CN107523571A - People EZH2 mutators and its application - Google Patents
People EZH2 mutators and its application Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6869—Methods for sequencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
Stomach cancer is that Chinese are most often suffered from and most easily lethal major cancers, its chemotherapeutic efficacy are assessed mainly by iconography means and serological index, but ageing poor and specific not strong.The invention provides people EZH2 mutators, mutator detection method and the application in chemotherapy of gastric cancer curative effect evaluation.The mutation of the EZH2 mutators betides the 1234th 1240 site of No. 19 intrones, preferable mutant gene sequence such as SEQ ID NO:Shown in 14, there is not been reported；This can be that parsing and drug development of stomach cancer occurrence and development mechanism etc. provide foundation and basis.In addition the application of EZH2 mutators and its detection method, it will help chemotherapy of gastric cancer curative effect evaluation and state of illness monitoring to guiding treatment scheme formulation.
Description
Technical field
The invention belongs to genetic test and application field, and in particular to EZH2 mutators, EZH2 mutators detection and
Its application in chemotherapy of gastric cancer curative effect evaluation.
Background technology
Stomach cancer is that Chinese most often suffer from and most easily lethal major cancers, its incidence of disease occupy the second of malignant tumour
Position, the death rate occupy the 3rd.What is clinically relied primarily at present for chemotherapy of gastric cancer curative effect evaluation is iconography means and blood
It is clear to learn index etc..But iconography means often have certain hysteresis, and the molecule essence of cancer stove can not be accurately reflected sometimes；
And the universal specificity of the serum markers such as CEA, CA72-4, CA19-9 is not strong, it monitors the Index for diagnosis and diagnosis and treatment for stomach cancer
Guiding value is limited.
EZH2 (Enhancer of Zeste Homolog 2) is a kind of human gene disclosed in recent years, and it is PcG
The core member of (Polycomb Group) gene family.PcG families include two kinds of polycomplexs of PRC1 and PRC2, rise respectively
And maintain gene to suppress the effect with initial gene silence.PRC2 compounds are by the common structure of several genes including EZH2
Into EZH2 is PRC2 catalytic active component, and its highly conserved SET domain can be to the 9th, 27 of nucleosome histone H 3
Lysine carries out the modification that methylates, so as to suppress to include the table of related hundreds of genes such as cell growth, differentiation, metastases
Reach.
EZH2 genes maintain the silence shape of homeotic gene in embryo development procedure by the modification to chromosome
State.In addition, the gene over-expresses in many type of malignancies of the mankind, it plays extremely important in the occurrence and development of tumour
Effect.Find to find that the high of EZH2 genes is expressed in Malignancy first, then again in prostate cancer, mammary gland
The gene high expression has been had been found that in cancer, carcinoma of urinary bladder, liver cancer and stomach cancer, and it is not expressed in corresponding normal structure or only few
Amount expression.EZH2 albumen has been used for the pernicious differentiation for monitoring prostate cancer, breast cancer etc. as a kind of new tumor marker.
EZH2 genes contain 20 extrons and 19 intrones, and extron length is 41-323bp, and opening code-reading frame is distributed
On 19 extrons；Length of intron is 0.15-17.7kb, it is known that transcription after have two spliceosomes：The He of transcript variant 1
Transcript variant 2；CDNA total length 2.7kb, encode 613 amino acid.The C-terminal of EZH2 genes because have 1 Su (var) 3-9,
Tri- kinds of albumen common structural domains of Enhancer ofzeste and trithorax and the highly conserved SET domains named；EZH2
Albumen institute mediate transcription suppresses to need to rely on complete SET regions, if thus the SET regions lost and cannot produce suppression
Tabulation type, and disinthibiting of gene can be made in some cases.And EZH2 No. 19 intrones are in the event of specific mutation,
Variable sheer is caused to occur that SET regions will be caused to lack, so as to which EZH2 modification tumor suppressor genes can not be relied in tumour cell.
At present, EZH2 genes are also fully disclosed and carried out far away for the clinical guidance of stomach cancer and application.Applicant
Early stage selects the ctDNA of patients with gastric cancer（Circulating tumor DNA, Circulating tumor DNA）Sample, it is included
The capture sequencing of gene set including EZH2 genes（That is two generation sequencing technologies of target gene capture joint）Research；With reference to sample
Clinical pathology information analysis, it is found that prognosis of the specific mutation of EZH2 No. 19 introne of gene to stomach cancer can realize good comment
Estimate and predict, there is the potential as chemotherapy of gastric cancer curative effect evaluation index, there is no this specific mutation and its application both at home and abroad at present
Report.For prognosis prediction, the trend and heat for being easy to establish that noninvasive detection method is current visiting of starting with from ctDNA samples
Door is very low yet with ctDNA contents and fragmentation is serious therefore traditional based on Sanger sequencings and real time fluorescent quantitative
PCR detection method is not suitable for.Two generation sequencing technologies can with it is sensitive effectively to rare mutation carry out high flux detection, and
It can be found that unknown mutation, therefore be a kind of preferable detection method.
The content of the invention
Deficiency for clinically lacking special effective chemotherapy of gastric cancer curative effect evaluation index at present, the invention provides with
The specific mutation feature and its detection method of this related EZH2 gene, and be applied in the chemotherapeutic efficacy assessment of stomach cancer.
These can be that parsing of chemotherapy of gastric cancer curative effect evaluation, state of illness monitoring and occurrence and development mechanism etc. provides foundation and basis.
The technical solution adopted for the present invention to solve the technical problems is as follows：
The purpose of the present invention 1 is to provide people's EZH2 mutators, and it is mutated the of No. 19 intrones betiding EZH2 genes
1234-1240 sites, containing the 1234th and the 1240th site（Referring to Fig. 1）；The mutation occurred includes missing, insertion or point mutation
But the one or more of not limited to this.Preferably, the EZH2 gene mutations refer to its No. 19 introne 1236-1238 sites
3 thymidylic acids missing（Corresponding mutation intron sequences such as SEQ ID NO:Shown in 1）, 1236-1239
The missing of 4 thymidylic acids in site（Corresponding mutation intron sequences such as SEQ ID NO:Shown in 2）, 1235-
The missing of 6 thymidylic acids in 1240 sites（Corresponding mutation intron sequences such as SEQ ID NO:Shown in 3）With
The insertion of 2 thymidylic acids in 1234-1235 sites（Corresponding mutation intron sequences such as SEQ ID NO:Shown in 4）In
But the one or more of not limited to this.SEQ ID NO:5 be No. 19 intron sequences of wild type EZH2 reference genes.
The purpose of the present invention 2 is to provide people's EZH2 mutator detection methods, and key step is as follows：
（1）The capture of EZH2 genes
Sample to be tested genomic DNA is extracted, EZH2 is carried out using the liquid phase capture probe group or PCR capture primer sets of design synthesis
The capture of gene region；
（2）The two generations sequencing parsing spectrum of mutation
Will（1）In capture product carry out the sequencing of two generations, and mutational site is found out by information analysis.
Wherein, step（1）Described in liquid phase capture probe group or PCR capture primer sets need comprising EZH2 can be captured
The probe collection or primer collection in No. 19 introne 1234-1240 sites of gene, described 1234-1240 sites include the
1234 and the 1240th site；Unrelated probe and primer can entrust commercialization biotech firm to design and synthesize.
Wherein, step（2）Described in capture product carry out two generations sequencing and information analysis, have two generation sequenators and match somebody with somebody
Cover analysis software then to carry out according to standard operation and analysis, or can be completed by commercialized high-flux sequence company；Final sequencing
As a result the mutational site obtained need to include No. 19 introne 1234-1240 sites of EZH2 genes（Include the 1234th and 1240
Site）Abrupt information.
Wherein, step（2）Described in mutational site, refer to the 1234-1240 positions of No. 19 intrones of EZH2 genes
Point, comprising the 1234th and the 1240th site, described mutation includes missing, insertion or point mutation but one kind of not limited to this or several
Kind；Preferably, the mutation refers to 3 thymidylic acids in No. 19 introne 1236-1238 sites of EZH2 genes
Lack, the missing of 4 thymidylic acids in 1236-1239 sites, 6 thymidine cores in 1235-1240 sites
Thuja acid missing and 2 thymidylic acids in 1234-1235 sites insertion in but not limited to this one or more, institute
Mutation intron sequences corresponding to this 4 kinds of mutation orders are arranged respectively such as SEQ ID NO:Shown in 1-4.SEQ ID NO:5 be wild
No. 19 intron sequences of type EZH2 reference genes.
The purpose of the present invention 3 is detecting the application in chemotherapy of gastric cancer curative effect evaluation in offer people EZH2 mutators, main
Want step as follows:
（1）Sample collection
Gather patients with gastric cancer 2-10 sample of chemotherapy；
（2）The capture sequencing of EZH2 genes
The genomic DNA of sample to be tested is extracted, is carried out using the liquid phase capture probe group or PCR capture primer sets of design synthesis
The capture of EZH2 gene regions, capture product is subjected to the sequencing of two generations afterwards, and mutational site is found out by information analysis；
（3）Chemotherapeutic efficacy is assessed
Progression of disease according to continuous chemotherapy sample EZH2 gene specific mutations testing result prediction patient：In previous sample
EZH2 gene specific mutations are detected, latter sample does not detect same mutation or detection but the frequency of mutation reduces, then predicts the state of an illness
Progress；EZH2 gene specific mutations, latter sample detection equally mutation but frequency of mutation rise are detected in previous sample, then is predicted
Stable disease or disease amelioration；EZH2 gene specific mutations are not detected in previous sample, latter sample detection mutation, then prediction is sick
Feelings stabilization or disease amelioration；Front and rear sample standard deviation does not detect EZH2 gene specific mutations, then does not give a forecast.
Wherein, step（1）Described in chemotherapy, its interval be no more than 3 months.Above gather for previous sample, after
Face collection for latter sample.
Wherein, step（1）Described in sample include but is not limited to flesh tissue, frozen tissue, formaldehyde immersion tissue, formaldehyde
Fixed paraffin-embedded tissue section, new blood, blood plasma and serum, hydrothorax, ascites and exfoliated tumor cells；For every kind of sample
Type, it is necessary to while gather cancer stove sample and normal structure sample is analyzed, cancer stove sample includes that cancer stove base can be obtained herein
Because of the sample such as blood of abrupt information（Determine its ctDNA）.Preferably, fresh anticoagulation 5-100mL is selected.
Wherein, step（2）Described in two generations sequencing, for cancer stove sample its be sequenced depth be 1000 × -10000 × it
Between.
Wherein, step（3）Described in EZH2 gene specific mutations refer to, betide the 1234- of its No. 19 intrones
Missing, insertion or the point mutation in 1240 this 7 sites but one or more of mutation of not limited to this；Preferably, it is described specific prominent
Become missing, the 1236-1239 for 3 thymidylic acids for referring to No. 19 introne 1236-1238 sites of EZH2 genes
The missing of 4 thymidylic acids in site, the missing of 6 thymidylic acids in 1235-1240 sites and
In the insertion of 2 thymidylic acids in 1234-1235 sites but not limited to this one or more, listed this 4 kinds mutation are suitable
Mutation intron sequences are respectively such as SEQ ID NO corresponding to sequence:Shown in 1-4.SEQ ID NO:5 be wild type EZH2 reference genes
No. 19 intron sequences.
Wherein, step（3）Described in be mutated, its detect frequency must be over 5%；The frequency of mutation reduces and rise, its
The range of decrease and increasing degree must be over 20%, be considered small echo dynamic stability less than 20%.The disease progression, stabilization and alleviate foundation
It is《NCCN clinical practice guidelines：Stomach cancer》（2012 editions）.
The specific mutation feature of No. 19 intrones of EZH2 genes provided by the invention has not yet to see report, and this can be
The parsing of stomach cancer occurrence and development mechanism and drug development etc. provide foundation and basis.Secondly, it is provided by the invention to be based on target base
Because of the EZH2 mutator detection methods of capture two generation sequencing technologies of joint, it can apply to ctDNA and FFPE genomes etc. and contain
Measure humble and serious fragmentation sample detection, realize it is sensitive it is special, efficiently comprehensively, fast and accurately abrupt climatic change.Finally
The application of EZH2 mutators and its detection method, it will help chemotherapy of gastric cancer curative effect evaluation and state of illness monitoring are to guiding treatment
The formulation of scheme.
Brief description of the drawings
Fig. 1 is No. 19 introne specific mutation schematic diagram of people EZH2 genes.Black box represents No. 19 in figure（It is left）With
20 exons（It is right）, horizontal line therebetween represents No. 19 intrones；Arrow points to site, and the starting of 1 No. 19 intrones of expression is
Turned right the 1st site in 19 exon low order end nucleotides, thick line area represents that mutation generation area is 1234-1240 sites
Place.
Embodiment
Below in conjunction with specific embodiment, the present invention is expanded on further.
Embodiment 1
Patients with gastric cancer 1 of the hospital through definitive pathological diagnosis where selection（Numbering 1#）, in the case where it endorsed informed consent form,
2 parts of blood sample in its therapeutic process is gathered, carries out gene trap and the sequencing of two generations of ctDNA samples, while collects its clinic and controls
Treatment and condition assessment information, finally carry out comprehensive analysis（Refer to table 1）.
（1）Blood specimen collection
Take the EDTA anticoagulations 5ml of patient's ulnar vein（Blood sampling time is shown in Table 1）, separated plasma and monocyte in 1 hour, its
Middle plasma sample can be used for the information of reflection cancer stove containing ctDNA, and monocyte group containing normal gene DNA is used to do reference.
（2）The capture sequencing of EZH2 genes
Will（1）Middle sample is sent to Hua Da gene with dry ice and carries out gene trap and the sequencing of two generations.Using QIAamp
Circulating Nucleic Acid Kit（German Qiagen companies）CtDNA is extracted, with QIAamp DNA Blood Mini
Kit（German Qiagen companies）Extract monocyte genomic DNA；Use CAN_panel_EZ_HX3 (1.7M) target area
Capture chip is captured, and the chip can capture No. 19 introne 1234-1240 sites of EZH2 genes（Include the 1234th
With the 1240th site）Region.
It is sequenced using the Hiseq2500 platforms of illumnia companies of the U.S., orthocytosis genomic DNA sample
Average sequencing depth is more than 200 ×, the average sequencing depths of blood plasma ctDNA samples is more than 1000 ×.It is supporting using Hiseq2500
Analysis software is to sequencing result initial analysis, using orthocytosis genomic DNA sample sequencing result as reference, then with thousand people's bases
Because a group database, snp database and COSMIC (version 64) database carry out blood plasma ctDNA samples mutational site solution
Analysis, as a result as shown in table 1.
（3）Chemotherapeutic efficacy is assessed
A blood sample before the patient（2015.5.28）No. 19 introne 1234-1240 sites for not detecting EZH2 genes occur
It is mutated, then a blood sample（2015.6.25）The insertion of 2 thymidylic acids in 1234-1235 sites is then detected i.e.
[1234-1235insTT] is mutated（Detection 2 times）, corresponding No. 19 intron sequences such as SEQ ID NO:Shown in 5, the mutation belongs to
Mutation on 1234-1240 sites, and the frequency of mutation is up to 60%.That is a preceding sample does not detect mutation, but latter
Secondary sample has detected mutation, therefore disease development can be predicted after the patient 2015.6.25 to be stable or alleviate.The patient in
2015.7.10 it is SD i.e. stable through the clinical assessment state of an illness, it is still SD that 2015.8.27 is assessed again；Thus demonstrate based on EZH2
The prediction of mutator.
The 1# patients with gastric cancer sample of table 1 is sequenced and clinical assessment result
Embodiment 2
Patients with gastric cancer 1 of the hospital through definitive pathological diagnosis where selection（Numbering 2#）, in the case where it endorsed informed consent form,
3 parts of blood sample in its therapeutic process is gathered, carries out gene trap and the sequencing of two generations of ctDNA samples, while collects its clinic and controls
Treatment and condition assessment information, finally carry out comprehensive analysis（Refer to table 2）.Detailed process is referring to embodiment 1.
A blood sample before the patient（2015.6.16）No. 19 introne 1236-1238 sites of EZH2 genes are detected
3 thymidylic acids missing be [1236-1238delTTT] mutation（Detection 9 times）, corresponding No. 19 intron sequences
Such as SEQ ID NO:Shown in 1, mutation that the mutation belongs on 1234-1240 sites, and the frequency of mutation is up to 52.63%；This
Outside, this blood sample（2015.6.16）4 thymus gland for also having detected No. 19 introne 1236-1239 sites of EZH2 genes are phonetic
The missing of pyridine nucleotides is [1236-1239delTTTT] mutation, corresponds to No. 19 intron sequences such as SEQ ID NO:, should shown in 2
The mutation belonged on 1234-1240 sites is mutated, and the frequency of mutation is 15%.A middle blood sample（2015.8.11）With it is latter
Secondary blood sample（2015.8.27）Mutation is not detected；That is a preceding sample has detected mutation, after twice sample again do not detect
Mutation, therefore can predict that the state of an illness is progress before and after the patient 2015.8.11.The patient is in 2015.8.10 through clinical assessment disease
Feelings are that PD is in progress, and thus demonstrate the prediction based on EZH2 mutators.
The 2# patients with gastric cancer sample of table 2 is sequenced and clinical assessment knot
Embodiment 3
Patients with gastric cancer 1 of the hospital through definitive pathological diagnosis where selection（Numbering 3#）, in the case where it endorsed informed consent form,
3 parts of blood sample in its therapeutic process is gathered, carries out gene trap and the sequencing of two generations of ctDNA samples, while collects its clinic and controls
Treatment and condition assessment information, finally carry out comprehensive analysis（Refer to table 3）.Detailed process is referring to embodiment 1.
A blood sample before the patient（2015.3.31）Do not detect mutation, and a middle blood sample（2015.5.15）Detect
The insertion of 2 thymidylic acids in No. 19 introne 1234-1235 sites of EZH2 genes is [1234-1235insTT]
Mutation（Detection 2 times）, corresponding No. 19 intron sequences such as SEQ ID NO:Shown in 5, the mutation belongs on 1234-1240 sites
Mutation, and the frequency of mutation is up to 50%；A blood sample afterwards（2015.7.21）Mutation is not detected.That is a preceding sample is not
Detection mutation, but a middle sample has detected mutation, and mutation is not detected again to a rear sample；Therefore the patient can be predicted
2015.5.15 disease development is stable or alleviation after, and disease development is progress after 2015.7.21.The patient in
2015.6.28 it is SD i.e. stable through the clinical assessment state of an illness, it is then PD that 2015.8.15 is assessed again, thus demonstrates and is based on
The prediction of EZH2 mutators.
The 3# patients with gastric cancer sample of table 3 is sequenced and clinical assessment result
Embodiment 4
Patients with gastric cancer 1 of the hospital through definitive pathological diagnosis where selection（Numbering 4#）, in the case where it endorsed informed consent form,
3 parts of blood sample in its therapeutic process is gathered, carries out gene trap and the sequencing of two generations of ctDNA samples, while collects its clinic and controls
Treatment and condition assessment information, finally carry out comprehensive analysis（Refer to table 4）.Detailed process is referring to embodiment 1.
A blood sample before the patient（2015.4.2）With a middle blood sample（2015.4.24）Mutation is not detected, then
Blood sample（2015.7.4）6 thymidine cores in No. 19 introne 1235-1240 sites of EZH2 genes are then detected
The missing of thuja acid is [1234-1240delTTTTTT] mutation（Detection 2 times）, the mutation belongs to prominent on 1234-1240 sites
Become, and the frequency of mutation is up to 50%；That is preceding sample twice does not detect mutation, and then a sample has detected mutation, therefore
Disease development can be predicted after the patient before and after once i.e. 2015.7.4 to be stable or alleviate.After though the patient is without 2015.7.4
Clinical state assessment result, but its in 2015.6.17 through the clinical assessment state of an illness be PR i.e. alleviate, and 2015.7.4 blood samples examine
Go out to be mutated, front and rear indication 7.4 is stabilization or alleviation, and 6.17 and 7.4 is very close in time, therefore this patient's result
Demonstrate the prediction based on EZH2 mutators.
The patients with gastric cancer sample of table 4 is sequenced and clinical assessment result
Embodiment 5
Patients with gastric cancer 1 of the hospital through definitive pathological diagnosis where selection（Numbering 5#）, in the case where it endorsed informed consent form,
3 parts of blood sample in its therapeutic process is gathered, carries out gene trap and the sequencing of two generations of ctDNA samples, while collects its clinic and controls
Treatment and condition assessment information, finally carry out comprehensive analysis（Refer to table 5）.Detailed process is referring to embodiment 1.
A blood sample before the patient（2015.6.2）No. 19 2, introne 1234-1235 sites of EZH2 genes are detected
The insertion of thymidylic acid is [1236-1237insTT] mutation（Detection 2 times）, the mutation belongs on 125-131 sites
Mutation, and the frequency of mutation is up to 50%；A middle blood sample（2015.7.3）With a rear blood sample（2015.8.4）Do not detect
Mutation；That is a preceding sample has detected mutation, after twice sample do not detect mutation again, therefore the patient can be predicted
2015.8.4 disease development is progress afterwards.The patient is that PD is in progress through the clinical assessment state of an illness in 2015.12.23, is thus tested
The prediction based on EZH2 mutators is demonstrate,proved.
The 5# patients with gastric cancer sample of table 5 is sequenced and clinical assessment result
In summary, EZH2 mutators provided by the invention and its detection method and its in chemotherapy of gastric cancer curative effect evaluation
Using being accurate and reliable, and because characterization of molecules changes earlier than cell tissue and iconography form, thus its have it is good
Good is ageing and perspective.Further expand patients with gastric cancer quantity validation, find the patient of the mutator containing EZH2 in sample
Number accounts for the 50% of total patient's number, it is known that the index that EZH2 mutators can be relatively broad and universal as one.
Due to the announcement based on No. 19 introne specific mutation information sequence of EZH2 genes provided by the invention, in order to open
Opening up the scientific research such as stomach cancer Related Mechanism Mechanism Study and the exploitation of check and evaluation method and clinical position needs, can be by where such mutation
Amplicon purified, be cloned into the commercialization carrier such as plasmid, be conducted into afterwards in the Host Strains such as Escherichia coli,
It is placed in -80 DEG C of refrigerators and preserves；Further, it is also possible to be prepared by the way of rite-directed mutagenesis by wild type gene, or pass through
The mode of sequent synthesis obtains.
Although the present invention is illustrated by preferable specific embodiment, come for those skilled in the art
Say, the present invention there can be various modifications and variations.Within the spirit and principles of the invention, it is any modification for being made, equivalent
Replace, improve etc., it should be included in the scope of the protection.
SEQUENCE LISTING
<110>Medical University Of Fujian
Chuan Xin bio tech ltd of Fujian Province
<120>People EZH2 mutators and its application
<130> 5
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1361
<212> DNA
<213>Artificial sequence
<400> 1
gttggtaaag tacatttcta gcatgatctc taagggcttt ttctactgga ttgtgaactc 60
tggacaaagg agggttttta gttctttgct tcttttgatg ggtcactttg ccatgagcat 120
tagtggggaa ttaggttaca ctttcctgtt atgtatttat tatccattta tatattatac 180
aaggcatgct tatttttaaa atagagtaaa atccatgcag aaagccccat ttctcaccct 240
gctgttgaca gctgggtgag tcctagacct tctcatatca tgccgcatgc tgcatgctca 300
ctcgtggagc gttttccaga taagtgcgat cacactgtcc tatagatttt tctgcaacat 360
ggttttactt gacaatatac tatgcatatc tttatggatt agatctactt aatttattgt 420
ttgctataat acttattaca catatcatta ccattttaaa aggggctgtt ggcaaatcgc 480
tgtctgatca ccctgtgggg atgcctggga cacgtgggcc accaatggta tctgtgtggt 540
taggcattag gctgccagtt gacactgtca agctctgtgg gatacattta agtcagccat 600
tgaatacaga tttaagtgca gtcagatgac tgtctagtta atgaactcct gtagccccgt 660
gtacatccca tccagaagct gtctttgagt actgtgttct tgcatattca ggctggcctt 720
taatggctgt tgcacagcag cgagctctca gtgaggcaga cagggtctga gtgaagggcc 780
cactttagca aaggccgctg ccttcactcc aggctggctc tgaatgaaga gtgtggggtg 840
gagcttcctt gggatgaccc ggtggcctca ccccccatgg tgggcttctt gtgctcctct 900
ctgggtgtcc gctgcactcc agttcttgct tcacctgggg gagcaactgg cgggctgctc 960
tttggccctc tttgctatgg tcatcatttt ttccagacca tggttctatt ttgccttccc 1020
agcgtcaggc ctaagcatcc tgatgtggac cagccttcct gggaccactt gcttcagagg 1080
cataggggag gctggtcagg ctctgggaat gcttgtagac aggtttgttc acttcagaac 1140
ttggcagcgt ctcaattaga tggccagcaa cacagaccta tgcattgcct tctatgaatg 1200
tgccgttatg caggcagctg tgtaattgga tgggtttttt tttttttttt tttaaagaca 1260
ttttaatgca cccactatct tcagcaggct ttgttgtgtt aagtctcagc acatgttgga 1320
tgggtggcca tccagcggac atctccttcc tgttgtttca g 1361
<210> 2
<211> 1360
<212> DNA
<213>Artificial sequence
<400> 2
gttggtaaag tacatttcta gcatgatctc taagggcttt ttctactgga ttgtgaactc 60
tggacaaagg agggttttta gttctttgct tcttttgatg ggtcactttg ccatgagcat 120
tagtggggaa ttaggttaca ctttcctgtt atgtatttat tatccattta tatattatac 180
aaggcatgct tatttttaaa atagagtaaa atccatgcag aaagccccat ttctcaccct 240
gctgttgaca gctgggtgag tcctagacct tctcatatca tgccgcatgc tgcatgctca 300
ctcgtggagc gttttccaga taagtgcgat cacactgtcc tatagatttt tctgcaacat 360
ggttttactt gacaatatac tatgcatatc tttatggatt agatctactt aatttattgt 420
ttgctataat acttattaca catatcatta ccattttaaa aggggctgtt ggcaaatcgc 480
tgtctgatca ccctgtgggg atgcctggga cacgtgggcc accaatggta tctgtgtggt 540
taggcattag gctgccagtt gacactgtca agctctgtgg gatacattta agtcagccat 600
tgaatacaga tttaagtgca gtcagatgac tgtctagtta atgaactcct gtagccccgt 660
gtacatccca tccagaagct gtctttgagt actgtgttct tgcatattca ggctggcctt 720
taatggctgt tgcacagcag cgagctctca gtgaggcaga cagggtctga gtgaagggcc 780
cactttagca aaggccgctg ccttcactcc aggctggctc tgaatgaaga gtgtggggtg 840
gagcttcctt gggatgaccc ggtggcctca ccccccatgg tgggcttctt gtgctcctct 900
ctgggtgtcc gctgcactcc agttcttgct tcacctgggg gagcaactgg cgggctgctc 960
tttggccctc tttgctatgg tcatcatttt ttccagacca tggttctatt ttgccttccc 1020
agcgtcaggc ctaagcatcc tgatgtggac cagccttcct gggaccactt gcttcagagg 1080
cataggggag gctggtcagg ctctgggaat gcttgtagac aggtttgttc acttcagaac 1140
ttggcagcgt ctcaattaga tggccagcaa cacagaccta tgcattgcct tctatgaatg 1200
tgccgttatg caggcagctg tgtaattgga tgggtttttt tttttttttt ttaaagacat 1260
tttaatgcac ccactatctt cagcaggctt tgttgtgtta agtctcagca catgttggat 1320
gggtggccat ccagcggaca tctccttcct gttgtttcag 1360
<210> 3
<211> 1358
<212> DNA
<213>Artificial sequence
<400> 3
gttggtaaag tacatttcta gcatgatctc taagggcttt ttctactgga ttgtgaactc 60
tggacaaagg agggttttta gttctttgct tcttttgatg ggtcactttg ccatgagcat 120
tagtggggaa ttaggttaca ctttcctgtt atgtatttat tatccattta tatattatac 180
aaggcatgct tatttttaaa atagagtaaa atccatgcag aaagccccat ttctcaccct 240
gctgttgaca gctgggtgag tcctagacct tctcatatca tgccgcatgc tgcatgctca 300
ctcgtggagc gttttccaga taagtgcgat cacactgtcc tatagatttt tctgcaacat 360
ggttttactt gacaatatac tatgcatatc tttatggatt agatctactt aatttattgt 420
ttgctataat acttattaca catatcatta ccattttaaa aggggctgtt ggcaaatcgc 480
tgtctgatca ccctgtgggg atgcctggga cacgtgggcc accaatggta tctgtgtggt 540
taggcattag gctgccagtt gacactgtca agctctgtgg gatacattta agtcagccat 600
tgaatacaga tttaagtgca gtcagatgac tgtctagtta atgaactcct gtagccccgt 660
gtacatccca tccagaagct gtctttgagt actgtgttct tgcatattca ggctggcctt 720
taatggctgt tgcacagcag cgagctctca gtgaggcaga cagggtctga gtgaagggcc 780
cactttagca aaggccgctg ccttcactcc aggctggctc tgaatgaaga gtgtggggtg 840
gagcttcctt gggatgaccc ggtggcctca ccccccatgg tgggcttctt gtgctcctct 900
ctgggtgtcc gctgcactcc agttcttgct tcacctgggg gagcaactgg cgggctgctc 960
tttggccctc tttgctatgg tcatcatttt ttccagacca tggttctatt ttgccttccc 1020
agcgtcaggc ctaagcatcc tgatgtggac cagccttcct gggaccactt gcttcagagg 1080
cataggggag gctggtcagg ctctgggaat gcttgtagac aggtttgttc acttcagaac 1140
ttggcagcgt ctcaattaga tggccagcaa cacagaccta tgcattgcct tctatgaatg 1200
tgccgttatg caggcagctg tgtaattgga tgggtttttt tttttttttt aaagacattt 1260
taatgcaccc actatcttca gcaggctttg ttgtgttaag tctcagcaca tgttggatgg 1320
gtggccatcc agcggacatc tccttcctgt tgtttcag 1358
<210> 4
<211> 1366
<212> DNA
<213>Artificial sequence
<400> 4
gttggtaaag tacatttcta gcatgatctc taagggcttt ttctactgga ttgtgaactc 60
tggacaaagg agggttttta gttctttgct tcttttgatg ggtcactttg ccatgagcat 120
tagtggggaa ttaggttaca ctttcctgtt atgtatttat tatccattta tatattatac 180
aaggcatgct tatttttaaa atagagtaaa atccatgcag aaagccccat ttctcaccct 240
gctgttgaca gctgggtgag tcctagacct tctcatatca tgccgcatgc tgcatgctca 300
ctcgtggagc gttttccaga taagtgcgat cacactgtcc tatagatttt tctgcaacat 360
ggttttactt gacaatatac tatgcatatc tttatggatt agatctactt aatttattgt 420
ttgctataat acttattaca catatcatta ccattttaaa aggggctgtt ggcaaatcgc 480
tgtctgatca ccctgtgggg atgcctggga cacgtgggcc accaatggta tctgtgtggt 540
taggcattag gctgccagtt gacactgtca agctctgtgg gatacattta agtcagccat 600
tgaatacaga tttaagtgca gtcagatgac tgtctagtta atgaactcct gtagccccgt 660
gtacatccca tccagaagct gtctttgagt actgtgttct tgcatattca ggctggcctt 720
taatggctgt tgcacagcag cgagctctca gtgaggcaga cagggtctga gtgaagggcc 780
cactttagca aaggccgctg ccttcactcc aggctggctc tgaatgaaga gtgtggggtg 840
gagcttcctt gggatgaccc ggtggcctca ccccccatgg tgggcttctt gtgctcctct 900
ctgggtgtcc gctgcactcc agttcttgct tcacctgggg gagcaactgg cgggctgctc 960
tttggccctc tttgctatgg tcatcatttt ttccagacca tggttctatt ttgccttccc 1020
agcgtcaggc ctaagcatcc tgatgtggac cagccttcct gggaccactt gcttcagagg 1080
cataggggag gctggtcagg ctctgggaat gcttgtagac aggtttgttc acttcagaac 1140
ttggcagcgt ctcaattaga tggccagcaa cacagaccta tgcattgcct tctatgaatg 1200
tgccgttatg caggcagctg tgtaattgga tgggtttttt tttttttttt ttttttttaa 1260
agacatttta atgcacccac tatcttcagc aggctttgtt gtgttaagtc tcagcacatg 1320
ttggatgggt ggccatccag cggacatctc cttcctgttg tttcag 1366
<210> 5
<211> 1364
<212> DNA
<213>Artificial sequence
<400> 5
gttggtaaag tacatttcta gcatgatctc taagggcttt ttctactgga ttgtgaactc 60
tggacaaagg agggttttta gttctttgct tcttttgatg ggtcactttg ccatgagcat 120
tagtggggaa ttaggttaca ctttcctgtt atgtatttat tatccattta tatattatac 180
aaggcatgct tatttttaaa atagagtaaa atccatgcag aaagccccat ttctcaccct 240
gctgttgaca gctgggtgag tcctagacct tctcatatca tgccgcatgc tgcatgctca 300
ctcgtggagc gttttccaga taagtgcgat cacactgtcc tatagatttt tctgcaacat 360
ggttttactt gacaatatac tatgcatatc tttatggatt agatctactt aatttattgt 420
ttgctataat acttattaca catatcatta ccattttaaa aggggctgtt ggcaaatcgc 480
tgtctgatca ccctgtgggg atgcctggga cacgtgggcc accaatggta tctgtgtggt 540
taggcattag gctgccagtt gacactgtca agctctgtgg gatacattta agtcagccat 600
tgaatacaga tttaagtgca gtcagatgac tgtctagtta atgaactcct gtagccccgt 660
gtacatccca tccagaagct gtctttgagt actgtgttct tgcatattca ggctggcctt 720
taatggctgt tgcacagcag cgagctctca gtgaggcaga cagggtctga gtgaagggcc 780
cactttagca aaggccgctg ccttcactcc aggctggctc tgaatgaaga gtgtggggtg 840
gagcttcctt gggatgaccc ggtggcctca ccccccatgg tgggcttctt gtgctcctct 900
ctgggtgtcc gctgcactcc agttcttgct tcacctgggg gagcaactgg cgggctgctc 960
tttggccctc tttgctatgg tcatcatttt ttccagacca tggttctatt ttgccttccc 1020
agcgtcaggc ctaagcatcc tgatgtggac cagccttcct gggaccactt gcttcagagg 1080
cataggggag gctggtcagg ctctgggaat gcttgtagac aggtttgttc acttcagaac 1140
ttggcagcgt ctcaattaga tggccagcaa cacagaccta tgcattgcct tctatgaatg 1200
tgccgttatg caggcagctg tgtaattgga tgggtttttt tttttttttt ttttttaaag 1260
acattttaat gcacccacta tcttcagcag gctttgttgt gttaagtctc agcacatgtt 1320
ggatgggtgg ccatccagcg gacatctcct tcctgttgtt tcag 1364
Claims (9)
1. people's EZH2 mutators, it is characterised in that mutation betides the 1234-1240 positions of No. 19 intrones of EZH2 genes
Point, include the 1234th and the 1240th site；The mutation occurred include missing, insertion or point mutation but one kind of not limited to this or
It is several.
2. people EZH2 mutators according to claim 1, it is characterised in that No. 19 intragenic mutations include its
The missing of 3 thymidylic acids in 1236-1238 sites, 4 thymidylic acids in 1236-1239 sites lack
Lose, the missing and 2,1234-1235 sites thymidylic acid of 6 thymidylic acids in 1235-1240 sites
Insertion in but not limited to this one or more；Mutation intron sequences are respectively such as SEQ corresponding to this listed 4 kinds of mutation orders
ID NO:Shown in 1-4.
3. the detection method of people EZH2 mutators as claimed in claim 1 or 2, it is characterised in that key step is as follows:
（1）The capture of EZH2 genes
Sample to be tested genomic DNA is extracted, EZH2 is carried out using the liquid phase capture probe group or PCR capture primer sets of design synthesis
The capture of gene region；
（2）The two generations sequencing parsing spectrum of mutation
Will（1）In capture product carry out the sequencing of two generations, and mutational site is found out by information analysis.
4. people EZH2 mutator detection methods according to claim 3, it is characterised in that step（1）Described in liquid phase
Capture probe group or PCR capture primer sets, it is necessary to include No. 19 introne 1234-1240 sites that can capture EZH2 genes
Probe collection or primer collection；Described 1234-1240 sites include the 1234th and the 1240th site.
5. application of the people EZH2 mutators as claimed in claim 1 or 2 in chemotherapy of gastric cancer curative effect evaluation, its feature exist
It is as follows in key step:
（1）Sample collection
Gather patients with gastric cancer 2-10 sample of chemotherapy；
（2）The capture sequencing of EZH2 genes
The genomic DNA of sample to be tested is extracted, is carried out using the liquid phase capture probe group or PCR capture primer sets of design synthesis
The capture of EZH2 gene regions, capture product is subjected to the sequencing of two generations afterwards, and mutational site is found out by information analysis；
（3）Chemotherapeutic efficacy is assessed
Progression of disease according to continuous chemotherapy sample EZH2 gene specific mutations testing result prediction patient：In previous sample
EZH2 gene specific mutations are detected, latter sample does not detect same mutation or detection but the frequency of mutation reduces, then predicts the state of an illness
Progress；EZH2 gene specific mutations, latter sample detection equally mutation but frequency of mutation rise are detected in previous sample, then is predicted
Stable disease or disease amelioration；EZH2 gene specific mutations are not detected in previous sample, latter sample detection mutation, then prediction is sick
Feelings stabilization or disease amelioration；Front and rear sample standard deviation does not detect EZH2 gene specific mutations, then does not give a forecast.
6. application of the people EZH2 mutators according to claim 5 in chemotherapy of gastric cancer curative effect evaluation, it is characterised in that
Step（1）Described in chemotherapy, its interval be no more than 3 months.
7. application of the people EZH2 mutators according to claim 5 in chemotherapy of gastric cancer curative effect evaluation, it is characterised in that
Step（1）Described sample is fresh anticoagulation, and blood volume is 5-100mL.
8. people EZH2 mutators according to claim 5 detect the application in chemotherapy of gastric cancer curative effect evaluation, its feature
It is step（2）Described in the sequencing of two generations, for cancer stove sample its be sequenced depth 1000 × -10000 × between.
9. people EZH2 mutators according to claim 5 detect the application in chemotherapy of gastric cancer curative effect evaluation, its feature
It is step（3）Described in mutation, its detect frequency must be over 5%；The frequency of mutation reduces and rise, its range of decrease or
Increasing degree must be over 20%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226513A (en) * | 2020-10-29 | 2021-01-15 | 华中科技大学 | Primer and kit for detecting variable shear site mutation of EZH2 gene and application of primer and kit |
| WO2022088692A1 (en) * | 2020-10-29 | 2022-05-05 | 武汉大学 | Ezh2 alternative spliceosome and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226513A (en) * | 2020-10-29 | 2021-01-15 | 华中科技大学 | Primer and kit for detecting variable shear site mutation of EZH2 gene and application of primer and kit |
| CN112226513B (en) * | 2020-10-29 | 2022-02-01 | 华中科技大学 | Primer and kit for detecting variable shear site mutation of EZH2 gene and application of primer and kit |
| WO2022088692A1 (en) * | 2020-10-29 | 2022-05-05 | 武汉大学 | Ezh2 alternative spliceosome and application thereof |
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