CN107164496A - The gene polymorphism sites related to thyroid cancer and its application - Google Patents

Abstract

The present invention provides the somatic mutation site of the pathogenic gene of thyroid cancer and its application. Specifically, the present invention provides a group of mutation sites of the pathogenic gene of thyroid cancer, including GNAS gene mutation site and NRAS gene mutation site. point, TSHR gene mutation site, etc., the gene mutation site provided by the present invention can be used as a marker for distinguishing benign from malignant thyroid nodules.

Description

Gene polymorphism sites related to thyroid cancer and its application

technical field

The invention belongs to the field of biomedicine, in particular, the invention relates to gene somatic cell-specific mutation sites related to thyroid nodules and applications thereof.

Background technique

Thyroid nodules are the most common clinical thyroid disease, and the prevalence of palpable thyroid nodules is about 4-7% in the American adult population. The application of sensitive thyroid B examination, in people over 65 years old, the prevalence of thyroid nodules is as high as 50%. In 2010, Professor Teng Weiping from the Endocrine Association of the Chinese Medical Association conducted an epidemiological investigation of thyroid disease among 15,181 residents in ten cities across the country. It was found that the prevalence of thyroid nodules in the Chinese population was as high as 18.6%, so it is speculated that our country's There are as many as 200 million patients with thyroid nodules. Most thyroid nodules are benign and can be treated conservatively, but 5-10% of thyroid nodules are malignant and require early surgical treatment in order to obtain a good prognosis. Therefore, the great challenge that a thyroid specialist faces is how to distinguish the benign and malignant thyroid nodules of a patient. Only in this way can the patient receive timely and reasonable treatment. If a large number of benign thyroid nodules that do not require surgery are inappropriately treated with surgery, the prevalence of such a large population will generate a huge medical insurance burden; on the contrary, if the real Malignant tumors will delay the treatment of these patients and endanger their lives and health.

At present, the methods commonly used clinically to differentiate benign and malignant thyroid nodules mainly include thyroid B-ultrasound, radionuclide scanning, and fine-needle aspiration biopsy (FNAB) of thyroid nodules. Among them, thyroid fine-needle aspiration biopsy is currently the gold standard for the differential diagnosis of benign and malignant thyroid nodules. According to the guidelines of the 2008 thyroid fine needle aspiration pathology meeting of the National Cancer Institute, the cytological diagnosis of thyroid nodules is currently divided into the following categories: 1. Inability to diagnose due to insufficient number of punctured thyroid cells; 2. Benign Nodules; 3. Nodules that cannot be diagnosed by pathological cytology; 4. Four types of malignant thyroid nodules. In recent years, with the widespread application of fine-needle aspiration under the guidance of B-ultrasound, the proportion of patients who cannot be diagnosed due to insufficient number of cells obtained by puncture has been significantly reduced, from as high as 15% in the past to less than 7% at present. . After obtaining enough thyroid follicular cells by fine needle aspiration, most benign and malignant thyroid nodules can be correctly diagnosed by cytopathological diagnostic methods. However, even in the best laboratory in the world for pathological diagnosis of thyroid cells, 20-40% of thyroid nodules with successful puncture cannot be determined to be benign or malignant, that is, nodules that cannot be diagnosed pathologically. Therefore, when patients undergo thyroid biopsy, the proportion of patients who cannot be correctly diagnosed due to puncture failure and pathological inability to judge benign and malignant is as high as 30-50%, which is affected by the technical level of thyroid pathologists and puncture physicians in each center larger.

Contents of the invention

The object of the present invention is to provide a pathogenic gene mutation site for the onset of thyroid cancer and its application.

The first aspect of the present invention provides the use of one or more gene mutation sites and/or detection reagents selected from the following group (I), for the preparation of reagents or kits, and the reagents or kits are used To differentiate benign and malignant thyroid nodules, the group (I) includes the following gene mutation sites:

GNAS gene:

NM_001077490:exon1:c.T1019C;

NRAS gene:

NM_002524:exon3:c.T284C;

TSHR gene:

NM_000369:exon10:c.A2098G.

In another preferred example, the group (I) also includes the following gene mutation sites:

CHEK2 gene:

NM_145862:exon11:c.A1250G.

In another preferred example, the group (I) also includes the following gene mutation sites:

PIK3CA gene:

NM_006218: exon12:c.1818 missing C.

In another preferred example, the group (I) also includes the following gene mutation sites:

GNAS gene:

NM_016592:exon1:c.C205A, NM_016592:exon1:c.C216T.

In another preferred example, the group (I) also includes the following gene mutation sites:

TSHR gene:

NM_000369:exon10:c.A2252G.

In another preferred example, the group (I) also includes the following gene mutation sites:

BRAF gene:

NM_004333:exon15:c.T1799A.

In another preferred example, the detected objects include: humans or non-human mammals (such as domestic animals, poultry, experimental animals, etc.).

In another preferred example, the reagents include primers, probes, chips, or antibodies.

In another preferred embodiment, the kit contains one or more reagents selected from the group consisting of:

(A) specific primers for genetic detection;

(B) specific probes for genetic testing;

(C) chips for genetic testing;

(D) Antibodies specific for detecting amino acid mutations corresponding to mutated genes.

In another preferred example, the reagent or kit is used for real-time fluorescent quantitative PCR detection.

In another preferred example, in the real-time fluorescence quantitative PCR, the annealing temperature is between 60-67°C, and the length of the PCR amplification product is 80-300bp.

In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature of the fluorescent probe is between 60-70°C.

In another preferred example, both ends of the probe are modified with chemical groups, the 5' end is modified with a fluorescence excitation group, and the 3' end is modified with a fluorescence quencher group.

In another preferred example, the detection is an auxiliary detection.

In a second aspect of the present invention, a kit is provided, which includes detection reagents for one or more gene mutation sites selected from the following group:

GNAS gene:

NM_001077490:exon1:c.T1019C;

NRAS gene:

NM_002524:exon3:c.T284C;

TSHR gene:

NM_000369:exon10:c.A2098G.

In another preferred example, the kit also includes detection reagents for the following gene mutation sites:

CHEK2 gene:

NM_145862:exon11:c.A1250G.

In another preferred example, the kit also includes detection reagents for the following gene mutation sites:

PIK3CA gene:

NM_006218: exon12:c.1818 missing C.

In another preference, the kit also includes detection reagents for one or more gene mutation sites selected from the following group:

GNAS gene:

NM_016592:exon1:c.C205A, NM_016592:exon1:c.C216T.

In another preferred example, the kit also includes detection reagents for the following gene mutation sites:

TSHR gene:

NM_000369:exon10:c.A2252G.

In another preference, the kit also includes detection reagents for one or more gene mutation sites selected from the following group:

BRAF gene:

NM_004333:exon15:c.T1799A, NM_004333:exon11:c.G1338A,

NM_004333:exon15:c.A1801G;

CHEK2 gene:

NM_145862:exon10:c.C1024T;

NRAS gene:

NM_002524:exon3:c.C181A, NM_002524:exon3:c.A182G;

AKT1 gene:

NM_001014431:exon3:c.G49A;

PPM1D gene:

NM_003620:exon1:c.C262T;

PTEN gene:

NM_000314:exon5:c.C328T;

RET gene:

NM_020630:exon11:c.T1888C, NM_020630:exon16:c.T2753C;

TP53 gene:

NM_001126115:exon3:c.C265T, NM_000546:exon8:c.G814T.

In another preference, the kit also includes detection reagents for one or more fusion genes selected from the following group:

ETV6-NTRK3 fusion gene:

ETV6{ENST00000396373}:r.1_737_NTRK3{ENST00000394480}:r.1719_19984;

NCOA4-RET fusion gene:

NCOA4{ENST00000452682}:r.1_1014_RET{ENST00000355710}:r.2369_5659;

CCDC6-RET fusion gene:

CCDC6{ENST00000263102}:r.1_535_RET{ENST00000355710}:r.2369_5659.

In another preferred example, the detection reagent is:

(A) specific primers for genetic detection;

(B) specific probes for genetic testing;

(C) chips for genetic testing;

(D) Antibody specific for detecting the amino acid mutation corresponding to the mutated gene.

A third aspect of the present invention provides a method for in vitro detection of whether there is a gene mutation in a sample, comprising the steps of:

(a) amplifying the polynucleotide of the sample with specific primers to obtain an amplification product; and

(b) Detect whether one or more of the following gene mutations exist in the amplified product:

GNAS gene:

NM_001077490:exon1:c.T1019C, NM_016592:exon1:c.C205A,

NM_016592:exon1:c.C216T;

NRAS gene:

NM_002524:exon3:c.T284C;

TSHR gene:

NM_000369:exon10:c.A2098G, NM_000369:exon10:c.A2252G.

In another preferred embodiment, the detection is non-diagnostic.

In another preferred example, the step (b) also includes detecting whether the following gene mutations exist in the amplified product:

CHEK2 gene:

NM_145862:exon11:c.A1250G.

In another preferred example, the step (b) also includes detecting whether the following gene mutations exist in the amplified product:

PIK3CA gene:

NM_006218: exon12:c.1818 missing C.

If one or more loci are present, the thyroid nodule is malignant.

In another preferred example, the sample is from a human.

In the fifth aspect of the present invention, an isolated gene polynucleotide sequence is provided, and the polynucleotide sequence is a fragment derived from a gene selected from the group consisting of: NRAS gene, GNAS gene, PIK3CA gene, CHEK2 gene, and TSHR gene, and the nucleotide sequence has a gene mutation site selected from the following group:

NRAS gene:

NM_002524:exon3:c.T284C;

GNAS gene:

NM_001077490:exon1:c.T1019C, NM_016592:exon1:c.C205A,

NM_016592:exon1:c.C216T;

PIK3CA gene:

NM_006218: exon12:c.1818 missing C;

CHEK2 gene:

NM_145862:exon11:c.A1250G;

TSHR gene:

NM_000369:exon10:c.A2098G, NM_000369:exon10:c.A2252G.

In another preferred example, the length of the polynucleotide sequence is 30-1000bp; preferably 50-500bp.

The present invention also provides the use of the polynucleotide sequence, which can be used as a positive control (standard).

The sixth aspect of the present invention provides the use of one or more or all genes selected from the following group and/or its detection reagents for the preparation of reagents or kits for the identification of thyroid nodules benign and malignant:

AKT1 gene, BRAF gene, CHEK2 gene, GNAS gene, NRAS gene, PIK3CA gene, PPM1D gene, PTEN gene, RET gene, TP53 gene, TSHR gene, ETV6-NTRK3 fusion gene, NCOA4-RET fusion gene, and CCDC6-RET fusion gene Gene.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

detailed description

Through extensive and in-depth research, the inventors unexpectedly obtained a group of mutation sites related to thyroid cancer pathogenic genes. Experimental results show that the gene mutation sites provided by the present invention can be used as markers for distinguishing benign from malignant thyroid nodules.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, the scope of the present invention being limited only by the appended claims.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are exemplified herein.

In this field, how to correctly diagnose these thyroid nodules before operation, so as to give patients a reasonable and timely treatment, has always been a hot spot in the field of thyroid research. With the development of molecular biology techniques, molecular diagnosis has gradually attracted people's attention in the differential diagnosis of benign and malignant thyroid nodules. The theoretical basis of the molecular differential diagnosis of benign and malignant nodules is based on the occurrence of specific somatic mutations or fusion genes in some thyroid cancer pathogenic genes only in cancer tissues of cancer patients. It is absent in the normal cells of the thyroid and in the thyroid tissue of non-thyroid cancer patients. Therefore, these molecular markers are very specific in the differential diagnosis of benign and malignant thyroid cancer. For example, it was found that 100% of patients with thyroid nodules with the Val600Glu mutation at the 600th amino acid position on the BRAF gene were malignant nodules. Although these gene mutations have very good specificity in the differential diagnosis of benign and malignant thyroid nodules, the mutation frequency of each pathogenic gene in thyroid cancer patients is not high. For detection, positive patients have important diagnostic significance, but negative patients will miss many real thyroid cancer patients and delay the treatment of patients. Therefore, to solve the dilemma of molecular diagnosis of thyroid cancer, it is necessary to simultaneously detect mutations in multiple genes that cause thyroid cancer, so as to improve the specificity of differential diagnosis of benign and malignant thyroid nodules and reduce the false negative rate.

However, in order to simultaneously detect somatic mutations and new fusion genes of multiple thyroid cancer-specific pathogenic genes in thyroid nodule samples, it is necessary to break through the following scientific and technical difficulties in order to achieve clinical application the goal of:

First: Although a large number of pathogenic gene mutations of thyroid cancer have been reported in the literature, it is not clear which of these reported mutated genes are real pathogenic genes and which are not causative of thyroid cancer. Secondly, among the currently reported thyroid cancer-causing genes, which gene combinations have a high positive rate in the differential diagnosis of thyroid nodules, and which are less likely to be missed in the negative diagnosis is currently unknown. Finding the combination of such a group of thyroid cancer pathogenic genes will be very important in the differentiation of benign and malignant thyroid nodules.

Second: Since the mutation (mutation and fusion gene) of the pathogenic gene of thyroid cancer is a somatic mutation in thyroid cancer cells, it is necessary to obtain thyroid nodule cells by fine needle aspiration for diagnosis, and fine needle aspiration The number of cells is very small (generally 10-200 cells), and it is impossible to detect a large number of gene mutations and fusion genes in such a small number of cells with traditional generation sequencing. New detection technology must be adopted to complete the establishment of molecular diagnostic methods for combined markers.

Third: Gene mutations in thyroid cancer mainly include two categories, one is gene somatic mutations; the other is new fusion genes appearing in tumor cells. Traditional methods for detecting these two types of mutations rely on the DNA and mRNA of the sample, respectively. The most adequate amount of thyroid nodule tissue needs to be collected twice. This is difficult to implement in clinical routine diagnosis. Therefore, how to use the same small biopsy sample to detect a large number of these gene variations is the bottleneck for the practical application of combined molecular markers.

Therefore, the present invention is mainly aimed at the above-mentioned 3 bottlenecks of molecular diagnosis of thyroid nodules, and mainly has the following originality:

First, the 19 pathogenic genes of papillary thyroid carcinoma, 8 different fusion genes, common TP53 and CTNNB1 genes in undifferentiated and differentially differentiated carcinomas, and the RET gene in medullary thyroid carcinoma were selected as molecular Markers, through the targeted amplification of the cDNA of these variable segments, combined with next-generation sequencing technology, gene mutation and fusion gene detection in thyroid cancer and thyroid tumors or normal thyroid tissues, a group of combined molecular markers were discovered, The overall positive rate in Chinese thyroid cancer population is as high as 81%, which is a group of molecular markers with good diagnostic value. The combination of 11 core genes with the most common mutations and 8 different fusion genes can make the positive rate of diagnosis as high as 93%.

Secondly, based on the target gene multiplex PCR amplification chip technology of Fludigam Company, a set of specific combined multiple target gene amplification technology was developed, which solved the problem of simultaneous targeted amplification of multiple genes in micro samples. Method; at the same time, multiple PCR primers are used to determine whether there are mutations or new fusion genes in each gene in each sample through the next-generation sequencing technology. Solve the technical bottleneck of simultaneous detection of multiple gene variations in trace samples.

In addition, the present invention selects mRNA as the sample for target gene amplification to detect gene variation of combined molecular markers, and realizes the purpose of detecting gene mutation and fusion gene in the same sample. The invention becomes possible in clinical application. At the same time, the detection of gene variation using mRNA as a template can evaluate the quality of puncture samples and objectively evaluate the reliability of diagnosis.

A combination of multiple molecular markers can be used to identify benign and malignant thyroid nodules. For example, using BRAFV600E, RAS, RET/PTC1, RET/PTC3, and PAX8/PPAR fusion genes as molecular markers can significantly increase the number of benign and malignant thyroid nodules. ability to diagnose malignancy. The sensitivity of this combined molecular marker for the differential diagnosis of benign and malignant thyroid nodules can reach 62%, and the specificity can reach 99.7%. , from 44% to 80% diagnosed by pathological cytology alone. The main reason for the low diagnostic sensitivity of this molecular marker is that all thyroid disease-causing genes have not been found so far. The above-mentioned common thyroid cancer-causing genes, in patients with thyroid cancer, are mutually exclusive among different genes. The mutation frequency is mostly around 70%. Therefore, using the combination of hot spots of common pathogenic genes as molecular markers to differentiate benign from malignant thyroid nodules has great limitations.

In order to improve the diagnostic sensitivity of molecular markers, it is first necessary to find the genes or molecular markers that cause thyroid cancer. According to the 2008 American Cancer Institute thyroid cytology diagnostic criteria, thyroid cancer is currently divided into papillary carcinoma (currently, follicular carcinoma is classified into this type of pathology, and it is classified as a variant papillary carcinoma), differential There are four types of carcinoma, undifferentiated carcinoma and medullary thyroid carcinoma, the most common of which is papillary cell carcinoma of the thyroid, accounting for more than 95% of all thyroid cancers. In different thyroid cancers, the pathogenic genes may be different, and the mutation frequency of the same pathogenic gene in different types of thyroid cancers is also different. For example, the common mutated gene in papillary thyroid carcinoma is BRAF gene, while in differentially differentiated and undifferentiated carcinoma, TP53 gene is a highly mutated gene. In undifferentiated carcinoma, more than 50% of patients carry TP53 gene mutation, but in thyroid Mutations in the P53 gene are rarely found in papillary carcinoma. The RET gene mutation is very common in patients with medullary thyroid carcinoma. For example, in familial medullary carcinoma, the mutation rate of this gene is as high as 95%, and in sporadic medullary thyroid carcinoma, the mutation rate of RET gene is as high as 40%. -50%. A study on whole-genome exome sequencing of a large sample of papillary thyroid carcinoma published in Cell in 2014 provided an exciting prospect for the application of combined molecular markers in the differential diagnosis of benign and malignant thyroid nodules. Through whole-genome exome sequencing of 496 cases of papillary thyroid carcinoma, they found 5 thyroid cancer-causing genes, including 3 possible new thyroid cancer-causing genes; and found 8 genes that were different from other genes Various types of fusion gene mutations formed. Among these thyroid cancers, 73.6% of the samples had at least one mutation in the five pathogenic genes, and 89.8% of the samples had more than one mutation in the pathogenic gene or fusion gene. In about 10% of thyroid cancers, there are neither point mutations nor fusion gene mutations found in these five pathogenic genes. They then analyzed the amplification and deletion variation of chromosomes in these tumor samples through SNP chips and other technologies, and found that large-scale amplification and deletion of chromosome segments (Arm-level chromosome variation) are very common in thyroid cancer samples, among which Chromosomal deletion of 22q occurs in about 14.4% of thyroid cancer patients, while large fragment amplification of 1q chromosome accounts for 14.8%. If these chromosomal arm-level amplification and deletion mutations are used as molecular markers, patients with at least one of the three types of mutations in pathogenic gene mutations, fusion gene mutations, or arm-level chromosomal amplification and deletion mutations increased to 96.6%. This shows that if we can use these three different types of mutations to carry out molecular diagnosis of thyroid nodules, the sensitivity of diagnosis may reach more than 95%, which will be the best among all the means of differential diagnosis of benign and malignant thyroid nodules. most reliable diagnostic method.

The multiplex PCR amplification of target genes combined with the emergence of next-generation sequencing using new molecular biology techniques makes it possible to simultaneously detect multiple gene variations using a small amount of RNA extracted from thyroid biopsy cells as a template, thus enabling the detection of combinatorial molecular markers. It becomes simple and easy to operate, and at the same time, it will not miss possible gene mutations. Fluidigm's targeted amplification chip can target multiple mutant genes and perform next-generation sequencing library construction. One chip can perform PCR amplification on 450 to 1,500 pairs of primers from 48 samples at the same time, meeting the requirements of these molecules in thyroid cancer. The need for sign diagnostics.

Therefore, the present invention adopts the target gene multiplex PCR amplification chip technology of Fluidigm Company, and selects 19 pathogenic genes and 8 different fusion genes found so far in papillary thyroid carcinoma, as well as TP53, TP53, CTNNB1 gene and RET gene in medullary thyroid carcinoma are used as molecular markers. Through targeted amplification of the cDNA of these mutated segments, combined with next-generation sequencing technology, it is possible to identify whether there are variations of these molecular markers in thyroid nodules, thereby Differential diagnosis of benign and malignant thyroid nodules. Finally, a batch of new gene polymorphism sites related to malignant thyroid nodules were found, and the specific information is shown in Table 1.

Table 1

gene name mutation 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

The gene sequence numbers in Table 1 refer to the GRCh37/hg19 version.

BRAF gene

The protein encoded by the BRAF gene belongs to the raf family of serine/threonine kinases. It plays a regulatory role in the MAPK/ERKs pathway and participates in cell division, differentiation and secretion. Mutations in the BRAF gene are thought to be associated with the development of a variety of cancers, including non-Hodgkin's lymphoma, colorectal cancer, malignant melanoma, thyroid cancer, and non-small cell lung cancer. In addition, studies have shown that mutations in the BRAF gene are associated with cardiofacial skin syndrome.

The sequence fragment of the BRAF gene (NM_004333) mutated at position 1799 is as follows:

CCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGT (SEQ ID NO. 1)

In a preferred embodiment of the present invention, the primer pair for detecting the 1799 mutation site of BRAF gene is as follows:

BRAF-E15-F GGAGCCTTGTATATAGACGG SEQ ID NO.2 BRAF-E15-R TGTATGTTCTAACAGGCACC SEQ ID NO.3

NRAS gene

The NRAS gene is an oncogene, and its encoded membrane protein can shuttle between the Golgi apparatus and the cell membrane. NRAS proteins have intrinsic GTPase activity, which can be activated by guanine nucleotide exchange factors and inhibited by GTPase activating proteins, respectively. Point mutations located on NRAS are thought to be associated with somatic rectal cancer, follicular thyroid cancer, autoimmune lymphoproliferative syndrome, Noonan syndrome, and leukemia.

The sequence fragment of the 284th mutated NRAS gene (NM_002524) is as follows:

ATAGCAAGTCATTTGCGGATATTAACCTC[T/C]ACAGGGAGCAGATTAAGCGAGTA AAAGACT (SEQ ID NO.4)

In a preferred embodiment of the present invention, the primer pair for detecting the 284th mutation site of NRAS gene is as follows:

NRAS-E3-F CAAGAAACCATATGCTCACC SEQ ID NO.5 NRAS-E3-R TTGGATTGTGTCCGTTGAGC SEQ ID NO.6

GNAS gene

GNAS is a protein-coding gene, and the related signaling pathways are G protein-coupled receptor (GPCR) signaling pathway and polypeptide ligand-binding receptor signaling pathway, which are involved in the activation of adenylyl cyclase and various cellular responses. There are multiple transcripts of this gene. Mutations in the GNAS gene have been associated with pseudohypoparathyroidism, hereditary osteodystrophy, McCune-Albright syndrome, progressive bone dysplasia, fibrous dysplasia polyostosis, and some pituitary tumors.

The sequence of the GNAS gene (NM_001077490) mutated at position 1019 is as follows:

GCCCAACAGCGCCGGAGCTTCCTTAACGCCCAC[C/A]ACCGCTCCGGCGCCCAGGTATTCCCTGAGTCCCCCG (SEQ ID NO. 7)

In a preferred embodiment of the present invention, the primer pair that is used to detect GNAS gene 1019 mutation sites is as follows:

GNAS-E1-1-F AGAGGCCGCCACCGTGTTAT SEQ ID NO.8 GNAS-E1-1-R AGGCCTCGCCATCATTCTC SEQ ID NO.9

TSHR gene

The protein encoded by the TSHR gene is a membrane protein, which is mainly involved in the metabolism of thyroid cells. It is a receptor for thyrotropin and can be activated by adenylyl cyclase. Diseases associated with TSHR include congenital hypothyroidism and non-autoimmune hyperthyroidism.

The sequence fragment of the TSHR gene (NM_000369) mutated at position 2252 is as follows:

TGAACTGATTGAAAACTCCCATCTAACCCCAAAGA[A/G]GCAAGGCCAAATCTCAGAAGAGTATATGCAAACGGTTTTGTAAG (SEQ ID NO. 10)

In a preferred embodiment of the present invention, the primer pair for detecting the 2252 mutation site of TSHR gene is as follows:

TSHR-E10-F AGGCAACTATGTTGAGCGTC SEQ ID NO.11 TSHR-E10-R TATGCCATCACCTTCGCCAT SEQ ID NO.12

CHEK2 gene

Regulation of cell cycle progression in the cell cycle plays a crucial role in DNA damage and replication. The protein encoded by the CHEK2 gene is a cell cycle check regulator and is considered a tumor suppressor gene. Mutations in this gene are associated with Li-Fraumeni syndrome, a familial, highly clustered cancer phenotype with TP53 mutations. In addition, those who carry this gene mutation have a tendency to develop sarcoma, breast cancer and brain tumors.

The sequence fragment of the CHEK2 gene (NM_145862) mutated at position 1250 is as follows:

GAAGGATCAGATCACCAGTGGAAAATACA[A/G]CTTCATTCCTGAAGTCTGGGCAGAAGTCTCAGAGAAAG (SEQ ID NO. 13)

In a preferred embodiment of the present invention, the primer pair for detecting the 1250 mutation site of CHEK2 gene is as follows:

CHEK2-E11-F TCCCACCACAGCACATACAC SEQ ID NO.14 CHEK2-E11-R CTTTTCTTTCTCTCTCTACC SEQ ID NO.15

PIK3CA gene

The PIK3CA gene is an oncogene, belonging to the PI3Ks family, responsible for coordinating various cell functions including survival and proliferation, and its gene mutation is related to the pathogenesis of cervical cancer and other tumors.

The sequence of the PIK3CA gene (NM_006218) mutated at position 1818 is as follows:

CTGAACAGGCTATGGAACTTCTGGACTGTAATTA[C/-]CCAGATCCTATGGTTCGAGGTTTTGCTGTTCGGTG (SEQ ID NO. 16)

In a preferred embodiment of the present invention, the primer pair for detecting the 1818 mutation site of PIK3CA gene is as follows:

PIK3CA-E12-F CACAAACTAGAGTCACACAC SEQ ID NO.17 PIK3CA-E12-R GCACGATTCTTTTAGATCTG SEQ ID NO.18

The main advantages of the present invention are:

(1) For the first time, a group of gene polymorphism sites that can be used as markers for distinguishing benign from malignant thyroid nodules are revealed, with high specificity;

(2) According to the combination of molecular markers (mutated gene sites) of the present invention, it has good diagnostic value for Chinese thyroid cancer population, and can make the positive rate of diagnosis as high as 90%.

(3) When collecting samples from clinical patients, it is only necessary to collect punctured cells of thyroid nodules and extract RNA as a template. Gene mutations and fusion genes can be detected at the same time, and the clinical feasibility is good.

Below in conjunction with specific embodiment, further elaborate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate detailed conditions in the following examples, usually according to the conditions described in "Molecular Cloning Laboratory Guide" (Huang Peitang et al. translation, Beijing: Science Press, 2002) such as U.S. Sambrook.J etc. , or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

method

The present invention provides a detection method of targeted gene next-generation sequencing for distinguishing between benign and malignant thyroid nodules, and determines the appropriate operation method and postoperative management for patients according to the molecular diagnosis results of thyroid nodules.

Specifically, the present invention provides a detection method for targeted gene next-generation sequencing for the differential diagnosis of thyroid nodules, comprising the following steps:

Step (1): Obtain information about genes known to cause thyroid cancer;

Step (2): deriving gene sequence and coding information through UCSC and NCBI databases;

Step (3): use Mass ARRAY Assay Design software to carry out AA chip primer design for these genes;

Step (4): The general steps of the AA chip experiment (can be divided into 5 steps);

①Sample loading: Use a row gun to suck the primers and templates in the 96-well plate onto the 48.48 Access Array IFC chip.

② Loading: Put the chip into the Pre-PCR IFC Controller AX instrument, and the primers and templates are automatically mixed.

③Thermal cycle: use FC1Cycler instrument for PCR amplification reaction.

④Harvest: Use the Post-PCR IFC Controller AX instrument to collect and harvest PCR products.

⑤Recovery: Use a discharge gun to suck out the PCR product at the sample loading site of the chip template.

Step (5): Using BWA, SAMTOOLS and GATK software to analyze the sequencing results.

Step (6): All measured SNVs and indels were filtered through ANNOVAR ( http:// annovar.openbioinformatics.org/ ) functional annotations and public databases (ExAC, 1000Genomes, dbSNP and ClinVar).

Step (7): ①Double hole SNV, alldepth>20, vaf>0.3; ②Single hole SNV, alldepth>1000, vaf>0.4; ③Double hole SNV, single hole measurement is not good, and double hole measurement Good (alldepth>1000) standard SNVs were confirmed by Sanger sequencing.

Step (8): Indels that meet the criteria of ① double-well INDEL, remove SNP; ② single-well INDEL, alldepth>25, vaf ≥0.4, remove SNP were confirmed by Sanger sequencing.

Preferably, the panel includes 19 genes, 8 fusion genes and related internal reference genes related to the onset of thyroid cancer, as follows:

Detect SNVs in 19 genes: BRAF, HRAS, NRAS, KRAS, EIF1AX, PPM1D, CHEK2, RET, CTNNB1, TP53, AKT1, GNAS, PIK3CA, PTEN, TSHR, CDKN2A, AXIN1, IDH1, VHL

Detect 8 fusion genes: CCDC6/RET, NCOA4/RET, ETV6/NTRK3, STRN/ALK, PAX8/PPARG, TPM3/NTRK3, EML4/ALK, PRKAR1A/RET

Thyroid-specific genes and reference genes: TG, TPO, GAPDH, β-actin, 18S rRNA, 28S rRNA, Tublin, RPLPO (human large ribosomal protein), TFRC (transferrin receptor)

Preferably, the primer design software used for the amplification of the Access ArrayTMSystem is Mass ARRAYAssay Design.

Preferably, the data analysis software after targeted sequencing is BWA, SAMTOOLS and GATK.

Preferably, the criteria for screening SNVs are ① double-well SNV, alldepth>20, vaf>0.3; ② single-well SNV, alldepth>1000, vaf>0.4; The hole test is good (alldepth>1000).

Preferably, the criteria for screening Indels are ① double-well INDEL, remove SNP; ② single-well INDEL, alldepth>25, vaf≥0.4, remove SNP.

The present invention is suitable for the clarification of the differential diagnosis of benign and malignant thyroid nodules, and makes up for the deficiencies and deficiencies in this field. According to the mutation genes and fusion genes that may lead to the onset of thyroid cancer, a combined Panel is established through the Access Array ^TM System to conduct targeted gene identification. Next generation sequencing. Through preliminary next-generation sequencing, data analysis and Sanger sequencing verification of 123 thyroid cancer patients, mutations were detected in 11 of the 19 genes, including hotspot mutations BRAF V600E, NRAS: Q61R, Q61K, RET M918T, PTEN, TSHR , TP53 and AKT1, fusion genes ETV6-NTRK3, NCOA4-RET and CCDC6-RET were detected. The diagnostic rate of the combined molecular markers in 123 patients with thyroid cancer was extremely high. Moreover, it was found that multiple disease-causing gene mutations or driver gene mutations and fusion genes existed in many patients, indicating that the onset of thyroid cancer is caused by the accumulation of multiple gene changes, and has tumor heterogeneity, further confirming that The need for precision medical diagnosis of such diseases. The present invention is suitable for clinical differential diagnosis of benign and malignant thyroid nodules, can perform high-throughput detection, and can be promoted clinically, helps to comprehensively select the treatment plan for patients, fully compensates for the deficiencies and deficiencies in this field, and has great practical value .

In summary, the results of this project confirm that the combined molecular markers based on next-generation sequencing technology play an important role in the diagnosis of benign and malignant thyroid nodules. Targeted next-generation sequencing of combined molecular markers covering most of the pathogenic genes of thyroid cancer can improve the diagnostic rate of thyroid malignancies, successfully make up for the current deficiency of thyroid cytology diagnosis, and guide the diagnosis and treatment of patients with thyroid nodules. It is perfect and avoids the waste of social medical resources and the potential risks of patients' surgery due to unnecessary diagnostic operations. At the same time, the results of this study provide new ideas for the role of genetic diagnosis in clinical practice.

Example 1 Screening and identification of gene polymorphism sites related to benign and malignant thyroid nodules

Sample Description: Thyroid fine needle aspiration cells, RNA extraction, and reverse transcription to obtain a cDNA library.

Specific implementation steps:

Step (1): According to the possible thyroid cancer-causing genes, including mutant genes and fusion genes, and the gene sequence in GRCh37/hg19 in UCSC Genome Browser, the chromosome position, coding, gene size, and pseudogene status of the gene were determined.

Step (2): According to the requirements of Access ArrayTMSystem amplification (the target sequence size is 240bp, there is overlap), design primers, and add tag sequences.

Step (3): After the primers are synthesized, dilute them, and prepare primer mixtures according to the amplification requirements of the Access ArrayTM System, and divide them into 96-well plates.

Step (4): Preparation of amplified template, quantified to 50ng/ul (if less than 50ng/ul is recorded as 50ng/ul), prepared template mixture, and divided into 96-well plates.

Step (5): Use a discharge gun to suck the primers and templates in the 96-well plate onto the 48.48Access Array IFC chip.

Step (6): Put the chip into the Pre-PCR IFC Controller AX instrument, the primers and templates are automatically mixed, and use the FC1Cycler instrument for PCR amplification reaction.

Step (7): Use the Post-PCR IFC Controller AX instrument to collect and harvest PCR products (note that the room needs to be changed during harvesting to prevent contamination)

Step (8): Prepare barcode mixture and add 1:100 diluted product to carry out PCR reaction.

Step (9): Purify the product with magnetic beads, run the gel to confirm that the barcode is added, and prepare for sequencing on the machine.

Step (10): NextSeq500 is sequenced on the machine, and the off-machine data is analyzed using BWA, SAMTOOLS and GATK software.

Step (11): The measured SNVs and indels are filtered through ANNOVAR ( http:// annovar.openbioinformatics.org/ ) functional annotations and public databases (ExAC, 1000Genomes, dbSNP and ClinVar).

Step (12): ①Double hole SNV, alldepth>20, vaf>0.3; ②Single hole SNV, alldepth>1000, vaf>0.4; ③Double hole SNV, single hole measurement is not good, and double hole measurement Good (alldepth>1000) standard SNVs were confirmed by Sanger sequencing.

Step (13): Indels that meet the criteria of ① double-well INDEL and remove SNP; ② single-well INDEL, alldepth>25, vaf≥0.4, and remove SNP were confirmed by Sanger sequencing.

The present invention is applicable to the molecular detection of the differential diagnosis of benign and malignant thyroid nodules, and makes up for the deficiencies and deficiencies in this field. Its principle is that the present invention designs primers through Mass ARRAYAssay Design software among the pathogenic genes related to the occurrence of thyroid cancer that have been reported so far. , on the 48.48Access Array IFC, after multiplex PCR amplification of targeted genes, next-generation sequencing analysis is performed, making it possible for high-throughput and larger-coverage gene diagnosis of the disease.

Through the study of 123 patients, it was found that mutations were detected in 11 of the 19 genes (Table 2), and 3 fusion genes were detected (Table 3), including 115 mutations, and the diagnostic rate was higher than 93%. Further, more driving genes and fusion genes of thyroid cancer in the Chinese population can be brought together in a diagnostic panel, which can perform genetic testing of a large number of samples more efficiently and quickly. The invention is very suitable for patients with thyroid nodules in the Chinese population, can make up for the deficiencies and deficiencies in this field, and has very practical value.

Table 2

table 3

fusion gene name sequence information number of mutation patients 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 has been analyzed, and the results show that each mutation site of the present invention has excellent tissue specificity, and the mutation specificity is detected in thyroid cancer tissue, but rarely in normal somatic cells. There was a mutation of Q61K in the NRAS gene detected only in 1 case of thyroid adenoma. It is worth noting that although thyroid adenoma is a benign disease, the pathological morphology of adenoma is sometimes very similar to papillary thyroid carcinoma. Therefore, diagnostic errors may sometimes occur based on pathology alone. The specific results are shown in Table 4 below:

Table 4

TA, Thyroid adenoma

NG, nodular goiter

PTC, Papillary thyroid carcinoma

FTC, follicular thyroid carcinoma (Follicular thyroid carcinoma, included in PTC)

MTC, Medullary thyroid carcinoma

PDTC, Poorly differentiated thyroid cancer

ATC, Anaplastic thyroid carcinoma

Preparation and effect verification of embodiment 2 kit

This embodiment provides a kit for detecting benign and malignant thyroid nodules.

This kit can detect gene mutations at the mutation sites in the above table 2 and the fusion genes in the above table 3:

Kit main reagents:

(1) Polymorphic site amplification primers

Upstream primer (F) and downstream primer (R) (designed by Mass ARRAY Assay Design software);

(2) Polymorphic site sequencing primers

Sequencing primer (S) (designed according to conventional methods in the art);

(3) Main reagents for PCR: Pfu high-fidelity enzyme, 10×PCR Buffer, dNTPMixtur, ddH ₂ O;

(4) Main reagents for pyrosequencing: 70% ethanol solution, magnetic beads, denaturing buffer, annealing buffer, binding buffer, washing buffer, substrate (ASP, fluorescein), enzyme mixed solution (DNA polymerase, Luciferase, ATP sulfurylase, ATPase), A/T/C/G bases.

Using the kit provided in this example, 300 patients with thyroid nodules were detected. For the specific method, refer to Example 1. A total of 176 patients were detected to carry the gene mutation of the present invention. Finally, the patients with thyroid cancer were confirmed to be 184 cases, the detection rate was as high as 95%. The specific test results are shown in Table 5.

Table 5 Results of targeted gene next-generation sequencing in 300 patients with thyroid cancer

All documents mentioned in this application are incorporated by reference in this application as if each individual document were individually indicated to be incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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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.