CN107475367A - A kind of mutator and its detection kit for assessing mammary cancer risk - Google Patents
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Abstract
The invention discloses a kind of mutator for assessing mammary cancer risk.The invention also discloses the specific primer and its detection kit of a kind of breast cancer mutator detection.The present invention assesses the detection kit of mammary cancer risk using the mutational site exploitation of ARHGEF18, RPTN and TTC21B gene, the kit has sensitiveness strong, high accuracy for examination, and method is simple, quick, early screening, prognosis evaluation to breast cancer have great importance, be also gene therapy, the clinical practice such as drug therapy provide important evidence.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to a kind of mutator for assessing mammary cancer risk and its inspection
Test agent box.
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 and tumor suppressor gene including oncogene etc..Therefore, gene mutation rises in the generation, evolution of breast cancer
Very important effect.
Breast cancer is a multifactor hereditary variability disease, is due to caused by single-gene defect only less than 10%.
It is more and more to be found with breast cancer related gene with the development of high flux gene technology, potential 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
May be affected in the presence of the metabolic pathway and pharmaceutically-active target gene for making antineoplastic, so affect the treatment and
Prognosis.
The most common mark of hereditary breast cancer is the mutation of BRCA1 and BRCA2 genes, but BRCA1/2 gene mutations
What detection breast cancer and treating cancer were generally inadequate, because these gene mutations do not occur in substantial amounts of accidental cancer,
The cause of disease of sub-fraction hereditary breast cancer can only be used for explaining.
The presence in mutational site is considered as assigning individual different phenotypic characters, and to environmental exposure, drug therapy etc.
Differential responses, therefore mutational site is probably the important hereditary basis for causing individual disease occurrence and development difference.By disease-susceptible humans
The spectrum of mutation is used for the auxiliary diagnosis of disease, is with a wide range of applications.If the base being present in accidental cancer can be filtered out
Because mutational site come diagnose with prognosis breast cancer, great importance will be produced to breast cancer individualized treatment, and and be its medicine
Screening and evaluating drug effect open up new approach.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned technical problem, there is provided a kind of mutator for assessing mammary cancer risk and its inspection
Test agent box.
The purpose of the present invention is realized by following technical proposal：
Present invention firstly provides a kind of mutator for detecting mammary cancer risk, it is characterised in that described mutation base
Because of one or several genes in following ARHGEF18, RPTN and/or TTC21B, the mutational site of the mutator is such as
Under：
(1)ARHGEF18:NM_001130955:exon12:c.C2101T:p.Q701X；
(2)RPTN:NM_001122965:exon3:c.C1834T:p.Q612X；
(3)TTC21B:NM_024753:exon1:c.20_21insGGTGAGCGGGTGAGCG:
p.K7_T8delinsKVSGX。
Wherein, X represents terminator codon.
The protein of ARHGEF18 gene codes is guanine nucleotide exchange factor, belongs to RhoGTPase GFE families.
Family member has common feature, and Dbl (DH) homeodomain, it is pleckstrin (PH) homeodomain.Rho GTP
Enzyme is the gtp binding protein for adjusting cell function.Rho GTP enzymes regulation cytoskeleton formed, genetic transcription, the cell cycle and
Played an important role in vesicular traffic, occur also have substantial connection with tumour and neurogenic disease, play molecule in vivo and open
The role of pass.Direct control of the activation of Rho GTP enzymes by guanine nucleotide exchange factor (GEFs).
RPTN genes are located on 1q21 chromosomes, and people RPTN belongs to S100 protein family newcomers, N-terminal have two can be with
Ca2+The tandem repetitive sequence rich in glutamine residue is contained in the EF hand domains of Reversible binding and inside, is a kind of and angle
Cell plastid envelope forms relevant precursor protein.RPTN people central nervous system wide expression, especially in hippocampus, prefrontal lobe
With the high expression in the region such as nucleus ceruleus, regions of these height expression and schizophrenia, depression it is in close relations.
TTC21B is protein coding gene, the member of coding TTC21 families, and it contains several tetrapeptides and repeats (TPR) domain.Should
Protein positioning plays a role in cilium aixs cylinder in may being transported in the flagellum that cilium drives in the wrong direction；The gene mutation and each fruit scale
Shape cell cancer, scrotum parasitosis are relevant with asphyxiating chest muscular dystrophy.
Preferably, it is described to sport nonsense mutation.
Preferably, the presence of the mutation is determined with following means：
(a)qPCR；
(b) cDNA microarray；And/or
(c) direct nucleic acid sequencing.
Preferably, the directly nucleic acid sequencing method：Advanced performing PCR amplification, obtain the PCR productions containing purposeful mutational site
Thing, then purified pcr product, then it is sequenced, according to sequencing peak figure interpretation genotype.
Preferably, the cDNA microarray：Advanced performing PCR amplification, obtain the PCR productions containing purposeful mutational site
Thing, then hybridizes PCR primer and mutant probe, wild probe, and the signal intensity hybridized by comparing two probes judges sample
Product genotype.Probe is fixed on nylon membrane, nitrocellulose filter, is referred to as solid phase chip on glass；Probe is fixed on small pearl
On son for liquid-phase chip, such as luminex, biocade detection platform.
Preferably, the qPCR methods include ARMS-PCR methods, HRM methods, Taqman-MGB sonde methods.
Further, the present invention provides a kind of specific primer of breast cancer mutator detection, and the primer sequence is：
(1) it is used for the primer for detecting mutational site described in ARHGEF18 mutators：
Sense primer SEQ ID No.1：5’-CCCCTTCCTTCCACAGTCGAG-3’；
Anti-sense primer SEQ ID No.2：5’-GTCTGGAGGGTTTCAGAAGGT-3’；
(2) it is used for the primer for detecting mutational site described in RPTN mutators：
Sense primer SEQ ID No.3：5’-GACAGACAGACAAGGCCAGAGC-3’；
Anti-sense primer SEQ ID No.4：5’-GGTGATGTTGGCTATCCTCTTCA-3’；
(3) it is used for the primer for detecting mutational site described in TTC21B mutators：
Sense primer SEQ ID No.5：5’-GCTAGGGGAGCTGAATTCTGC-3’；
Anti-sense primer SEQ ID No.6：5’-GACCAGCAATTCAGAAACACCG-3’.
Preferably, the primer in the mutational site of above-mentioned mutator is expanded respectively according to ARHGEF18 (NC_ in NCBI
000019.10), RPTN (NC_000001.11) and TTC21B (NC_000002.12) genome sequence design, the primer
Synthesized by Shanghai life work design, the primer specificity is strong, and expanding effect is good.
Further, the present invention provides a kind of detection kit for assessing mammary cancer risk, it is characterised in that the reagent
Box contains the one or more in above-mentioned primer.
Preferably, the kit also includes：DNTP solution, Taq enzyme are positive for the reagent of isolated or purified nucleic acid
Control or negative control.
Beneficial effect of the present invention：
The present invention assesses the detection of mammary cancer risk using the mutational site exploitation of ARHGEF18, RPTN and TTC21B gene
Kit, the kit have that sensitiveness is strong, high accuracy for examination, and method is simple, quick, and the early stage of breast cancer is sieved
Look into, prognosis evaluation has great importance, be also gene therapy, the clinical practice such as drug therapy provide important evidence.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means.
Embodiment 1 prepares the detection kit for assessing mammary cancer risk
The making of kit of the present invention and operating process are based on Sanger sequencing Scanning Detction typing methods.It is described
Kit includes DNA extracts reagents；The kit also includes specific amplification ARHGEF18:NM_001130955:exon12:
The primer of c.C2101T gene mutations is that reverse primer sequences are SEQ ID shown in SEQ ID No.1 such as forward primer sequence
Shown in No.2；The kit can also include the conventional reagent of PCR reactions, such as Taq enzyme, dNTP mixed liquors, MgCl2Solution, go
Ionized water etc.；These common agents are all well known to those skilled in the art, it can in addition contain including control group normal health female
Property peripheral blood DNA.PCR reaction systems used are 25 μ L：Taq Buffer 2.5 μ L, DNA 1 μ L, the μ L of forward primer 0.5, reversely
0.5 μ L, 10mM dNTP of primer 0.5 μ L, Taq enzyme 0.2 μ L, ddH2O 19.8μL.PCR response procedures：95 DEG C of 3min, 35 are followed
Ring (94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min), 72 DEG C of 10min.The value of this kit be only to need peripheral blood without
Other tissue samples, mutational site is detected with special primer pair by most simplifying, is not only stablized, it is easy to detect, and accurately, greatly
The big Sensitivity and Specificity for improving medical diagnosis on disease, guidance and help are provided for diagnosis and individualized treatment.
Kit of the present invention can also include the one of specific amplification ARHGEF18, RPTN and/or TTC21B mutator
Kind or a variety of primers, the primer are：
(1) it is used for the primer for detecting mutational site described in ARHGEF18 mutators：
Sense primer SEQ ID No.1：5’-CCCCTTCCTTCCACAGTCGAG-3’；
Anti-sense primer SEQ ID No.2：5’-GTCTGGAGGGTTTCAGAAGGT-3’；
(2) it is used for the primer for detecting mutational site described in RPTN mutators：
Sense primer SEQ ID No.3：5’-GACAGACAGACAAGGCCAGAGC-3’；
Anti-sense primer SEQ ID No.4：5’-GGTGATGTTGGCTATCCTCTTCA-3’；
(3) it is used for the primer for detecting mutational site described in TTC21B mutators：
Sense primer SEQ ID No.5：5’-GCTAGGGGAGCTGAATTCTGC-3’；
Anti-sense primer SEQ ID No.6：5’-GACCAGCAATTCAGAAACACCG-3’.
The kit includes specific amplification ARHGEF18 with above-mentioned:NM_001130955:exon12:C.C2101T bases
Because the preparation method of the kit of mutant primer is identical.
The use of detection kit prepared by the embodiment 1 of embodiment 2
1st, DNA extraction
The blood sample that 46 clinical detections are patient with breast cancer is taken, extracts DNA.
Concretely comprise the following steps：
(1) to the peripheral blood addition hemolyzing reagent being stored in 2mL cryopreservation tubes, (i.e. lysate, 40 deal collocation methods are such as
Under：After sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20mL are mixed, with TrisHcl solution
2000mL is settled to, similarly hereinafter), it is transferred to completely after reverse mixing.
(2) red blood cell is removed：5mL centrifuge tubes are mended to 4mL with hemolyzing reagent, overturns and mixes, 4000rpm centrifuges 10 points
Clock, abandon supernatant.4mL hemolyzing reagents are added into precipitation, overturns mix cleaning once again, 4000rpm is centrifuged 10 minutes, is abandoned
Clearly.
(3) DNA is extracted：Into precipitation plus 1mL extracts (contain 122.5mL0.2M sodium chloride, 14.4mL in per 300mL
0.5M ethylenediamine tetra-acetic acids, 15mL10% lauryl sodium sulfate, 148.1mL distilled waters, similarly hereinafter) and 8 μ L Proteinase Ks, vibration
Fully vibration is mixed on device, and 37 DEG C of water-baths are stayed overnight.
(4) isolating protein is removed：1mL saturated phenols are added fully to mix (hand jog 15 minutes), 4000rpm is centrifuged 10 minutes, is taken
Supernatant is transferred in new 5mL centrifuge tubes.Isometric chloroform and isoamyl alcohol mixed liquor (chloroform are added in supernatant：Isoamyl alcohol=
24:1, v/v, similarly hereinafter), after fully mixing (hand 15 minutes), 4000rpm is centrifuged 10 minutes, takes supernatant (to be divided into two 1.5mL
Centrifuge tube).
(5) DNA is precipitated：The 3M μ L of sodium acetate 60 are added in supernatant, it is anhydrous to add the ice isometric with supernatant
Ethanol, upper and lower jog, it is seen that white flock precipitate thing, then 10min is centrifuged with 12000rpm.
(6) DNA is washed：Ice absolute ethyl alcohol 1mL, 12000rpm centrifugation 10min is added in precipitation, vacuum is taken out after abandoning supernatant
Do or be placed in and be evaporated in cleaning dry environment.
(7) concentration is measured：Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D：2800D) in 1.8-2.0.
2nd, it is sequenced
It is 25 μ L with PCR reaction systems：Taq Buffer 2.5 μ L, DNA 1 μ L, the μ L of forward primer 0.5, reverse primer
The μ L of 0.5 μ L, 10mM dNTP 0.5, Taq enzyme 0.2 μ L, ddH2O 19.8μL.PCR response procedures：95 DEG C of 3min, 35 circulations
(94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min), 72 DEG C of 10min.1% agarose gel electrophoresis detects, with ultraviolet bale cutting instrument incision
Take PCR primer gel and purify.All PCR primers are sequenced with forward and reverse primer sequence respectively, and sequencing result is carried out
Further analysis, is sequenced and is completed by Shanghai bioengineering；Sequencing result is by Chromas sequence analysis softwares, by sequencing result
It is compared with standard sequence, and combines healthy women control group sequence, finds mutational site.
3rd, interpretation of result
Check whether breast cancer ARHGEF18 gene carrier mutational site changes according to sequencer map, if in sequencer map
The site is unimodal C, then wild type (CC) is normal not ill；If the site is bimodal, simultaneously containing C and T, then heterozygosis is dashed forward
Modification (CT) is illness；If the site is unimodal T, homozygous mutant (TT) is illness.
As a result show, in 46 samples, mutation 18, mutation occur in the extrons of ARHGEF18 (NM_001130955) the 12nd
Rate 39.1%, wherein heterozygous mutant have 12, and homozygous mutant has 6.
Comparative example 1 utilizes Taqman-MGB detection methods detection breast cancer
1st, DNA extraction
The blood sample that 46 clinical detections are patient with breast cancer is taken, DNA is the same as embodiment 2 for extraction.
2nd, PCR is expanded
(1) design of primers
According to ARHGEF18 (No. GenBank：NC_000019.10), expanded by Primer Express3.0 Software for Design
Increase ARHGEF18:NM_001130955:exon12:The specific primer and probe in c.C2101T mutational sites, and given birth to by Shanghai
Work design synthesis, the primer and probe sequence are as follows：
Sense primer SEQ ID NO.7：ACAGTCGAGGGCATCCAGAGC
Anti-sense primer SEQ ID NO.8：CTGTGGAGCTGCCTCCGTCTT
Wild-type probe SEQ ID NO.9：VIC-CCTGATCTGCAGGCAGCT-NFQ
Saltant type probe SEQ ID NO.10：FAM-CCTGATCTGCAGGTAGCT-NFQ
(2) PCR reaction systems
Universal PC R amplification premix reagents (being purchased from ABI), each 400nM of primer, 2 of fluorescence labeling
Taqman-MGB probes each 200nM, DNA 150ng.
(3) PCR reaction conditions
The PCR system configured is put into fluorescent PCR instrument, carries out fluorescent PCR augmentation detection；PCR amplification programs are：
50℃2min；95℃10min；95 DEG C of 15s, 60 DEG C of 1min, carry out 40 circulations.
Fluorescent PCR instrument：With the real-time fluorescence quantitative PCR instrument (ABI) of ABI 7500.
3rd, Genotyping
By the fluorescence signal intensity of the FAM and VIC probes shown on quantitative real time PCR Instrument, it is determined that the SNP positions detected
The genotype of point.Genotype interpretation：It is homozygous mutant genotypes only to have sample caused by amplified signal in FAM channels；Only exist
It is wild-type genotype to have sample caused by amplified signal in VIC channels；Have in FAM and VIC channels caused by amplified signal
Sample is heterozygous mutant gene type；The ARHGEF18 genes of subject with homozygous mutant genotypes and heterozygous mutant gene type
Abrupt climatic change result is the positive.
As a result show, in 46 samples, mutation 18, mutation occur in the extrons of ARHGEF18 (NM_001130955) the 12nd
Rate 39.1%, wherein heterozygous mutant have 12, and homozygous mutant has 6.As a result the method institute used with kit of the present invention
The result obtained is consistent.
Conclusion：ARHGEF18 detection kit sensitiveness prepared by the present invention is strong, high accuracy for examination, and method letter
Single, quick, early screening, prognosis evaluation to breast cancer have great importance, and are also gene therapy, the clinic such as drug therapy
Using providing important evidence.
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, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>BeiJing University ShenZhen Hospital (Peking University's Shenzhen Clinical Medical Institute) Medical University Of Anhui
<120>A kind of mutator and its detection kit for assessing mammary cancer risk
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<170> PatentIn version 3.5
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Claims (5)
1. it is a kind of detect mammary cancer risk mutator, it is characterised in that described mutator be selected from following ARHGEF18,
One or several genes in RPTN and/or TTC21B, the mutational site of the mutator are as follows：
(1)ARHGEF18:NM_001130955:exon12:c.C2101T:p.Q701X；
(2)RPTN:NM_001122965:exon3:c.C1834T:p.Q612X；
(3)TTC21B:NM_024753:exon1:c.20_21insGGTGAGCGGGTGAGCG:
p.K7_T8delinsKVSGX。
2. mutator as claimed in claim 1, it is characterised in that described to sport nonsense mutation.
3. the specific primer of a kind of breast cancer mutator detection, it is characterised in that the primer sequence is：
(1) it is used for the primer for detecting mutational site described in ARHGEF18 mutators：
Sense primer SEQ ID No.1：5’-CCCCTTCCTTCCACAGTCGAG-3’；
Anti-sense primer SEQ ID No.2：5’-GTCTGGAGGGTTTCAGAAGGT-3’；
(2) it is used for the primer for detecting mutational site described in RPTN mutators：
Sense primer SEQ ID No.3：5’-GACAGACAGACAAGGCCAGAGC-3’；
Anti-sense primer SEQ ID No.4：5’-GGTGATGTTGGCTATCCTCTTCA-3’；
(3) it is used for the primer for detecting mutational site described in TTC21B mutators：
Sense primer SEQ ID No.5：5’-GCTAGGGGAGCTGAATTCTGC-3’；
Anti-sense primer SEQ ID No.6：5’-GACCAGCAATTCAGAAACACCG-3’.
4. a kind of detection kit for assessing mammary cancer risk, it is characterised in that the kit contains the primer of claim 3
In one or more.
5. kit as claimed in claim 4, it is characterised in that the kit also includes：DNTP solution, Taq enzyme, is used for
The reagent of isolated or purified nucleic acid, positive control or negative control.
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| ANNE R. BRESNICK ET AL.: "S100 proteins in cancer", 《NAT REV CANCER》 * |
| BERNARD PEREIRA ET AL.: "The somatic mutation profiles of 2,433 breast cancers refine their genomic and transcriptomic", 《NATURE COMMUNICATIONS》 * |
| JN ANASTAS ET AL.: "protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression", 《ONCOGENE》 * |
| REFERENCE SNP(RS) REPORT: "rs551931470", 《NCBI-DBSNP》 * |
| REFERENCE SNP(RS) REPORT: "rs779468719", 《NCBI-DBSNP》 * |
| SERENA NIK-ZAINAL ET AL.: "Landscape of somatic mutations in 560 breast cancer whole-genome sequences", 《NATURE》 * |
| SHANTANU BANERJI ET AL.: "Sequence analysis of mutations and translocations across breast cancer subtypes", 《NATURE》 * |
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