 12000rpm.

6th, DNA washing：Ice absolute ethyl alcohol 1mL, 12000rpm is added to be centrifuged 10min in precipitation, after abandoning supernatant, vacuum is taken out Do or be placed in cleaning dry environment and be evaporated.

7th, measure concentration：Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D：2800D) in 1.8-2.0.

The full extron group detection of embodiment 3SNP

Two groups of crowds in embodiment 2 are detected through full Exon chip and obtains correlated results.

1st, library construction

Beijing Nuo Hezhi source Science and Technology Co., Ltd. adopts the liquid-phase chip capture systems of Agilent, entirely outer to people Aobvious sub-district domain dna carries out efficiently concentrating, then carries out high flux, high depth sequencing on Illumina Hiseq platform.Build storehouse Adopt Agilent SureSelect Human All ExonV5 kit with capture experiment, strict operation instructions are recommended Reagent and consumptive material, and operated with reference to the up-to-date experiment flow through optimizing.

Experiment basic procedure：Genomic DNA is crushed instrument through Covaris and is broken into the piece that length is 180-280bp at random Section, through end repair and plus A tail after fragment two ends connect respectively top connection preparation DNA library.Library with special index With up to 543 after pooling, the probe of 872 biotin labelings carries out solution hybridization, and reusing the magnetic bead with streptomysin will The 334 of 20,965 genes, 378 exon trappings get off, and through the laggard style of writing storehouse quality inspection of PCR linear amplification, qualified can enter Machine sequencing on row.

2nd, storehouse inspection

After the completion of library construction, first carry out preliminary quantitation using Qubit2.0, dilution library, to 1ng/ μ L, subsequently uses Agilent 2100 detects to the insert size in library, after insert size meets expection, using Q-PCR method pair The valid density in library carries out accurate quantitative analysis (library valid density>2nM), to ensure Library Quality.

3rd, upper machine sequencing

Storehouse inspection is qualified, and the valid density according to library and data output demand carry out the sequencing of Illumina Hiseq platform.

4th, data analysis and process

Through data screening, deep processing and bioinformatics sequence alignment, final determination " breast cancer case " is organized and " strong Genotype distribution frequency 53 SNP site that there were significant differences finding in health female control " group are preferred sensitivity level site.Its In, positioned at gene C 12orf45 nucleotide sequence, from 5 ' ends, the 8187th bit base is the SNP site of G, and described site is C12orf45:NM_152318:exon4:C.C368G, this Mutation is as follows to albumen influence value：

ljb23_sift：0,1.00,D

ljb23_pp2hvar：0.951,D

ljb23_pp2hdiv：0.999,D

ljb23_mt：0.996,0.004,N.

This site, through bioinformatic analysis, can confirm that as breast cancer candidate markers.

Embodiment 4 analyzes the onset risk of SNP and breast cancer further using MELD method

The present inventor is by 2 groups of samples (" breast cancer case group " and " healthy women control group ") genotype distribution frequency Comparison, select positive association SNP, with full extron scanned samples, single SNP regression coefficient, as weight, is tried to achieve further Dangerous score value, draws ROC to evaluate sensitivity and the specificity of diagnosis, and then diagnoses the judgement to pathogenesis of breast carcinoma for these SNP Ability.Conjoint Analysis to all SNP marks finds, positioned at gene C 12orf45:NM_152318:The 368th alkali of exon4 Base is the mutation of G/C, and its sensitivity and specificity all reach more than 60%.

Therefore, inventors demonstrated that this site mark can be well by healthy women comparison and patient with breast cancer area Point.

The Genotyping of the single SNP of embodiment 5

1st, 5 patient with breast cancers and 5 healthy women comparison DNA samples are taken with embodiment 2；

2nd, PCR amplification

Using Primer Premier 5 software to C12orf45:NM_152318:exon4:C.C368G designs single SNP Specificity amplification primer as shown in table 1.

Table 1 primer sequence

PCR reaction system is as shown in table 2.PCR amplification program is：95 DEG C of denaturations 10min, 94 DEG C of denaturation 15s, 61 DEG C are moved back Fiery 15s, 72 DEG C of extension 30s, carry out 30 circulations, last 72 DEG C of extension 30min, preserve in 4 DEG C, overnight need -20 DEG C of placement cold Freeze.

Table 2 reaction system

Component	Addition
		2×mix	25μL
Upstream primer (10uM)	3.0μL
		Downstream primer (10uM)	3.0μL
Template	5μL
		Add sterile purified water	To 50 μ L

3rd, it is sequenced

After PCR amplification terminates, take 5 μ L amplified productions, 1% agarose gel electrophoresis, electrophoresis 30min, dye 20min, so Afterwards gel piece is placed in gel imaging instrument and observes, according to the clip size situation comparing Marker, tentatively judge amplified fragments Whether correct.And then satisfactory amplified production is purified：Using Mag- BindOligonucleotidePurificationKit kit, and operated by kit requirement.Loading is sequenced：Using ABI company BigDye3.1SequencingKit kit, and operated by kit requirement；Surveyed with ABI company 3730 type Sequence instrument is sequenced.

4th, interpretation of result

By Chromas sequence analysis software, sequencing result is compared with standard sequence, find SNP site, pass through At analysis SNP site, the type of base is it is possible to obtain the genotype of SNP site.Result shows：5 patient with breast cancer's sequencings As shown in SEQ ID NO.1, it is CG, GG in the 239th bit base to the nucleotide sequence of fragment obtaining 392bp；And 5 health Female control sequencing obtains the nucleotide sequence of the fragment of 392bp as shown in SEQ ID NO.2, and it is CC in the 239th bit base； Confirm that this site is the susceptible genotype being judged as breast cancer during CG, GG genotype, this site is to be judged as during CC genotype The non-susceptible genotype of breast cancer, thus further confirm that described C12orf45：NM_152318:exon4:The SNP of c.C368G Site can be used for the auxiliary diagnosis such as the detection of breast cancer, treatment, diagnosis, prognosis evaluation.

Embodiment 6 is used for the making of Computer-aided Diagnosis of Breast Cancer SNP kit

The primer sets being obtained based on embodiment 5, assemble the kit for breast cancer of the present invention, described kit Primer pair such as SEQ ID NO including nucleotide sequence as shown in SEQ ID NO.1 for the specific amplified:3 and SEQ ID NO:4 institutes Show.Described kit can also have the common agents needed for corresponding round pcr, such as：DNTPs, MgCl₂, distilled water, Taq enzyme etc., These common agents are all well known to those skilled in the art, it can in addition contain have standard items and comparison (as determined genotype Standard items and blank etc.).The value of this kit is to only need to peripheral blood without other tissue samples, by Simplify and detect SNP with special primer pair, then auxiliary judgment breast cancer is composed by SNP, not only stable, easy to detect, and accurately, Greatly improve the Sensitivity and Specificity of medical diagnosis on disease, therefore by this kit put into practice, can help instruct diagnosis and more Effectively individualized treatment.

Although, above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Sequence table

<110>Shenzhen City Second People's Hospital

<120>A kind of SNP marker related to breast cancer

<130> P1690

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 392

<212> DNA

<213>Artificial sequence

<400> 1

ttgctcatca actcccaacc taagtccaga aagacctcca ctcttcaaac agttcggata 60

gagaggagtc ccttattgga ccaggtacag acatttctcc cacagatggc acgggcaaat 120

gaaaagctaa gaaaagaaat ggcagctgca ccacctggtc gtttcaatat tgaaaacatt 180

gatgggcctc atagtaaagt tatacaaatg gatgtggctt tgtttgagat gaatcagtcg 240

gattcaaaag aagtggacag ttcagaagag agttcacaag acagttcaga gaacagttca 300

gaatcagaag acgaagatga cagcatccca tctgaagtca ccatagataa cattaagctt 360

cccaattctg aaggtggaaa aggcaagatt ga 392

<210> 2

<211> 392

<212> DNA

<213>Artificial sequence

<400> 2

ttgctcatca actcccaacc taagtccaga aagacctcca ctcttcaaac agttcggata 60

gagaggagtc ccttattgga ccaggtacag acatttctcc cacagatggc acgggcaaat 120

gaaaagctaa gaaaagaaat ggcagctgca ccacctggtc gtttcaatat tgaaaacatt 180

gatgggcctc atagtaaagt tatacaaatg gatgtggctt tgtttgagat gaatcagtcg 240

gattcaaaag aagtggacag ttcagaagag agttcacaag acagttcaga gaacagttca 300

gaatcagaag acgaagatga cagcatccca tctgaagtca ccatagataa cattaagctt 360

cccaattctg aaggtggaaa aggcaagatt ga 392

<210> 3

<211> 23

<212> DNA

<213>Artificial sequence

<400> 3

ttgctcatca actcccaacc taa 23

<210> 4

<211> 23

<212> DNA

<213>Artificial sequence

<400> 4

cttccacctt ttccgttcta act 23

Claims

1. a kind of biomarker related to breast cancer is it is characterised in that described biomarker is included positioned at gene C12orf45 nucleotide sequence the 8187th bit base from 5 ' ends is the SNP site of G, and described site is C12orf45:NM_ 152318:exon4:c.C368G:p.S123W.

2. application in prediction breast cancer diagnosis reagent for the biomarker described in claim 1.

3. application as claimed in claim 2 is it is characterised in that described reagent is included for expanding drawing of described SNP site Thing pair, or include the primer pair for expanding described SNP site and restriction enzyme.

4. application as claimed in claim 3 is it is characterised in that the primer pair expanding described SNP site has SEQIDNO：3-4 Shown nucleotide sequence.

5. application as claimed in claim 4 is it is characterised in that the nucleotide sequence such as SEQIDNO of described primer pair amplifies：1 Shown.

6. a kind of kit of Computer-aided Diagnosis of Breast Cancer is located at gene C 12orf45 nucleotides it is characterised in that it includes detection The reagent of sequence the 8187th SNP site genotype from 5 ' ends.

7. kit as claimed in claim 6 is it is characterised in that include the primer pair for expanding described SNP site, or Including primer pair and restriction enzyme for expanding described SNP site.

8. kit as claimed in claim 6 is it is characterised in that described primer pair has SEQIDNO：Nucleosides shown in 3-4 Acid sequence.

9. kit as claimed in claim 7 or 8 is it is characterised in that described kit also includes dNTPs, Taq enzyme, Mg²⁺With PCR reaction buffer.