CN107164496A - The gene polymorphism sites related to thyroid cancer and its application - Google Patents
The gene polymorphism sites related to thyroid cancer and its application Download PDFInfo
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Abstract
The invention provides the somatic mutation site of thyroid cancer morbidity Disease-causing gene and its application, specifically the invention provides the mutational site of one group of thyroid cancer morbidity Disease-causing gene, including GNAS gene mutation sites, NRAS gene mutation sites, TSHR gene mutation sites etc., the mark that the gene mutation site that the present invention is provided can be pernicious as Benign Thyroid Nodules are differentiated.
Description
Technical field
The invention belongs to biomedicine field, specifically, the present invention relates to the genosome related to thyroid nodule is thin
Born of the same parents' specific mutation sites and its application.
Background technology
Thyroid nodule is a kind of clinical most common thyroid disease, palpation can with discovery thyroid nodule
Illness rate is about 4-7% in US adult crowd.And apply sensitive thyroid gland B to check, and more than 65 years old in crowd, first
The illness rate of shape gland tubercle is up to more than 50%.China is taken the lead for 2010 by endocrine association of Chinese Medical Association professor Teng Weiping
The result of study of the thyroid disease epidemiology survey of the resident of national ten city 15,181 of progress is found, in population of China
The illness rate of thyroid nodule is up to 18.6%, therefore speculates that the patient of the thyroid nodule of our countries is up to 200,000,000 people.Greatly
Most thyroid nodules are benign, can take expectant treatment, but the thyroid nodule for having 5-10% be it is pernicious, it is necessary to
Operation in early stage, could obtain good prognosis.Therefore, the doctor of a thyroid gland training, the huge challenge faced is exactly
How to differentiate patient thyroid nodule it is benign and malignant, only in this way, patient is obtained rational treatment in time.
If the substantial amounts of human thyroid benign nodules that need not be performed the operation, operative treatment is inadequately given, so big crowd is ill
Rate, it will produce huge medical insurance burden；, whereas if can not recognize that identification is really pernicious from substantial amounts of benign protuberance
Tumour, will delay the treatment to these patients, jeopardizes the life and health of patient.
At present clinically the means of the conventional pernicious discriminating of Benign Thyroid Nodules mainly have thyroid gland B ultrasound, radioisotope scanning and
Thyroid nodule FNA pathological biopsy (Fine-needle Aspiration Biopsy, FNAB).Wherein, thyroid gland is thin
Pin puncture cytology biopsy is the goldstandard of current Benign Thyroid Nodules differential diagnosis of malignant.Studied according to National Cancer
The pathological special meeting of fine-needle aspiration of thyroid nodules of 2008 guide, thyroid nodule Puncture cytology is examined at present
It is disconnected to be divided into following a few classes：1. because puncturing thyroid cell lazy weight, it is impossible to diagnose；2. benign protuberance；3. pathocytology without
The tubercle that method is made a definite diagnosis；4. the pernicious class of thyroid nodule four.In recent years, as B ultrasound guides extensively should for lower progress FNA
With that is not enough because puncturing the cell quantity obtained, it is impossible to which the ratio of the patient of diagnosis is significantly reduced, and is up to from former
15%, drop to current less than 7%.Obtained in FNA after enough thyroid follicular cells, most thyroid gland knot
Good pernicious equal can be obtained by the diagnostic method of cell pathology of section correctly diagnoses.Even if best in the world at present
The laboratory of thyroid cell pathological diagnosis, also has 20-40% to puncture successful thyroid nodule and not can determine that its is good pernicious,
The tubercle that i.e. pathology can not be made a definite diagnosis.Therefore, when patient carries out thyroid needle pathological biopsy, because of puncture failure and pathology
It can not judge good pernicious so as to the ratio up to 30-50% that can not be correctly diagnosed to patient, by the thyropathy at each center
The technical merit influence for managing diagnostician and puncture doctor is larger.
The content of the invention
It is an object of the invention to provide a kind of Disease-causing gene mutational site of thyroid cancer morbidity and its application.
The first aspect of the present invention there is provided be selected from the group one or more of (I) gene mutation site and/or its
The purposes of detection reagent, for reagent preparation or kit, the reagent or kit are used for the good evil for differentiating thyroid nodule
Property, described group (I) includes following gene mutation site：
GNAS genes：
NM_001077490:exon1:c.T1019C；
NRAS genes：
NM_002524:exon3:c.T284C；
TSHR genes：
NM_000369:exon10:c.A2098G。
In another preference, described group (I) also includes following gene mutation site：
CHEK2 genes：
NM_145862:exon11:c.A1250G。
In another preference, described group (I) also includes following gene mutation site：
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C.
In another preference, described group (I) also includes following gene mutation site：
GNAS genes：
NM_016592:exon1:c.C205A、NM_016592:exon1:c.C216T。
In another preference, described group (I) also includes following gene mutation site：
TSHR genes：
NM_000369:exon10:c.A2252G。
In another preference, described group (I) also includes following gene mutation site：
BRAF gene：
NM_004333:exon15:c.T1799A。
In another preference, detected object includes：People or non-human mammal (such as domestic animal, poultry, experimental animal
Deng).
In another preference, described reagent includes primer, probe, chip or antibody.
In another preference, described kit contains one or more reagents being selected from the group：
(A) it is used for the specific primer of genetic test；
(B) it is used for the specific probe of genetic test；
(C) it is used for the chip of genetic test；
(D) it is used for the specific antibody for detecting the amino acid mutation corresponding to the gene of mutation.
In another preference, the reagent or kit are detected for real-time fluorescence quantitative PCR.
In another preference, in the real-time fluorescence quantitative PCR, annealing temperature is between 60-67 DEG C, PCR amplifications produces
Thing length is 80-300bp.
In another preference, in the real-time fluorescence quantitative PCR, fluorescence probe annealing temperature is between 60-70 DEG C.
In another preference, double ends of the probe carry out the modification of chemical group, and 5' is terminal modified fluorescence excitation
Group, 3 terminal modified have fluorescent quenching group.
It is described to be detected as complementary detection in another preference.
The second aspect of the present invention is there is provided a kind of kit, and the kit is one or more including what is be selected from the group
The detection reagent of gene mutation site：
GNAS genes：
NM_001077490:exon1:c.T1019C；
NRAS genes：
NM_002524:exon3:c.T284C；
TSHR genes：
NM_000369:exon10:c.A2098G。
In another preference, the kit also includes the detection reagent of following gene mutation site：
CHEK2 genes：
NM_145862:exon11:c.A1250G。
In another preference, the kit also includes the detection reagent of following gene mutation site：
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C.
In another preference, the kit also includes the inspection for the one or more gene mutation sites being selected from the group
Test agent：
GNAS genes：
NM_016592:exon1:c.C205A、NM_016592:exon1:c.C216T。
In another preference, the kit also includes the detection reagent of following gene mutation site：
TSHR genes：
NM_000369:exon10:c.A2252G。
In another preference, the kit also includes the inspection for the one or more gene mutation sites being selected from the group
Test agent：
BRAF gene：
NM_004333:exon15:c.T1799A、NM_004333:exon11:c.G1338A、
NM_004333:exon15:c.A1801G；
CHEK2 genes：
NM_145862:exon10:c.C1024T；
NRAS genes：
NM_002524:exon3:c.C181A、NM_002524:exon3:c.A182G；
AKT1 genes：
NM_001014431:exon3:c.G49A；
PPM1D genes：
NM_003620:exon1:c.C262T；
PTEN genes：
NM_000314:exon5:c.C328T；
RET genes：
NM_020630:exon11:c.T1888C、NM_020630:exon16:c.T2753C；
TP53 genes：
NM_001126115:exon3:c.C265T、NM_000546:exon8:c.G814T。
In another preference, the kit also includes the detection examination for the one or more fusions being selected from the group
Agent：
ETV6-NTRK3 fusions：
ETV6{ENST00000396373}:r.1_737_NTRK3{ENST00000394480}:r.1719_19984；
NCOA4-RET fusions：
NCOA4{ENST00000452682}:r.1_1014_RET{ENST00000355710}:r.2369_5659；
CCDC6-RET fusions：
CCDC6{ENST00000263102}:r.1_535_RET{ENST00000355710}:r.2369_5659。
In another preference, described detection reagent is：
(A) it is used for the specific primer of genetic test；
(B) it is used for the specific probe of genetic test；
(C) it is used for the chip of genetic test；
(D) it is used for the specific antibody for detecting the amino acid mutation corresponding to the gene of mutation.
There is provided a kind of method that vitro detection sample whether there is gene mutation, including step for the third aspect of the present invention
Suddenly：
(a) with the polynucleotides of primer amplified sample, amplified production is obtained；With
(b) it whether there is following one or more gene mutations in detection amplified production：
GNAS genes：
NM_001077490:exon1:c.T1019C、NM_016592:exon1:c.C205A、
NM_016592:exon1:c.C216T；
NRAS genes：
NM_002524:exon3:c.T284C；
TSHR genes：
NM_000369:exon10:c.A2098G、NM_000369:exon10:c.A2252G。
In another preference, the nondiagnostic that is detected as.
Also include dashing forward with the presence or absence of following gene in detection amplified production in another preference, in the step (b)
Become：
CHEK2 genes：
NM_145862:exon11:c.A1250G。
Also include dashing forward with the presence or absence of following gene in detection amplified production in another preference, in the step (b)
Become：
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C.
If with one or more gene locis, it is pernicious to illustrate the thyroid nodule.
In another preference, the sample comes from people.
There is provided a kind of gene polynucleotides sequence of separation, described polynucleotide sequence for the fifth aspect of the present invention
For the fragment derived from the gene being selected from the group：NRAS genes, GNAS genes, PIK3CA genes, CHEK2 genes and TSHR bases
Cause, and described nucleotide sequence has the gene mutation site being selected from the group respectively：
NRAS genes：
NM_002524:exon3:c.T284C；
GNAS genes：
NM_001077490:exon1:c.T1019C、NM_016592:exon1:c.C205A、
NM_016592:exon1:c.C216T；
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C；
CHEK2 genes：
NM_145862:exon11:c.A1250G；
TSHR genes：
NM_000369:exon10:c.A2098G、NM_000369:exon10:c.A2252G。
In another preference, the polynucleotide sequence length is 30-1000bp；Preferably 50-500bp.
Present invention also offers the purposes of polynucleotide sequence, described sequence can be used as positive control (standard items).
There is provided the one or more or full gene being selected from the group and/or its detection reagent for the sixth aspect of the present invention
Purposes, for reagent preparation or kit, the reagent or kit be used for differentiate the good pernicious of thyroid nodule：
AKT1 genes, BRAF gene, CHEK2 genes, GNAS genes, NRAS genes, PIK3CA genes, PPM1D genes,
PTEN genes, RET genes, TP53 genes, TSHR genes, ETV6-NTRK3 fusions, NCOA4-RET fusions and
CCDC6-RET fusions.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited,
No longer tire out one by one herein and state.
Embodiment
The present inventor's in-depth study by extensive, unexpectedly obtains the mutation of one group and thyroid cancer Disease-causing gene
Site, test result indicates that, the mark that the gene mutation site that the present invention is provided can be pernicious as Benign Thyroid Nodules are differentiated
Thing.
Before describing the present invention, it should be understood that the invention is not restricted to described specific method and experiment condition, because this
Class method and condition can change.It should also be understood that its purpose of term used herein is only that description specific embodiment, and
And it is not intended to be restricted, the scope of the present invention will be limited only by the claims which follow.
Although can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention
And material, herein place enumerate preferred method and material.
In the art, how these thyroid nodules correctly to be diagnosed in the preoperative, so as to give patient one conjunction
Reason is timely treated, and is always the focus of thyroid gland research field.With the progress of Protocols in Molecular Biology, molecule diagnosis exists
In the pernicious antidiastole of Benign Thyroid Nodules, gradually paid close attention to by people.The theory of the good pernicious molecular diagnosis of tubercle
Basis is the Disease-causing gene based on some thyroid cancers, and specific body cell only occurs in the cancerous tissue of cancer patient and dashes forward
Change or the appearance of fusion, this mutation or new fusion are in the normal cell of patient and the first of Non-thyrogenous cancer patient
It is non-existent in shape glandular tissue.Therefore, these molecular markers, in the malignant and benign lesion antidiastole of thyroid cancer, have
It is very special.As it is found that the thyroid gland knot that the Val600Glu of the 600th amino acids is mutated on BRAF gene
The patient of section 100% is Malignant Nodules.Although these gene mutations have very good in Benign Thyroid Nodules differential diagnosis of malignant
Specificity, but the frequency of mutation of each Disease-causing gene in thyroid cancer patient is not high, therefore, is depended merely on so to list
The molecular mutation of individual gene detected, positive patients have an important diagnostic significance, but patients with negative can fail to pinpoint a disease in diagnosis it is many really
Thyroid cancer patients, delay the treatment of patient.Therefore to solve thyroid cancer molecule diagnoses this predicament, it is necessary to causing first
Multiple genes of shape gland cancer carry out abrupt climatic change simultaneously, can improve the specificity of Benign Thyroid Nodules differential diagnosis of malignant,
Reduce false negative rate.
But, want in thyroid nodule sample, while the body for carrying out multiple thyroid cancer specific pathogenetic genes is thin
Cytoplasmic process becomes the detection with new fusion, it is necessary to break through the Science and Technology difficult point of the following aspects, can be only achieved clinic
The purpose of application：
First：Although the Disease-causing gene mutation of the substantial amounts of thyroid cancer of document report, in the mutator of these reports
In, which is real Disease-causing gene, and which is not causing a disease for thyroid cancer, and these problems are unclear.Secondly, in mesh
In the thyroid cancer Disease-causing gene of preceding report, the combination of which gene is high in the positives rate of thyroid nodule antidiastole, and cloudy
The possibility that property is failed to pinpoint a disease in diagnosis is small, is not know at present.The combination of such a group thyroid cancer Disease-causing gene is found, will be in first shape
It is very important in gland tubercle malignant and benign lesion.
Second：Because the variation (mutation and fusion) of the Disease-causing gene of thyroid cancer is in thyroid carcinoma cell
Interior somatic mutation, it is therefore necessary to which obtaining thyroid nodule cell by FNA could be diagnosed, and FNA
Cell quantity seldom (general cell number 10-200), want in so few cell, carry out lots of genes mutation and
The detection of fusion, is impossible with traditional generation sequencing.The new detection technique ability that must be used, completes combination
The foundation of the molecular diagnosis method of type mark.
3rd：Because genetic mutation mainly includes two major classes in thyroid cancer, a class is gene somatic mutation；It is another
Class is that new fusion occur in tumour cell.This two classes variation method of traditional detection, respectively rely on sample DNA and
mRNA.Need to gather the thyroid nodule tissue of most enough amounts twice.This performs difficulty in clinical routine diagnostics, therefore how
It can utilize with once a small amount of biopsy sample, carry out the detection of these a large amount of genetic mutations, be that combined molecular marker moves towards real
The bottleneck of border application.
Therefore, the bottleneck that the present invention is diagnosed primarily directed to above-mentioned 3 thyroid nodule molecules mainly has come what is carried out
There is following originality:
First, Disease-causing gene, the 8 different fusions for 19 thyroid papillary carcinomas that selection has now been found that, with
And the RET genes in undifferentiated carcinoma and differencing cancer in common TP53, CTNNB1 gene and medullary carcinoma of thyroid gland, it is used as molecule
Mark, is expanded by the targeting of the cDNA to these variation sections, with reference to two generation sequencing technologies, in thyroid cancer and thyroid gland
In knurl or normal thyroid tissue, gene mutation and fusion detection are carried out, this group's mould assembly molecular marker is found,
Total positive rate is up to 81% in Chinese thyroid cancer crowd, is the molecular marker that a group has fine diagnostic value.Wherein
The combination of 11 core genes of most common mutation fusion different with 8, can make the positive rate of diagnosis be up to 93%
More than.
Secondly, the target gene multiplexed PCR amplification chip technology based on Fludigam companies is developed a set of specific
The technology of the multiple target genes amplification of combination, is solved in trace sample, while carrying out the side of the targeting amplification of multiple genes
Method；Simultaneously to multiple PCR primer, by two generation sequencing technologies, so that in each clear and definite sample, the specific base of each gene
The mutation of cause or new fusion whether there is.Solve in trace sample, while the skill of the detection to multiple genetic mutations
Art bottleneck.
In addition, present invention selection mRNA is combined the gene of type molecular marker as the object for carrying out target gene amplification
Make a variation the sample detected, realizes and gene mutation is detected in same sample, and fusion is detected
Purpose, is that the invention is possibly realized in clinical practice.Meanwhile, the detection of genetic mutation is carried out by template of mRNA, having to comment
Valency punctures the quality of sample, and the reliability to diagnosis carries out objective appraisal.
It can apply the combination of different kinds of molecules mark is pernicious to Benign Thyroid Nodules to differentiate, such as apply BRAF
V600E, RAS and RET/PTC1, RET/PTC3, and PAX8/PPAR fusions etc. can substantially increase as molecular marker
Plus the ability of Benign Thyroid Nodules differential diagnosis of malignant.This associativity molecular marker is used for the pernicious discriminating of Benign Thyroid Nodules
The sensitiveness of diagnosis can reach 62%, and specificity can reach 99.7%；Use in conjunction such as is diagnosed with pathocytology, can
So that the sensitiveness of the pernicious discriminating of Benign Thyroid Nodules, 80% is brought up to from the 44% of the diagnosis of alone pathocytology.And cause
The not high main cause of this molecular marker diagnostic sensitivity, is that there is presently no find all thyroid pathogenic bases
Cause, the Disease-causing gene of above-mentioned Common Thyroid cancer, in thyroid cancer patients, the mutually exclusive frequency of mutation between different genes
Mostly 70% or so, therefore, with the combination in the focus site of Disease-causing gene common at present, as molecular marker, carry out
The pernicious antidiastole of Benign Thyroid Nodules has larger limitation.
Want to improve the sensitiveness that molecular marker is diagnosed, first have to find thyroid cancer pathogenic gene or molecule mark
Will.According to american cancer research institute thyroid cell diagnostic criteria in 2008, thyroid cancer is divided into papillary carcinoma at present
It is (filter blocking cancer point is classified as one kind of variability papillary carcinoma in this kind of histological type at present), differencing cancer, undifferentiated
Four kinds of cancer and medullary carcinoma of thyroid gland, most common of which is papillary thyroid cell cancer, accounts for the 95% of all thyroid cancers
More than.In different thyroid cancers, pathogenic gene may be different, and same Disease-causing gene is in different types of thyroid gland
The frequency of mutation in cancer is also different.Mutator as common in thyroid papillary carcinoma is BRAF gene, and differencing and not
Break up in cancer, TP53 genes are the mutators of high frequency, more than 50% patient carries TP53 genes and dashed forward in undifferentiated carcinoma
Become, but in thyroid papillary carcinoma, seldom find the mutation for there are P53 genes.And RET genes in medullary carcinoma of thyroid gland patient
Mutation be very common, such as in familial cephaloma, the mutation rate of the gene is up to 95%, in sporadic thyroid gland
In cephaloma, the mutation rate of RET genes also has 40-50%.A large sample thyroid gland nipple being published on Cell for 2014
The research of the full-length genome extron sequencing of shape cancer, the pernicious mirror of Benign Thyroid Nodules is carried out for application associativity molecular marker
Infusive prospect Zhen Duan not provided.They are surveyed by the full-length genome extron to 496 thyroid papillary carcinomas
Sequence, finds the Disease-causing gene of 5 thyroid cancers, including 3 thyroid cancer Disease-causing genes that may be new；And it is found that 8
Gene and various types of fusions of other different genes formation make a variation.In these thyroid cancers, at least provided with this 5
The sample of more than one gene mutation accounts for 73.6% in individual Disease-causing gene, finds to become with Disease-causing gene mutation or fusion
The sample that more than one in different makes a variation accounts for 89.8%.In also 10% or so thyroid cancer, both without this 5 Disease-causing genes
Point mutation, also do not find fusion variation.They are then by technologies such as SNP chips, in these tumor samples
The amplification deletion mutation of chromosome is analyzed, and finds large fragment amplification and missing (the Arm-level dye of chromosome segment
Colour solid makes a variation) it is very common in thyroid cancer sample, wherein 22q chromosome deficiency is about in 14.4% thyroid cancer patient
Occur, and the amplification of the large fragment of 1q chromosomes accounts for 14.8%.If by the amplification of these chromosomes arm-level levels and lacked
Variation is lost, as molecular marker, the chromosome amplification with Disease-causing gene mutation, fusion change XOR arm-level will be made
Lacking the patient of at least one of three classes variation variation increases to 96.6%.This shows, if we can be different with this three class
Variation, to thyroid nodule carry out molecule diagnosis if, it would be possible to be diagnosis sensitiveness reach more than 95%, this will be
Most reliable diagnostic method in all means of current Benign Thyroid Nodules differential diagnosis of malignant.
The multiplexed PCR amplification of new Protocols in Molecular Biology target gene combines the appearance being sequenced in two generations so that utilize first shape
The a small amount of RNA extracted in gland puncture cell is template, while detecting that several genes variation is possibly realized, so that associativity point
The detection of son mark becomes simple, easy operation, while being unlikely to missing inspection gene mutation that may be present again.Fluidigm is public
The targeting amplification chip of department, which can expand to various mutations gene target and carry out the sequencing of two generations, builds storehouse, and chip piece can be simultaneously right
450 to 1500 pairs of primers of 48 samples enter performing PCR amplification, meet the demand of thyroid cancer these molecular markers diagnosis.
Therefore, the present invention uses the target gene multiplexed PCR amplification chip technology of Fluidigm companies, and selection has now been found that
It is common in the Disease-causing gene of 19 thyroid papillary carcinomas, 8 different fusions, and undifferentiated carcinoma and differencing cancer
RET genes in TP53, CTNNB1 gene and medullary carcinoma of thyroid gland, as molecular marker, by these variation sections
CDNA targeting amplification, with reference to two generation sequencing technologies, whether there is the variation of these molecular markers in identification thyroid nodule,
So as to carry out the pernicious antidiastole of Benign Thyroid Nodules.The related base of a collection of new pernicious thyroid nodule is finally found that
Because of pleomorphism site, specifying information is as shown in table 1.
Table 1
| Gene Name | Mutational site |
| BRAF | NM_004333:exon15:c.T1799A |
| CHEK2 | NM_145862:exon11:c.A1250G:p.N417S |
| GNAS | NM_001077490:exon1:c.T1019C:p.L340P |
| GNAS | NM_016592:exon1:c.C205A:p.H69N |
| GNAS | NM_016592:exon1:c.C216T:p.G72G |
| NRAS | NM_002524:exon3:c.T284C:p.L95P |
| PIK3CA | NM_006218:exon12:c.1818delC:p.Y606fs |
| TSHR | NM_000369:exon10:c.A2098G:p.K700E |
| TSHR | NM_000369:exon10:c.A2252G:p.K751R |
Gene order numbering is with reference to GRCh37/hg19 versions in table 1.
BRAF gene
The albumen of BRAF gene coding belongs to serine/threonine kinase raf families.Played in MAPK/ERKs paths
Adjustment effect, participates in cell division, differentiation and secretion etc..The generation that BRAF gene mutation is considered as with kinds cancer is relevant, bag
Include NHL, colorectal cancer, malignant mela noma, thyroid cancer and non-small cell lung cancer.In addition, research shows
BRAF gene mutation is relevant with heart face skin syndrome.
The sequence fragment of 1799th BRAF gene undergone mutation (NM_004333) is as follows：
CCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGATGGAG
TGGGTCCCATCAGTTTGAACAGTTGT(SEQ ID NO.1)
One in the present invention is preferably carried out being used for the primer pair for detecting 1799 mutational sites of BRAF gene in mode
It is as follows：
| BRAF-E15-F | GGAGCCTTGTATATAGACGG | SEQ ID NO.2 |
| BRAF-E15-R | TGTATGTTCTAACAGGCACC | SEQ ID NO.3 |
NRAS genes
NRAS genes are oncogene, and its memebrane protein encoded can shuttle between golgiosome and cell membrane.NRAS eggs
It is white that there is inherent GTP enzymatic activitys, it can be activated and pressed down by GTP enzyme activation albumen by guanine nucleotide exchange factor respectively
System.Point mutation on NRAS is considered as and the body cell carcinoma of the rectum, follicular thyroid cancer, LADA lymphocytic hyperplasia
The relevant of generation of syndrome, Noonan syndromes and leukaemia
284th sequence fragment of NRAS genes (NM_002524) undergone mutation is as follows：
ATAGCAAGTCATTTGCGGATATTAACCTC[T/C]ACAGGGAGCAGATTAAGCGAGTA AAAGACT(SEQ
ID NO.4)
One in the present invention is preferably carried out being used for the primer pair for detecting 284 mutational sites of NRAS genes in mode
It is as follows：
| NRAS-E3-F | CAAGAAACCATATGCTCACC | SEQ ID NO.5 |
| NRAS-E3-R | TTGGATTGTGTCCGTTGAGC | SEQ ID NO.6 |
GNAS genes
GNAS is a protein coding gene, related signal path for g protein coupled receptor (GPCR) signal path and
Polypeptide ligand bind receptor signal path, participates in the activation and various kinds of cell reaction of adenyl cyclase.The gene has a variety of turns
Record version.GNAS gene mutations and pseudohypoparathyroidism, albright syndrome disease, McCune-
Albright syndromes, progressivity osteodysplasty, polyostotic fibrous dysplasia and some pituitary tumors are related.
1019th sequence of GNAS genes (NM_001077490) undergone mutation is as follows：
GCCCAACAGCGCCGGAGCTTCCTTAACGCCCAC[C/A]ACCGCTCCGGCGCCCAGGT
ATTCCCTGAGTCCCCCG(SEQ ID NO.7)
One in the present invention is preferably carried out being used for the primer pair for detecting 1019 mutational sites of GNAS genes in mode
It is as follows：
| GNAS-E1-1-F | AGAGGCCGCCACCGTGTTAT | SEQ ID NO.8 |
| GNAS-E1-1-R | AGGCCTCGCCATCATTCTC | SEQ ID NO.9 |
TSHR genes
The albumen of TSHR gene codes be memebrane protein, be primarily involved in the metabolism of thyroid cell, be thyroid-stimulating hormone by
Body, can be activated by adenyl cyclase.The disease related to TSHR has congenital hypothyroidism and non-self immune
Property hyperthyroidism.
2252nd sequence fragment of TSHR genes (NM_000369) undergone mutation is as follows：
TGAACTGATTGAAAACTCCCATCTAACCCCAAAGA[A/G]GCAAGGCCAAATCTCAG
AAGAGTATATGCAAACGGTTTTGTAAG(SEQ ID NO.10)
One in the present invention is preferably carried out being used for the primer pair for detecting 2252 mutational sites of TSHR genes in mode
It is as follows：
| TSHR-E10-F | AGGCAACTATGTTGAGCGTC | SEQ ID NO.11 |
| TSHR-E10-R | TATGCCATCACCTTCGCCAT | SEQ ID NO.12 |
CHEK2 genes
Cell cycle regulation process plays vital effect to DNA damage and in replicating in the cell cycle.
The albumen of CHEK2 gene codes is the cell cycle to check adjuster, it is considered to be tumor suppressor gene.This gene mutation and Li-
Fraumeni syndromes, a kind of cancerous phenotype of the high aggregation of the familial of carrying TP53 mutation.In addition, carrying this gene
Mutation has generation sarcoma, breast cancer and brain tumor tendency.
1250th sequence fragment of CHEK2 genes (NM_145862) undergone mutation is as follows：
GAAGGATCAGATCACCAGTGGAAAATACA[A/G]CTTCATTCCTGAAGTCTGGGCAG
AAGTCTCAGAGAAAG(SEQ ID NO.13)
One in the present invention is preferably carried out being used for the primer for detecting 1250 mutational sites of CHEK2 genes in mode
To as follows：
| CHEK2-E11-F | TCCCACCACAGCACATACAC | SEQ ID NO.14 |
| CHEK2-E11-R | CTTTTCCTTTCTCTCTCTACC | SEQ ID NO.15 |
PIK3CA genes
PIK3CA genes are oncogene, belong to PI3Ks families, are responsible for coordinating a variety of cell functions including giving birth to
Deposit and breed, the morbidity of the tumour such as its gene mutation and cervical carcinoma is relevant.
1818th sequence of PIK3CA genes (NM_006218) undergone mutation is as follows：
CTGAACAGGCTATGGAACTTCTGGACTGTAATTA[C/-]CCAGATCCTATGGTTCGAG
GTTTTGCTGTTCGGTG(SEQ ID NO.16)
One in the present invention is preferably carried out being used for the primer for detecting 1818 mutational sites of PIK3CA genes in mode
To as follows：
| PIK3CA-E12-F | CACAAACTAGAGTCACACAC | SEQ ID NO.17 |
| PIK3CA-E12-R | GCACGATTCTTTTAGATCTG | SEQ ID NO.18 |
Main advantages of the present invention are：
(1) disclose first one group can as the gene polymorphism sites for differentiating the pernicious mark of Benign Thyroid Nodules,
It is specific high；
(2) combined, had for Chinese thyroid cancer crowd according to the molecular marker (gene loci of mutation) of the present invention
Fine diagnostic value, can make the positive rate of diagnosis be up to more than 90%.
(3) collect clinical patient sample when, only need to collect thyroid nodule puncture cell, extracting RNA as template,
Gene mutation and fusion can be detected simultaneously, and Clinical feasibility is preferable.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.The experimental method of unreceipted detailed conditions in the following example, generally according to routine
Condition such as U.S. Sambrook.J etc. writes《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002
Year) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number press weight
Amount is calculated.Experiment material and reagent used can be obtained from commercially available channel unless otherwise instructed in following examples.
Method
The present invention provides a kind of detection method for the generation of target gene two sequencing for differentiating that Benign Thyroid Nodules are pernicious, and root
The suitable art formula of patient and management after operation are determined according to the molecule diagnostic result of thyroid nodule.
Specifically, the invention provides a kind of inspection of the generation of target gene two sequencing for thyroid nodule antidiastole
Survey method, comprises the following steps：
Step (1)：It may cause the related gene information of thyroid cancer known to obtaining；
Step (2)：Gene order and coding information are exported by UCSC and ncbi database；
Step (3)：AA chip design of primers is carried out to these genes using Mass ARRAY Assay Design softwares；
Step (4)：AA array experiments substantially step (can be divided into 5 steps)；
1. loading：Primer and template in 96 orifice plates are drawn onto on 48.48 Access Array IFC chips with the volley of rifle fire.
2. load：Put chip into Pre-PCR IFC Controller AX instruments, primer and template are mixed automatically.
3. thermal cycle：Pcr amplification reaction is carried out using FC1Cycler instruments.
4. harvest：Collect and harvest using Post-PCR IFC Controller AX instruments progress PCR primer.
5. recover：PCR primer at chip template loading is suctioned out with the volley of rifle fire.
Step (5)：Using BWA, SAMTOOLS and GATK softwares are analyzed sequencing result.
Step (6)：All SNV measured and indel by ANNOVAR (http:// annovar.openbioinformatics.org/) functional annotation and public database (ExAC, 1000Genomes, dbSNP
And ClinVar) filtering.
Step (7)：Meet 1. duplicate hole SNV, alldepth>20,vaf>0.3；2. single hole SNV, alldepth>1000,
vaf>0.4；3. duplicate hole SNV, it is bad that single hole is surveyed, the good (alldepth that multiple holes are surveyed>1000) the SNV warps of standard
Sanger sequencings are confirmed.
Step (8)：Meet 1. duplicate hole INDEL, remove SNP；2. single hole INDEL, alldepth>25,vaf ≥0.4,
Remove the Indel of SNP standards is confirmed through Sanger sequencings.
It is preferred that including 19 genes relevant with thyroid cancer morbidity, 8 fusions and related internal reference in panel
Gene, it is specific as follows：
Detect the SNV of 19 genes:BRAF,HRAS,NRAS,KRAS,EIF1AX,PPM1D,CHEK2, RET,CTNNB1,
TP53,AKT1,GNAS,PIK3CA,PTEN,TSHR,CDKN2A,AXIN1,IDH1, VHL
Detect 8 fusions：CCDC6/RET,NCOA4/RET,ETV6/NTRK3,STRN/ALK, PAX8/PPARG,
TPM3/NTRK3,EML4/ALK,PRKAR1A/RET
Thyroid gland specific gene and reference gene：TG, TPO, GAPDH, β-actin, 18S rRNA, 28S rRNA,
Tublin, RPLPO (mankind's large ribosomal albumen), TFRC (TfR)
It is preferred that the described primer-design software for being used for Access ArrayTMSystem amplifications is Mass ARRAY
Assay Design。
It is preferred that the DAS after described targeting sequencing is BWA, SAMTOOLS and GATK..
It is preferred that screening SNV standard is 1. duplicate hole SNV, alldepth>20,vaf>0.3；2. single hole SNV,
alldepth>1000,vaf>0.4；3. duplicate hole SNV, it is bad that single hole is surveyed, the good (alldepth that multiple holes are surveyed>1000).
It is preferred that screening Indel standard is 1. duplicate hole INDEL, SNP is removed；2. single hole INDEL, alldepth>
25, vaf >=0.4, remove SNP.
The present invention is clear and definite suitable for Benign Thyroid Nodules differential diagnosis of malignant, compensate for the deficiency and missing in the field,
According to the mutator and fusion that thyroid cancer may be caused to fall ill, pass through Access ArrayTMSystem sets up combination
Property Panel carry out target gene two generations sequencing.Pass through the sequencing of two generations, the data analysis tentatively to 123 thyroid cancer patients
And Sanger sequence verifications, there are 11 genes to measure mutation, including hot spot mutation BRAF V600E, NRAS in 19 genes:
Q61R, Q61K, RET M918T, PTEN, TSHR, TP53 and AKT1, fusion detect ETV6-NTRK3, NCOA4-RET
And CCDC6-RET.This time associativity molecular marker applies the diagnosis in 123 thyroid cancer patients high.And hair
Occur the feelings that multiple Disease-causing genes occur being mutated or there is driving gene mutation and fusion simultaneously in present many cases patient
Condition, the morbidity for illustrating thyroid cancer is that several genes change accumulation is caused, and with Tumor Heterogeneity, further confirmation pair
The necessity of accurate medical diagnosis is carried out in such disease.The present invention is examined suitable for the malignant and benign lesion of clinical thyroid tubercle
It is disconnected, high flux detection can be carried out, and clinical expansion is carried out, contribute to the therapeutic scheme of selection patient comprehensively, fully make up more
The deficiency and missing in the field are mended, very with practical value.
In summary, the result of this project confirms to exist based on the associativity molecular marker on the basis of two generation sequencing technologies
Played a significant role in the good pernicious diagnosis of thyroid nodule.Forgive the associativity molecule of most of thyroid cancer Disease-causing genes
The generation of targeting two sequencing of mark can improve the diagnosis of thyroid malignancy, successfully compensate for current thyroid cell
Learn the deficiency of diagnosis, and guide thyroid nodule patient diagnosis and treatment be able to it is perfect, it is to avoid nonessential diagnostic operation is to society
The waste of the medical resource of meeting and the potential risk of sufferer operation.Meanwhile, the result of this research is gene diagnosis in clinical practice
In play a role there is provided new thinking.
The screening and identification of the pernicious related gene polymorphism sites of the Benign Thyroid Nodules of embodiment 1
Sample explanation：Fine-needle aspiration of thyroid nodules cell, extracting RNA, reverse transcription obtains cDNA library.
Specific implementation step：
Step (1)：According to possible thyroid cancer Disease-causing gene, including mutator and fusion, according to UCSC
Gene order in Genome Browser in GRCh37/hg19, specify gene chromosome position, coding, gene size with
And pseudogene situation etc..
Step (2)：(there is weight in target sequence size 240bp for the requirement expanded according to Access ArrayTMSystem
It is folded), primer is designed, and add sequence label.
Step (3)：After primer synthesis, it is diluted, requirement is expanded by Access ArrayTMSystem, prepares primer
Mixture, divides in 96 orifice plates.
Step (4)：The preparation of template is expanded, quantitatively (is remembered to 50ng/ul less than 50ng/ul with 50ng/ul), with molding
Plate mixture, divides in 96 orifice plates.
Step (5)：Primer and template in 96 orifice plates are drawn onto on 48.48Access Array IFC chips with the volley of rifle fire.
Step (6)：Put chip into Pre-PCR IFC Controller AX instruments, primer and template are mixed automatically
Close, and pcr amplification reaction is carried out using FC1Cycler instruments.
Step (7)：PCR primer, which is carried out, using Post-PCR IFC Controller AX instruments collects and harvest (attention
Need to change room operations during harvest, to prevent pollution)
Step (8)：Prepare barcode mixtures and add 1：100 cut backs, enter performing PCR reaction.
Step (9)：Magnetic beads for purifying product, after race glue confirms that barcode is added, machine is sequenced in preparation.
Step (10)：The upper machine sequencings of NextSeq500, using BWA, SAMTOOLS and GATK softwares are carried out to lower machine data
Analysis.
Step (11)：Measured SNV and indel by ANNOVAR (http:// annovar.openbioinformatics.org/) functional annotation and public database (ExAC, 1000Genomes, dbSNP
And ClinVar) filtering.
Step (12)：Meet 1. duplicate hole SNV, alldepth>20,vaf>0.3；2. single hole SNV, alldepth>
1000,vaf>0.4；3. duplicate hole SNV, it is bad that single hole is surveyed, the good (alldepth that multiple holes are surveyed>1000) the SNV warps of standard
Sanger sequencings are confirmed.
Step (13)：Meet 1. duplicate hole INDEL, remove SNP；2. single hole INDEL, alldepth>25,vaf≥0.4,
Remove the Indel of SNP standards is confirmed through Sanger sequencings.
The present invention be applied to Benign Thyroid Nodules differential diagnosis of malignant Molecular Detection, compensate for the field deficiency with
Missing, its principle in the thyroid cancer reported at present morbidity related genes, passes through Mass ARRAY to be of the invention
Assay Design software Design primers, on 48.48Access Array IFC, carry out target gene multiplexed PCR amplification
Afterwards, carry out two generation sequencing analysis, make it possible for the disease the bigger coverage rate of high flux gene diagnosis.
By the research to 123 patients, find there are 11 genes to measure mutation (table 2) in 19 genes, detect 3
Fusion (table 3) is planted, 115 kinds of mutation are contained, diagnosis is higher than 93%.Further can be by first in more Chinese populations
The driving gene and fusion of shape gland cancer converge in one diagnosis panel in, can with it is more efficient, rapidly carry out a large amount of samples
This genetic test.The present invention is very suitable for Chinese population thyroid nodule patient, can make up the field it is not enough with it is scarce
Lose, very with practical value.
Table 2
Table 3
| Fusion title | Sequence information | It is mutated patient's number |
| ETV6-NTRK3 | ETV6{ENST00000396373}:r.1_737_NTRK3{ENST00000394480}:r.1719_19984 | 10 |
| NCOA4-RET | NCOA4{ENST00000452682}:r.1_1014_RET{ENST00000355710}:r.2369_5659 | 3 |
| CCDC6-RET | CCDC6{ENST00000263102}:r.1_535_RET{ENST00000355710}:r.2369_5659 | 2 |
The tissue specificity of the gene mutation of the present invention is analyzed, as a result shown, each mutational site of the invention
With excellent tissue specificity, mutation specific has detection in human thyroid carcinoma, few in normal somatic cell
Detection, only detects the mutation of the Q61K on NRAS genes in 1 thyroid adenoma.Although it is worth noting that, first shape
Gland adenoma is benign disease, but the pathomorphism of adenoma is sometimes much like with thyroid papillary carcinoma, therefore depends merely on pathology sometimes
May occur Error Diagnostics, concrete outcome is as shown in table 4 below：
Table 4
TA, thyroid adenoma (Thyroid adenoma)
NG, nodular goiter (Nodular goiter)
PTC, thyroid papillary carcinoma (Papillary thyroid carcinoma)
FTC, thyroid follcular carcinoma (Follicular thyroid carcinoma, included in PTC)
MTC, medullary carcinoma of thyroid gland (Medullary thyroid carcinoma)
PDTC, low differentiation thyroid cancer (Poorly differentiated thyroid cancer)
ATC, undifferentiated thyroid carcinoma (Anaplastic thyroid carcinoma)
The preparation of the kit of embodiment 2 and compliance test result
Present embodiments provide a kind of kit for detecting that Benign Thyroid Nodules are pernicious.
This kit can carry out detection in Gene Mutation to the fusion in the mutational site in upper table 2 and upper table 3：
Kit main agents：
(1) polymorphic site amplimer
Sense primer (F) and anti-sense primer (R) (Mass ARRAY Assay Design Software for Design)；
(2) polymorphic site sequencing primer
Sequencing primer (S) (according to this area conventional design)；
(3) PCR main agents：Pfu high-fidelity enzymes, 10 × PCR Buffer, dNTPMixtur, ddH2O；
(4) pyrosequencing main agents：70% ethanol solution, magnetic bead, denaturation buffer, annealing buffer, with reference to slow
Fliud flushing, lavation buffer solution, substrate (ASP, fluorescein), enzyme mixed solution (archaeal dna polymerase, luciferase, atriphos sulphur
Phosphorylase, apyrase), A/T/C/G bases.
300 thyroid nodule patients are detected by the kit provided using the present embodiment, specific method reference
Embodiment 1, has detected the gene mutation that 176 patients carry the present invention, thyroid gland is confirmed finally by routine clinical method altogether
Cancer patient is 184, and recall rate is up to more than 95%, and specific testing result is shown in Table 5.
The result of 5 300 generation of thyroid cancer patients target gene two sequencings of table
All documents referred in the present invention are all incorporated as reference in this application, just as each document coverlet
Solely it is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
Sequence table
<110>Town in Shanghai first bio tech ltd
<120>The gene polymorphism sites related to thyroid cancer and its application
<130> P2017-0424
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 80
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<213>People
<220>
<221> misc_feature
<222> (39)..(39)
<223>N=T, or A
<400> 1
cctcacagta aaaataggtg attttggtct agctacagng aaatctcgat ggagtgggtc 60
ccatcagttt gaacagttgt 80
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggagccttgt atatagacgg 20
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<211> 20
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<400> 3
tgtatgttct aacaggcacc 20
<210> 4
<211> 60
<212> DNA
<213>People
<220>
<221> misc_feature
<222> (30)..(30)
<223>N=T, or C
<400> 4
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<210> 5
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caagaaacca tatgctcacc 20
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ttggattgtg tccgttgagc 20
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<210> 8
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tgaactgatt gaaaactccc atctaacccc aaagangcaa ggccaaatct cagaagagta 60
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<400> 11
aggcaactat gttgagcgtc 20
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<400> 12
tatgccatca ccttcgccat 20
<210> 13
<211> 68
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<223>N=A, or G
<400> 13
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agagaaag 68
<210> 14
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tcccaccaca gcacatacac 20
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cttttccttt ctctctctac c 21
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<222> (35)..(35)
<223>N=C, or nothing
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ctgttcggtg 70
<210> 17
<211> 20
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Claims (10)
1. one or more of (I) gene mutation site and/or the purposes of its detection reagent are selected from the group, for reagent preparation
Or kit, the reagent or kit are for differentiating the good pernicious of thyroid nodule, and described group (I) includes following gene mutation
Site：
GNAS genes：
NM_001077490:exon1:c.T1019C；
NRAS genes：
NM_002524:exon3:c.T284C；
TSHR genes：
NM_000369:exon10:c.A2098G。
2. purposes as claimed in claim 1, it is characterised in that described group (I) also includes following gene mutation site：
CHEK2 genes：
NM_145862:exon11:c.A1250G；And/or
Described group (I) also includes following gene mutation site：
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C；And/or
Described group (I) also includes following gene mutation site：
GNAS genes：
NM_016592:exon1:c.C205A、NM_016592:exon1:c.C216T；And/or
Described group (I) also includes following gene mutation site：
TSHR genes：
NM_000369:exon10:c.A2252G；And/or
Described group (I) also includes following gene mutation site：
BRAF gene：
NM_004333:exon11:c.G1338A。
3. purposes as claimed in claim 1, it is characterised in that described reagent includes primer, probe, chip or antibody.
4. a kind of kit, it is characterised in that the kit includes the one or more gene mutation sites being selected from the group
Detection reagent：
GNAS genes：
NM_001077490:exon1:c.T1019C；
NRAS genes：
NM_002524:exon3:c.T284C；
TSHR genes：
NM_000369:exon10:c.A2098G。
5. kit as claimed in claim 4, it is characterised in that the kit also includes the inspection of following gene mutation site
Test agent：
CHEK2 genes：
NM_145862:exon11:c.A1250G；And/or
The kit also includes the detection reagent of following gene mutation site：
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C；And/or
The kit also includes the detection reagent for the one or more gene mutation sites being selected from the group：
GNAS genes：
NM_016592:exon1:c.C205A、NM_016592:exon1:c.C216T；And/or
The kit also includes the detection reagent of following gene mutation site：
TSHR genes：
NM_000369:exon10:c.A2252G。
6. kit as claimed in claim 4, it is characterised in that the kit is one or more also including what is be selected from the group
The detection reagent of gene mutation site：
BRAF gene：
NM_004333:exon15:c.T1799A、NM_004333:exon11:c.G1338A、
NM_004333:exon15:c.A1801G；
CHEK2 genes：
NM_145862:exon10:c.C1024T；
NRAS genes：
NM_002524:exon3:c.C181A、NM_002524:exon3:c.A182G；
AKT1 genes：
NM_001014431:exon3:c.G49A；
PPM1D genes：
NM_003620:exon1:c.C262T；
PTEN genes：
NM_000314:exon5:c.C328T；
RET genes：
NM_020630:exon11:c.T1888C、NM_020630:exon16:c.T2753C；
TP53 genes：
NM_001126115:exon3:c.C265T、NM_000546:exon8:c.G814T；
And/or, the kit also includes the detection reagent for the one or more fusions being selected from the group：
ETV6-NTRK3 fusions：
ETV6{ENST00000396373}:r.1_737_NTRK3{ENST00000394480}:r.1719_19984；
NCOA4-RET fusions：
NCOA4{ENST00000452682}:r.1_1014_RET{ENST00000355710}:r.2369_5659；
CCDC6-RET fusions：
CCDC6{ENST00000263102}:r.1_535_RET{ENST00000355710}:r.2369_5659。
7. a kind of method that vitro detection sample whether there is gene mutation, it is characterised in that including step：
(a) with the polynucleotides of primer amplified sample, amplified production is obtained；With
(b) it whether there is following one or more gene mutations in detection amplified production：
GNAS genes：
NM_001077490:exon1:c.T1019C、NM_016592:exon1:c.C205A、
NM_016592:exon1:c.C216T；
NRAS genes：
NM_002524:exon3:c.T284C；
TSHR genes：
NM_000369:exon10:c.A2098G、NM_000369:exon10:c.A2252G。
8. method as claimed in claim 7, it is characterised in that also include in the step (b) in detection amplified production whether
There is following gene mutation：
CHEK2 genes：
NM_145862:exon11:c.A1250G；And/or
Also include whether there is following gene mutation in detection amplified production in the step (b)：
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C.
9. the gene polynucleotides sequence of a kind of separation, it is characterised in that described polynucleotide sequence is derived under
The fragment of the gene of group：NRAS genes, GNAS genes, PIK3CA genes, CHEK2 genes and TSHR genes, and described nucleosides
Acid sequence has the gene mutation site being selected from the group respectively：
NRAS genes：
NM_002524:exon3:c.T284C；
GNAS genes：
NM_001077490:exon1:c.T1019C、NM_016592:exon1:c.C205A、
NM_016592:exon1:c.C216T；
PIK3CA genes：
NM_006218:exon12:C.1818 position lacks C；
CHEK2 genes：
NM_145862:exon11:c.A1250G；
TSHR genes：
NM_000369:exon10:c.A2098G、NM_000369:exon10:c.A2252G。
10. the one or more or full gene and/or the purposes of its detection reagent that are selected from the group, it is characterised in that for making
Standby reagent or kit, the reagent or kit are used to differentiate the good pernicious of thyroid nodule：
AKT1 genes, BRAF gene, CHEK2 genes, GNAS genes, NRAS genes, PIK3CA genes, PPM1D genes, PTEN bases
Cause, RET genes, TP53 genes, TSHR genes, ETV6-NTRK3 fusions, NCOA4-RET fusions and CCDC6-RET
Fusion.
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