CN110484622A - A kind of chromaffin tissue oncogene Panel and its application - Google Patents
A kind of chromaffin tissue oncogene Panel and its application Download PDFInfo
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- CN110484622A CN110484622A CN201910753776.3A CN201910753776A CN110484622A CN 110484622 A CN110484622 A CN 110484622A CN 201910753776 A CN201910753776 A CN 201910753776A CN 110484622 A CN110484622 A CN 110484622A
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- 108700020796 Oncogene Proteins 0.000 title claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 12
- 101000951145 Homo sapiens Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial Proteins 0.000 claims description 5
- 102100038014 Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial Human genes 0.000 claims description 5
- 102100037249 Egl nine homolog 1 Human genes 0.000 claims description 4
- 102100029974 GTPase HRas Human genes 0.000 claims description 4
- 101000584633 Homo sapiens GTPase HRas Proteins 0.000 claims description 4
- 101000685323 Homo sapiens Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Proteins 0.000 claims description 4
- 101000874160 Homo sapiens Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Proteins 0.000 claims description 4
- 102100037248 Prolyl hydroxylase EGLN2 Human genes 0.000 claims description 4
- 102100023155 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Human genes 0.000 claims description 4
- 102100035726 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Human genes 0.000 claims description 4
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 claims description 3
- 101000881648 Homo sapiens Egl nine homolog 1 Proteins 0.000 claims description 3
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 claims description 3
- 101000971697 Homo sapiens Kinesin-like protein KIF1B Proteins 0.000 claims description 3
- 101000881650 Homo sapiens Prolyl hydroxylase EGLN2 Proteins 0.000 claims description 3
- 101000934888 Homo sapiens Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Proteins 0.000 claims description 3
- 101000637950 Homo sapiens Transmembrane protein 127 Proteins 0.000 claims description 3
- 102100021524 Kinesin-like protein KIF1B Human genes 0.000 claims description 3
- 102100031715 Succinate dehydrogenase assembly factor 2, mitochondrial Human genes 0.000 claims description 3
- 108050007461 Succinate dehydrogenase assembly factor 2, mitochondrial Proteins 0.000 claims description 3
- 102100025393 Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Human genes 0.000 claims description 3
- 102100032072 Transmembrane protein 127 Human genes 0.000 claims description 3
- 102100031634 Cold shock domain-containing protein E1 Human genes 0.000 claims description 2
- 101000940535 Homo sapiens Cold shock domain-containing protein E1 Proteins 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 101150020330 ATRX gene Proteins 0.000 claims 1
- 101000582631 Homo sapiens Menin Proteins 0.000 claims 1
- 102100030550 Menin Human genes 0.000 claims 1
- 102000056014 X-linked Nuclear Human genes 0.000 claims 1
- 108700042462 X-linked Nuclear Proteins 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 13
- 201000010099 disease Diseases 0.000 abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 6
- 238000013461 design Methods 0.000 abstract description 5
- 238000005457 optimization Methods 0.000 abstract description 5
- 108700024394 Exon Proteins 0.000 abstract description 3
- 108091093088 Amplicon Proteins 0.000 description 14
- 238000012163 sequencing technique Methods 0.000 description 9
- 230000035772 mutation Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
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- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical group [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 101710111663 Egl nine homolog 1 Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101000579425 Homo sapiens Proto-oncogene tyrosine-protein kinase receptor Ret Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- -1 MET Proteins 0.000 description 1
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- 108010085793 Neurofibromin 1 Proteins 0.000 description 1
- 101710170760 Prolyl hydroxylase EGLN2 Proteins 0.000 description 1
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- 238000011529 RT qPCR Methods 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
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- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The present invention relates to a kind of chromaffin tissue oncogene Panel and its applications, including 20 target genes.The target gene Panel that the present invention designs covers chromaffin tissue tumour overwhelming majority Disease-causing gene, and whole exons that Panel can be designed after optimization with Successful amplification have had dependable with function, can be applied to further study.
Description
Technical field
The invention belongs to chromaffin tissue tumor area, in particular to a kind of chromaffin tissue oncogene Panel and its application.
Background technique
Pheochromocytoma and Chromaffionoma (PPGLs) are on the kidney of catecholamine secretion parahormone for originate from neural ridge
Gland medullary substance tumour and the outer chromaffin tissue tumour of adrenal gland, are 0.6% adult and the direct cause of disease of 1% children hypertension.Thermophilic chromium group
It knits tumor development history and the progress of molecular diagnosis is closely related, the development of the following chromaffin tissue tumour will also be established deeper
On the basis of the molecular diagnosis and mechanism that enter.And chromaffin tissue tumour it is traditional generation sequencing means it is complicated in chromaffin tissue tumour
Clinical phenotypes and more and more Disease-causing genes in face of seem more insufficient, the appearance of high throughput sequencing technologies of new generation is solution
Certainly this problem provides opportunity.The two generation sequencing technologies that present maturation uses can be divided into three classes, and be Illumina principle respectively
(the reversible termination+laser scanning imaging of bridge-type PCR+4 color fluorescence), (the Water-In-Oil PCR+4 kind dNTP combination+inspection by turns of Roche 454
Pyrophosphoric acid hydrolysis is surveyed to shine) and Ion Torrent principle (Water-In-Oil PCR+4 kind dNTP combination+microelectrode PH detection by turns).And
Ion Torren platform has the characteristics that easy to operate, detection cycle is short, accuracy rate is high, and does not need expensive physics imaging
Equipment, be otherwise known as " two generations half " sequencing technologies, is particularly suitable for the exon sequencing of mini gene group and specific gene, flat based on this
The target gene Panel of platform has the characteristics that high throughput, convenient for customization, can be very good to meet research needs.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of chromaffin tissue oncogene Panel and its application, designs
Target gene Panel cover chromaffin tissue tumour overwhelming majority Disease-causing gene, by optimization after Panel can be with Successful amplification
Whole exons of design, have had dependable with function, can be applied to further study.
The present invention provides a kind of chromaffin tissue oncogene Panel, including following target gene: ATRX, MEN1,
CSDE1、EGLN1、EGLN2、FH、HRAS、KIF1B、MAX、MET、NF1、RET、SDHA、SDHAF2、SDHB、SDHC、SDHD、
SETD2、TMEM127、VHL。
Exon 1 and neighbour by the multipair primer amplification of Ion AmpliSeq Designer software design this 20 genes
Nearly montage area multiplies PCR amplification primer pond Pool1 and Pool2 with sub-district, synthesis is included more.
The target gene amplicon length is 265~280bp.
The present invention also provides the applications of chromaffin tissue oncogene Panel a kind of, detect chromaffin tissue tumour in preparation
Product in application.
Target gene of the present invention and corresponding transcript number:
Beneficial effect
The target gene Panel that the present invention designs covers chromaffin tissue tumour overwhelming majority Disease-causing gene, by optimization
Whole exons that Panel can be designed with Successful amplification afterwards, have had dependable with function, can be applied to further grind
Study carefully.
Detailed description of the invention
Fig. 1 is that (on) afterwards (under) sequencing result compares before optimization.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
1. library testing process is built in the sequencing of two generations
Include: peripheral blood DNA extract, build library DNA sample purifying, Qubit 3.0 detect DNA concentration, multiplexed PCR amplification,
Connecting detection Barcode, connection product, qPCR quantify library concentration, Water-In-Oil PCR, be enriched with the template positive ISPs microballon, on
Machine testing.
Wherein, multiplexed PCR amplification, using hifi enzyme, the entire primer pond segment of high-pass expanding.This Panel shares 2 and draws
Object pond is denoted as Pool1, Pool2 respectively, selects 10ul amplification system, expands stencil-chosen gDNA.
After sample-adding, vortex need to be used slightly to shake and mix sample, bubble removing is removed in centrifugation, then upper machine.
It is loaded according to reaction system:
Reagent | 10ul system |
2×primer Pool | 5μL |
5 × HiFi enzyme | 2μL |
DNA | 10ng |
Without enzyme water | Complement to 10 μ L |
Multi-PRC reaction condition:
2. selection through sanger method confirm genotype specific PPGLs patient 7, Disease-causing gene be respectively RET, VHL,
SDHB,SDHD,SDHA,MAX,FH.It is sequenced using PPGLs gene Panel, observes corresponding target gene fragment and mutation position
The amplification efficiency and recall rate of point.
3. optimizing Panel primer pond
1) separation expands inefficient (average reading < 100x) amplicon, and amplified fragments primer low in Pool1 is integrated into
Amplified fragments primer low in Pool2 is integrated into Pool4. by Pool3
2) 3.3 steps are re-started using new primer pond and multiplies PCR amplification more.A total of 4 primer Pool, remember respectively
It is 10ul amplification system for Pool1, Pool2, Pool3, Pool4 are 5ul amplification systems.
4. result
1) efficiency that original BAM file measures each gene and amplicon is sequenced by analysis.Using multiplier < 100x as standard,
The lower amplicon of amplification efficiency in further screening first edition Panel primer pond.Mainly comprising EGLN1exon1,
EGLN2exon2, RET exon1 and exon11, SDHD exon3, HRAS exon5, MAX exon1, MET exon1,
TMEM127exon2,VHL exon1.PANEL design just has two pieces of 384 orifice plates for carrying each amplicon primer, will be above right
The amplicon primer answered is chosen, and illustrates to prepare Pool3 and Pool4 primer pond (because of Pool1 and Pool2 primer according to protocol
Pond is competitive, so Pool3 Primer Source is Pool1 in new primer pond, Pool4 then corresponds to Pool2).Pool1 primer pond
Total 6 amplicon amplification efficiencies are low (being shown in Table 1), and Pool2 primer pond amounts to 8 amplicon low efficiencys (being shown in Table 2).
The low amplification efficiency amplicon of 1 Pool1 of table and exon
The low amplification efficiency amplicon of 2 Pool2 of table
2) optimize Panel result
Expand sample again using new primer pond, by analysis original document find each amplicon it is equal > 100x, it is average
235.9.Amplicon reads multiplier > 100x amplicon accounting 94.53%, and (preceding 92.17%) of optimization, amplicon overall length expand ratio
90.31% (optimizes preceding 54.29%) amplification homogeneity 97.81% and (optimizes preceding 95.06%), index meets germline gene screening and grinds
Study carefully needs.Other information is shown in Fig. 1.
3) two generations sequencing positive rate verifying
7 patient's two generations sequencing results are consistent with generation result (being shown in Table 3), mutation type cover point mutation, frameshift mutation,
Montage region mutation.For mutational site reading more than 100x, Quality Control effect is ideal, is conducive to mutational site detection and result relatively can
It leans on.
Patient known to 37 germline mutations of table targets Panel sequencing result and Quality Control
The present invention had chosen from indispensable gene FH, MAX, NF1, RET, SDHA, SDHB, SDHC, SDHD, TMEM127,
The genes such as VHL have chosen the genes such as EGLN1/PHD2, KIF1B, MET, SDHAF2 from extending in alternative gene;In addition it is also included in
It is some only in tumor susceptibility gene ATRX, EGLN2/PHD1, HRAS, SETD2 etc. of somatic mutation report.By screening these bases
Cause, to which in studies and clinical application, the patients for detecting to carry mutation as far as possible provide timely hereditary more for patient
Consulting and prognosis management.
Claims (3)
1. a kind of chromaffin tissue oncogene Panel, it is characterised in that: including following target gene: ATRX, MEN1, CSDE1,
EGLN1、EGLN2、FH、HRAS、KIF1B、MAX、MET、NF1、RET、SDHA、SDHAF2、SDHB、SDHC、SDHD、SETD2、
TMEM127、VHL。
2. a kind of chromaffin tissue oncogene Panel according to claim 1, it is characterised in that: the target gene expands
Increasing sub- length is 265~280bp.
3. a kind of application of chromaffin tissue oncogene Panel as described in claim 1, it is characterised in that: detected in preparation
Application in the product of chromaffin tissue tumour.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116516009A (en) * | 2023-05-06 | 2023-08-01 | 中国医学科学院北京协和医院 | Amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108588224A (en) * | 2018-06-14 | 2018-09-28 | 大连医科大学附属第医院 | A kind of biomarker and its application for pheochromocytoma/Chromaffionoma early diagnosis and preoperative evaluation |
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2019
- 2019-08-15 CN CN201910753776.3A patent/CN110484622A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108588224A (en) * | 2018-06-14 | 2018-09-28 | 大连医科大学附属第医院 | A kind of biomarker and its application for pheochromocytoma/Chromaffionoma early diagnosis and preoperative evaluation |
Non-Patent Citations (4)
Title |
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JOAKIM CRONA等: "New Perspectives on Pheochromocytoma and Paraganglioma: Toward a Molecular Classification", 《ENDOCRINE REVIEWS》 * |
NORIKO KIMURA等: "Risk Stratification on Pheochromocytoma and Paraganglioma from Laboratory and Clinical Medicine", 《JOURNAL OF CLINICAL MEDICINE》 * |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116516009A (en) * | 2023-05-06 | 2023-08-01 | 中国医学科学院北京协和医院 | Amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof |
CN116516009B (en) * | 2023-05-06 | 2023-10-13 | 中国医学科学院北京协和医院 | Amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof |
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