CN106636415A - Detection kit for detecting MPN related gene group - Google Patents

Detection kit for detecting MPN related gene group Download PDF

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CN106636415A
CN106636415A CN201611241367.8A CN201611241367A CN106636415A CN 106636415 A CN106636415 A CN 106636415A CN 201611241367 A CN201611241367 A CN 201611241367A CN 106636415 A CN106636415 A CN 106636415A
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贾玉娇
寇坤元
覃莉
陈龙
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Tianjin Xiehe Medical Diagnostic Technology Co Ltd
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Abstract

The invention discloses a sequencing kit used for screening MPN related gene mutation and a subsequent medical interpretation database. An MPN related gene group comprises 17 genes including JAK2, CALR and the like. The sequencing kit comprises multi-PCR primers used for amplification of all exons of the 17 genes related to MPN. The kit for screening MPN gene mutation has the advantages of being high in detection efficiency, wide in mutant gene coverage, high in detection rate and the like, and a given medical interpretation report has the advantages of being accurate and authoritative.

Description

A kind of detection kit of detection MPN related gene groups
Technical field
The present invention relates to technical field of molecular biology, further to the detection reagent of examination MPN associated gene mutations Box and its medical science unscrambling data storehouse, and in particular to a kind of detection kit of detection MPN related gene groups.
Background technology
Bone marrow proliferative tumour (MPN), is disease that a class myelosis is crossed many cells and formed.Such disease and marrow Hyperplasia exception syndrome or acute myelogenous leukemia are relevant or produce, and myeloproliferative disease is having compared with other diseases More good prognosis.Bone marrow proliferative tumour is a kind of rare disease, and the people of incidence 0.02-2.8/10 ten thousand is most commonly in 60-70 year, Year mild case about 20% during diagnosis.Morbidity is without feature, and half patient is asymptomatic when diagnosing.It belongs to inertia tumour, and WHO is strict The ET life cycles close normal person of definition, 15 years survival rates 80% of ET, conversion AML or MF risks are less than 1%, PV10 within 10 years Estimated survival rate is more than 75%, and leukemic transformation and fibrosis progression are respectively smaller than 5% and 10%.Major complications are blood vessel Event (thrombosis and aberrant angiogenesis)
At present the method for detection MPN relates generally to cellular morphology, flow cyctometry, cytogenetics, molecular biology.Its The experimental technique of middle molecular biology mainly has nest-type PRC, generation sequencing etc..PCR method intuitively can not specifically be dashed forward Become sequence, and the method for generation sequencing is affected by sequencing throughput, it is impossible to disposably detect whole extrons of multiple genes Region.And two generations were sequenced as a kind of high-throughout detection method, the extron sequence of multiple genes disposably can be accurately measured Row, are possibly realized precisely treatment.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of detection reagent of detection MPN related gene groups Box, to solve prior art in the diagnostic method of MPN more limit to, lack from the angle of molecular biology and diagnose the foundation of MPN.
The invention solves the problems that another technical problem be prior art MPN diagnostic method accuracy it is relatively low.
To realize above technical purpose, the present invention is employed the following technical solutions：
A kind of detection kit of detection MPN related gene groups, the kit includes following 17 genes：JAK2, CALR, CBL, TP53, SETBP1, NRAS, SF3B1, DNMT3A, ASXL1, TET2, PTPN11, KRAS, SRSF2, KIT, NPM1, MPL, FLT3。
Above-mentioned 17 genes are made up of 28 hot spot mutations, and its mutational site is the conventional mutational site in this area, preferably Include indel mutational sites (insertion or the sequence that causes of disappearance change insertion or deletion, indel), nothing Adopted mutational site, splice region mutational site or missense mutation site, 17 gene extron subregions more preferably of the present invention Hot spot mutation it is as shown in table 1.
The hot spot mutation list of table 1
Mutator Mutational site Mutator Mutational site Mutator Mutational site
JAK2 p.V617F SETBP1 p.D868N ASXL1 p.Y591X
CALR p.L367fs SETBP1 p.G870S PTPN11 p.A72T
CALR p.K385fs NRAS p.Q61R KRAS p.A146T
CBL p.H398Q NRAS p.G12D SRSF2 p.95_103del
CBL p.F418L SF3B1 p.L747W KIT p.D816V
CBL p.R420Q SF3B1 p.N626H NPM1 p.W288fs
TP53 R248W DNMT3A p.R882H MPL p.W515K
TP53 p.Y220C DNMT3A p.R882C FLT3 ITD
TP53 p.V173M TET2 p.N1266S
TP53 p.P151S TET2 p.P1962S
The hot spot mutation of 17 gene extron subregions of the present invention can be used to make MPN auxiliary diagnosis, ability Domain MPN conventional diagnostic method has morphology, cytogenetics and flow cyctometry, and the method for the invention is preferably from molecule Biology angle makes diagnosis to MPN.
The multiple PCR primer of the present invention one group of detection of offer MPN related genes exon region of the present invention, it is described to draw The amplification scope of thing includes the exon region of 17 and MPN related genes, and it is that 250bp is left to expand the individual chip length for obtaining It is right.The multiple PCR primer provided using the present invention, with reference to two generation sequencing technologies, can detect mutator group of the present invention In whole exon regions.
Two wherein described generation sequencing reagents are reagent commonly used in the art, and gained sequence is entered as long as disclosure satisfy that The requirement of the generation of row two sequencing.The preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention more preferably also includes that the genomic DNA that will be extracted in detection object is made and is available for surveying The reagent in the library that sequence is used, this kind of reagent is reagent commonly used in the art, as long as disclosure satisfy that multiplex PCR to genome The requirement of DNA mass.The preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention preferably also includes：DNTP solution, archaeal dna polymerase, for isolated or purified core Acid is Backman magnetic beads.
Sample of the present invention is the tissue of detection object, as long as the gene of detection object can be extracted from detection sample Group DNA.The detection sample is preferably one or more in marrow, blood, solid tumor sample, it is therefore preferable to marrow Sample.
A kind of using method of kit of the present invention, it is comprised the following steps：
(1) marrow of detection object is extracted using detection kit of the present invention, genomic DNA is extracted；
(2) exon region of 17 MPN related genes described in invention in genomic DNA is entered with multiple PCR primer Row amplification；
(3) fragment obtained by amplification in step (2) is made the library for being available for Ion Torrent microarray datasets to be sequenced；
(4) gained DNA library in step (3) is sequenced, obtains lower machine data；
(5) to (4) in lower machine data carry out bioinformatic analysis, then understood using medical science unscrambling data storehouse Make diagnosis.
Above the concrete operation method of each step refers to kit specification.
Reagent and raw material used by the present invention is commercially available.
The present invention positive effect be：Can be used for examination MPN associated gene mutations using what the present invention was provided Gene group, with reference to high throughput sequencing technologies, it is possible to achieve auxiliary diagnosis are carried out to MPN outside morphology and flow cyctometry, Assessment is made especially for disease prognosis to make anticipation to the action effect for related target medicine there is special advantage.This The detection kit of the gene group for examination MPN associated gene mutations of bright offer has detection efficiency height, recall rate height etc. Advantage.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details, Will not be described in detail to belonging to known structure or function in following examples.In addition to being defined, institute in following examples Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Material and method：
In blood disease hospital of the Chinese Academy of Medical Sciences and consonance magnificent medical diagnosis center in August, 2015 in July, 2016 In the patient of diagnosis, according to following standard, we enter Line Continuity and enter group (63).
Inclusion criteria is：Jing cytomorphologies and flow cyctometry are diagnosed as the patient of bone marrow proliferative tumour (MPN).
The diagnostic result of 63 patients is as shown in table 2：
The patient's diagnostic result list of table 2
Collection 3mL Bone Marrow of Patients extraction genomic DNAs are to be measured, and the detection is through the human relations of blood disease hospital of the Chinese Academy of Medical Sciences The reason committee ratifies.
Sample process：Genome utilizes Tiangeng DNA extraction kit (article No. in sample：DP318-03) it is stripped, has Operation instructions of the body method of operating referring to kit.
Genomic DNA is expanded with multiple PCR primer, use of the Examination on experimental operation referring to Life companies kit Specification.
The structure in library：The DNA fragmentation obtained to amplification builds storehouse examination using the Ion Torrent platforms that Life companies produce Agent box carries out the structure of sequencing library, operation instructions of the Examination on experimental operation referring to Life companies kit.
High-flux sequence：The sequencing library for building is carried out into height Jing after Water-In-Oil process on Ion Torrent platforms Flux is sequenced, Water-In-Oil processing method and high-flux sequence method refer to high-flux sequence instrument Ion Torrent and it is matched somebody with somebody The operation instructions of complete equipment One Touch.
Bioinformatic analysis：First, using Ion Report softwares (v4.6, Thermo Fisher, Carlsbad, CA, USA) low-quality sequencing fragment is filtered, and qualified sequencing fragment is compared to mankind's reference gene group hg19 (http://hgdownload.cse.ucsc.edu/downloads.html), using Torrent Variant Caller (v4.6.0.7) subprogram detection mutational site, software parameters use the setting of acquiescence.This software has been optimized for Process the distinctive type of error of Ion Torrent microarray datasets.The finally mutation to finding out is used including SNP and Indel ANNOVAR(http://annovar.openbioinformatics.org/en/latest/) software annotated, in annotation Appearance includes being mutated the position in genome, the gene of association, gene extron numbering, nucleotide level variation, corresponding egg White level makes a variation, and the mutation is in dbSNP (http://www.ncbi.nlm.nih.gov/snp/), 1000Genomes databases (http://www.1000genomes.org/) and cancer mutation database COSMIC (http:// Cancer.sanger.ac.uk/cosmic the annotation in), PolyPhen (http://genetics.bwh.harvard.edu/ ) and SIFT (http pph2/://sift.jcvi.org/) function prediction result etc..Further caused a disease using the screening of principle once Mutational site：(1) genomic locations being located according to mutation and type, filter out and the prominent of impact are not produced on protein product sequence Become；(2) using 1000Genomes data, each ratio of mutation in crowd is annotated, if ratio is less than or equal to 1% not It is considered pleomorphism site；(3) cancer databases COSMIC are retrieved, whether one mutation of inquiry is on the books in COSMIC, its In occurring in hematological system tumor；(4) predict whether the mutation affects egg using protein function forecasting software PolyPhen-2 Contour painting energy；(5) if after (2) (3) (4), a mutation meets " non-polymorphism ", " crossing described in hematological system tumor " At least two in " impact protein function " this three, will be judged to may be related to disease.Finally, if a though mutation So it is unsatisfactory for (5) but is recorded as being detected in tumour in COSMIC, then is read the mutation for interrogatory.Finally If one mutation be expressly recited in the literature not with disease height correlation, be directly judged to hot spot mutation.
Statistical analysis：The ratio that each gene carries pathogenic mutation is counted, mutation rate highest gene is filtered out, is used Software is Excel.
Medical science is understood：Molecular pathology is made to the genic mutation type that bioinformatic analysis draw with medical science unscrambling data storehouse Diagnosis.Medical science unscrambling data storehouse used is as follows：
(1) being currently known JAK2exon12/14 abrupt climatic changes contributes to MPN diagnosis, and V617F mutation are found in 90-95% PV patient and 50-60% ET and PMF patients.JAK2 albumen is the non-acceptor related to cytokine receptor signal path Type tyrosine kinase, participates in the growth of cell, development, differentiation.Ruxolitinib is JAK2 inhibitor, can improve MPN patient The symptoms such as splenomegaly.(PMID：24740812)
(2) the MPN patient of ET and myelofibrosis is more common in CALR gene mutations, it is known that CALR-Exon9 abrupt climatic changes Contribute to MPN diagnosis.There are some researches show that Patient leukemic's conversion that CALR is mutated is relatively low with thrombotic possibility, thus be The preferable index of prognosis.(PMID：27376361)
(3) CBL is a tumor suppressor gene, and the gene mutation has been reported that in AML, MDS in MPN patient.Have been reported that Show the overall survival preferably (PMID of the AML patient of CBL gene mutations：23783394).
(4) TP53 gene mutations are mainly seen in the diseases in the blood system such as MDS, AML, CLL and solid tumor.TP53 genes are dashed forward The MDS survivals of change are reduced and most prognosis mala.Acting on the related medicine of TP53 and its path has Cisplatin, Fluorouracil, Docetaxel, Paclitaxel, Aspirin.(PMID：21714648)
(5) SETBP1 gene coded proteins participate in the regulation and control of DNA replication dna.SETBP1 somatic mutations turn with marrow series leukemia Change related, the prognosis mala in MDS and CMML.(PMID：23832012)
(6) NRAS gene mutations are mainly seen in the diseases in the blood system such as AML, CML, ALL, MPN and solid tumor.RAS eggs White NRAS is primarily involved in RAS signal path.In AML, if patient carries NRAS, DNMT3A and NPM1 mutation simultaneously, then carry Show prognosis bona.RAS mutation are still not enough to become the independent prognostic factor in AML.But nearest research prompting, for carrying The AML patient of RAS mutation, it is probably beneficial to carry out after treatment using the cytarabine of high dose after alleviation.(PMID： 26376137)
(7) SF3B1 gene mutations are mainly seen in CLL, MDS and MPN patients, the prognosis mala in CLL patient.In MDS In, it is that refractory anemia increases (RARS) with ringed sideroblasts that SF3B1 is mutated modal hypotype, and overall survival is carried It is high.(PMID：21995386)
(8) DNMT3A gene mutations are mainly seen in AML, especially the AML of normal karyotype, are also found in other blood diseases Disease, or normal the elderly (occurring in normal person, general load is relatively low, and independent mutation).DNMT3A is used as a table The gene of genetic correlation is seen, the Preleukemia stage is typically occurred in, its mutational load is general higher.DNMT3A gene Primary mutations Form is point mutation, is common in the amino acid of R882 positions more, and modal mutant form is R882H.DNMT3A gene mutations are suffered from The most prognosis mala of person.DNMT3A mutation patients may be benefited using the daunorubicin of high dose, and DNA methylation transferase is pressed down Preparation Decitabine has sound response.(PMID：25426837；26755712；22417203；19776405)
(9) TET2 gene mutations are mainly seen in AML patient, also exist in CML, MDS, MPN and Lymphoma, at present TET2 gene mutations are the preferable index of prognosis in CMML, and the prognosis mala in MDS/MPN, Secondary cases AML.
(10) ASXL1 gene mutations have been reported that in AML, CML, MDS in MPN patient, with AML overall patient's survival rates Reduce correlation (PMID:22417203).
(11) PTPN11 gene mutations are mainly seen in CML, AML, ALL and MPN patient.PTPN11 is protein tyrosine phosphatase Enzyme, plays an important role in cell growth, differentiation, mitotic cycle and oncogenic transformation.Dodecane- Trimethylamine can target PTPN11 (in grinding).
(12) KRAS is found in the diseases in the blood system such as MDS, AML, MPN, MM and solid tumor.RAS albumen KRAS It is primarily involved in RAS signal path.KRAS will not inducing cell morphology and transfer ability change, but can strengthen ERK, The phosphorylation of PDK1 and AKT.RAS mutation are still not enough to become the independent prognostic factor in AML.But nearest research prompting, For the AML patient for carrying RAS mutation, after treatment is carried out using the cytarabine of high dose after alleviation possibly beneficial 's.
(13) SRSF2 mutation are mainly seen in the diseases such as MDS, t-AML and CML, the MDS overall patients life of SRSF2 mutation Rate reduction is deposited, the risk to AML conversions is improved.(PMID：22389253)
(14) KIT mutation are mainly seen in AML, and KIT mutation are considered as occurring in the progressive stage of MDS, the junket of KIT gene codes Histidine kinase acceptor plays an important role in the growth of hematopoietic cell and propagation.The AML patient of KITExon17/8 mutation is mostly pre- It is bad afterwards, for caryogram is t (8；21) patient, KIT mutation prognosis is also bad.Imatinib is the targeted inhibition of KIT kinases Agent, can be used to treat the patient for carrying KIT activated mutants.(PMID：26376137)
(15) NPM1 mutation are mainly seen in AML, especially the AML of normal karyotype.NPM1 mutation mostly are the frameshit of Exon 12 Mutation, is mutated most commonly seen with A types.NPM1 is individually mutated prompting patient prognosis bona；Dash forward when other prognosis mala genes are merged (such as FLT3, DNMT3A) during change, patient's prognosis mala；Document report is mutated not for the elder patients more than 65 years old, NPM1 Reresent prognosis bona.NPM1 mutation can be additionally used in the monitoring of minimal residual disease.The patient of NPM1 mutation uses heavy dose of soft Erythromycin for treating may benefit.(PMID：26755712；22417203；26376137；25713434)
(16) MPL W515L mutation are found in the MF patient of 9% JAK2 mutation feminine genders.Platelet receptor MPL is one Proto-protein, participates in the relevant signal path of thrombocytopoiesis.MPL exon10 mutation contribute to the diagnosis of MPN.(PMID： 16834459)
(17) FLT3 gene mutations are mainly seen in AML, especially the AML of normal karyotype.FLT3 gene mutations are modal Form is that FLT3-ITD and FLT3-TKD (such as D835Y) is mutated.Prognosis mala is pointed out in FLT3 gene mutations, and can be independently of Outside caryogram.At present Sunitinib is the most frequently used targeted drugs of FLT3.Another document report, the daunorubicin of high dose for The AML patient of FLT3-ITD mutation may be beneficial.(PMID：26755712；27268085)
Using kit testing result of the present invention：
The positive rate of 17 genes is as shown in table 3 below detected by 63 patients：
3 17 gene masculine rate lists of table
Show Jing after statistics：(being summarized according to above table) as can be seen here, using two generations be sequenced method to marrow Proliferating tumor carries out auxiliary diagnosis, with diagnosis accurately, obtain the characteristics of containing much information.
Embodiments of the invention have been described in detail above, but the content is only presently preferred embodiments of the present invention, Not to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should It is included within protection scope of the present invention.

Claims (7)

1. a kind of detection kit of detection MPN related gene groups, it is characterised in that the kit includes following 17 genes： JAK2, CALR, CBL, TP53, SETBP1, NRAS, SF3B1, DNMT3A, ASXL1, TET2, PTPN11, KRAS, SRSF2, KIT, NPM1, MPL, FLT3.
2. a kind of detection kit of detection MPN related gene groups according to claim 1, it is characterised in that the detection Kit also includes the primer of 17 genes above, and the amplification scope of the primer includes whole extrons of 17 genes above Region.
3. the detection kit of a kind of detection MPN related gene groups according to claim 2, it is characterised in that described outer aobvious Subregion includes 28 hot spot mutations, 28 hot spot mutations and its corresponding relation such as following table institute with 17 genes Show：
4. a kind of detection kit of detection MPN related gene groups according to claim 3, it is characterised in that the reagent Box includes the multiple PCR primer for expanding the mutator group exon region, and for the reagent of two generations sequencing.
5. a kind of detection kit of detection MPN related gene groups according to claim 3, it is characterised in that the reagent Box include by extract in detection object genomic DNA make be available for be sequenced library reagent.
6. a kind of detection kit of detection MPN related gene groups according to claim 3, it is characterised in that the reagent Box includes dNTP solution, archaeal dna polymerase, for the reagent of isolated or purified nucleic acid, positive control or negative control.
7. a kind of detection kit of detection MPN related gene groups according to claim 3, it is characterised in that the reagent Box includes the database of gene group examining report, and the database contains the medical explanation to the hot spot mutation.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164538A (en) * 2017-07-11 2017-09-15 复旦大学附属华山医院 A kind of the internal reference amplimer composition and its amplification system of detection CALR gene mutations
CN107365841A (en) * 2017-07-13 2017-11-21 广州金域医学检验中心有限公司 For detecting pcr amplification primer thing, kit and the detection method of MPL genes W515 mutation
CN108949971A (en) * 2017-05-23 2018-12-07 曹国君 1 type abrupt climatic change of calprotectin gene closing PNA probe
CN110819710A (en) * 2018-08-10 2020-02-21 珠海铂华生物工程有限公司 High-throughput sequencing detection of myeloid tumors
CN112410407A (en) * 2020-12-04 2021-02-26 李建新 Primer group for simultaneously detecting two or three MPN pathogenic gene mutations and kit containing primer group

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