CN110484622A - A kind of chromaffin tissue oncogene Panel and its application - Google Patents

A kind of chromaffin tissue oncogene Panel and its application Download PDF

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Publication number
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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Prior art keywords
panel
chromaffin tissue
application
chromaffin
oncogene
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CN201910753776.3A
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Inventor
宁光
王卫庆
叶蕾
韩如来
谢晓雁
赵萸
宋蔚
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SHANGHAI INSTITUTE OF ENDOCRINE AND METABOLIC DISEASES
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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SHANGHAI INSTITUTE OF ENDOCRINE AND METABOLIC DISEASES
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Priority to CN201910753776.3A priority Critical patent/CN110484622A/en
Publication of CN110484622A publication Critical patent/CN110484622A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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

A kind of chromaffin tissue oncogene Panel and its application
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.
CN201910753776.3A 2019-08-15 2019-08-15 A kind of chromaffin tissue oncogene Panel and its application Pending CN110484622A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Title
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》 *
张富勋等: "嗜铬细胞瘤与副神经节瘤基因组学新进展", 《中国肿瘤临床》 *
谢晓雁等: "嗜铬细胞瘤7个致病基因的高通量基因检测方法的建立", 《上海交通大学学报(医学版)》 *

Cited By (2)

* Cited by examiner, † Cited by third party
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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Application publication date: 20191122