WO2018103679A1 - 良性甲状腺结节特异基因 - Google Patents

良性甲状腺结节特异基因 Download PDF

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WO2018103679A1
WO2018103679A1 PCT/CN2017/114889 CN2017114889W WO2018103679A1 WO 2018103679 A1 WO2018103679 A1 WO 2018103679A1 CN 2017114889 W CN2017114889 W CN 2017114889W WO 2018103679 A1 WO2018103679 A1 WO 2018103679A1
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gene
seq
mutation
kit
primer
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French (fr)
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宁光
王卫庆
叶蕾
周晓艺
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宁光
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment

Definitions

  • the present invention relates to the field of medical testing, and more particularly to three benign thyroid nodule specific genes.
  • thyroid nodules With the popularity of conventional thyroid ultrasound, the detection rate of thyroid nodules has increased significantly. A large-scale population study found that the prevalence of thyroid nodules was highest in women and the elderly, reaching 19-68%. Most new nodules are benign nodules, with less than 5% of nodules diagnosed as malignant. Although high-resolution ultrasound combined with fine-needle aspiration cytology results in a diagnosis rate of 85% of benign and malignant thyroid nodules, patients and clinicians are always anxious about whether malignant nodules have malignant potential. Therefore, in 2009, the American Thyroid Association (ATA) recommended regular follow-up of benign nodules every 12-18 months, resulting in huge medical resources and psychosocial burden.
  • ATA American Thyroid Association
  • kits for detecting a benign thyroid nodule comprising one or more pairs of primers selected from the group consisting of:
  • a primer for specifically amplifying a SPOP gene or a transcript the primer amplifying an amplification product having a length of 80 to 2000 bp and containing the 281th position of SEQ ID NO.: 1;
  • a primer for specifically amplifying an EZH1 gene or a transcript the primer amplifying an amplification product having a length of 80 to 2000 bp and containing the 1712th position of SEQ ID NO.: 3;
  • a primer for specifically amplifying a ZNF148 gene or a transcript which amplifies an amplification product having a length of 1000 to 3000 bp and containing positions 1273 to 2871 of SEQ ID NO.: 5.
  • nucleotide sequence of the primer for specifically amplifying the SPOP gene or transcript is as shown in SEQ ID NO.: 7 and 8.
  • nucleotide sequence of the primer for specifically amplifying the EZH1 gene or transcript is as shown in SEQ ID NO.: 9 and 10.
  • the primer that specifically amplifies the ZNF148 gene or transcript is selected from the group consisting of:
  • the kit further comprises a reagent selected from the group consisting of:
  • the mutation includes a single-stranded mutation and a double-stranded mutation.
  • the kit further comprises a reagent selected from the group consisting of:
  • the kit is used for the auxiliary judgment of benign thyroid nodules.
  • the kit is for the detection of a thyroid nodule tissue sample and/or a blood sample.
  • the detection is pre-detection.
  • the blood sample comprises serum and plasma.
  • the detection is performed on an Asian population.
  • the detection is to detect a Chinese population.
  • the detection is for determining whether the thyroid nodule is benign.
  • the test is for determining that the thyroid nodule is not a malignant thyroid nodule, and preferably for determining that the thyroid nodule is not papillary thyroid cancer.
  • a polynucleotide molecule for the preparation of a kit for detecting benign thyroid nodules; wherein said polynucleotide molecule comprises:
  • a SPOP gene (i) a SPOP gene, a primer that specifically amplifies a SPOP gene or a transcript, a probe or a chip that specifically binds to a nucleotide sequence of the SPOP gene, that is, a 281th C ⁇ G mutation in SEQ ID NO.: And/or detecting a specific antibody at position 94 of the P ⁇ R mutation in SEQ ID NO.: 2;
  • an EZH1 gene (ii) an EZH1 gene, a primer that specifically amplifies the EZH1 gene or transcript, a probe or a chip that specifically binds to the nucleotide sequence of the EZH1 gene, that is, the 1712th A ⁇ G mutation in SEQ ID NO.: And/or detecting a specific antibody of the 571th Q ⁇ R mutation in SEQ ID NO.: 4; and/or
  • a primer which specifically amplifies a ZNF148 gene or a transcript and a probe which specifically binds to a nucleotide sequence of the ZNF148 gene, that is, a mutation of the 1273-2871 mutation in SEQ ID NO.: 5.
  • the kit is used for the auxiliary judgment of benign thyroid nodules.
  • the kit further includes a specification in which the following is described:
  • the thyroid nodules of the test subject are prompted to be benign.
  • a benign thyroid nodule related gene for preparing a reagent or a kit for detecting a benign thyroid nodule
  • the benign thyroid nodule related gene comprises a SPOP gene, EZH1 Gene, and / or ZNF148 gene.
  • the reagent or kit is used to detect the following single nucleotide mutations:
  • the nucleotide sequence of the SPOP gene the 281th C ⁇ G in SEQ ID NO.:1.
  • the reagent comprises a primer that specifically amplifies a SPOP gene or a transcript, an amplification product containing the mutation site, a probe that specifically binds to the mutation site, and specificity A nucleic acid chip that detects the mutation site.
  • the kit includes instructions for use and one or more of the following reagents:
  • a container (a) and a primer located within the container that specifically amplifies a SPOP gene or transcript;
  • the SPOP gene is used as a standard or control.
  • the reagent or kit is used to detect the following single nucleotide mutations:
  • the nucleotide sequence of the EZH1 gene that is, the 1712th A ⁇ G in SEQ ID NO.:3.
  • the reagent comprises a primer that specifically amplifies an EZH1 gene or a transcript, an amplification product containing the mutation site, a probe that specifically binds to the mutation site, and specificity A nucleic acid chip that detects the mutation site.
  • the kit includes instructions for use and one or more of the following reagents:
  • the EZH1 gene is used as a standard or control.
  • the reagent or kit is used to detect the following mutations:
  • the nucleotide sequence of the ZNF148 gene the mutation at position 1273 to 2871 in SEQ ID NO.: 5.
  • the ZNF148 gene is used as a standard or control.
  • a method for non-diagnostic detection of a sample of a benign thyroid nodule related gene mutation in vitro comprising the steps of:
  • the nucleotide sequence of the SPOP gene the 281th C ⁇ G in SEQ ID NO.:1;
  • the nucleotide sequence of the EZH1 gene the 1712th A ⁇ G in SEQ ID NO.: 3;
  • the nucleotide sequence of the ZNF148 gene the mutation at position 1273 to 2871 in SEQ ID NO.: 5.
  • the amplification product is 80-2000 bp in length and comprises position 281 in SEQ ID NO: 1, position 1712 in SEQ ID NO.: 3, and/or SEQ ID NO.: 5th in the 1273-2871.
  • the amplified sample is a thyroid nodule tissue sample.
  • a method of detecting a benign thyroid nodule of a subject comprising the steps of:
  • SPOP genes, transcripts and/or proteins and compared to normal SPOP genes, transcripts and/or proteins,
  • the difference indicates that the individual's thyroid nodules are benign.
  • the gene, transcript, and/or protein in the nodule sample of the individual to be tested is detected and compared to the gene, transcript, and/or protein in the blood sample of the individual.
  • the difference is that the following mutations are present:
  • the nucleotide sequence of the SPOP gene is the 281th C ⁇ G in SEQ ID NO.:1;
  • the nucleotide sequence of the EZH1 gene is the 1712th A ⁇ G in SEQ ID NO.:3;
  • the nucleotide sequence of the ZNF148 gene is mutated at positions 1273 to 2871 in SEQ ID NO.: 5.
  • the individual's thyroid nodule tissue sample is tested to detect whether the individual's thyroid nodule is benign.
  • the inventors have extensively and intensively studied, and for the first time, unexpectedly found genes associated with three sex nodules, namely SPOP gene, EZH1 gene and ZNF148 gene.
  • SPOP, EZH1 and ZNF148 are mutually dissociated gene mutations, which occur in 29.2% of benign nodules, in paired PTC (papillary Adenocarcinoma, papillary thyroid carcinoma) does not occur in tumor tissue.
  • the above three benign nodule-related genes provide "excluded" information for malignant thyroid nodules and have important diagnostic significance in gene mutation detection.
  • the protein encoded by the SPOP gene regulates the transcriptional inhibitory activity of the death-associated protein (DAXX), which interacts with histone deacetylase, core histone, and other histone-associated proteins.
  • DAXX death-associated protein
  • mice the SPOP-encoded protein binds to the leucine zipper domain of macroH2A1.2, which is an isoform of the H2A histone, enriched on the inactive X chromosome.
  • the BTB/POZ domain of this protein interacts with other proteins, regulates transcriptional repression activity, and interacts with components of the co-inhibition complex of histone deacetylase. Selective splicing of the SPOP gene produces many transcript variants encoding the same protein.
  • the protein encoded by the EZH1 gene (NM_001991) is a component of a non-canonical polycombine inhibitor complex 2 (PRC-2) that regulates the methylation of histone H3 27th lysine (H3K27) during maintenance of the embryo Stem cells play an important role in pluripotency and plasticity.
  • PRC-2 non-canonical polycombine inhibitor complex 2
  • the protein encoded by the ZNF148 gene belongs to a class of Krüppel-like transcription factors, which both have transcriptional activation and transcription inhibition.
  • the low expression of ZNF148 is associated with poor prognosis in colorectal cancer, and the expression of ZNF148 overexpressing clones is significantly reduced in hepatocellular carcinoma cell lines.
  • Thyroid nodules are masses in the thyroid gland that move up and down with the thyroid gland as they swallow. They are common clinical conditions and can be caused by a variety of causes. There are many thyroid diseases in the clinic, such as thyroid degeneration, inflammation, autoimmunity and new organisms, which can be expressed as nodules. Thyroid nodules can be single or multiple, and multiple nodules have a higher incidence than single nodules, but the incidence of single nodular thyroid cancer is higher.
  • Thyroid nodules are classified into benign thyroid nodules and malignant thyroid nodules. Most new nodules are benign nodules, and less than 5% of malignant nodules are clearly diagnosed.
  • the present invention provides a method for detecting a benign thyroid nodule in an individual by detecting a SPOP gene, an EZH1 gene, and a ZNF148 gene in a thyroid nodule, and comparing it with a corresponding gene in a blood sample. In comparison, predict whether the thyroid nodules are benign in advance.
  • the method of the invention can be used to aid in diagnostic typing, especially early assisted diagnosis.
  • kits of the invention detect the following mutations:
  • the test sample used in the present invention is not particularly limited, and for detecting a mutation site, it may be DNA or mRNA extracted from a sample such as a cell or a tissue. Since the mutation of the present invention is mainly present in thyroid nodule cells, it is usually not present in peripheral blood cells. Therefore, the preferred test sample is thyroid nodule cells, and peripheral blood cells can be used as a control.
  • a part or all of the gene sequence detection of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as a "gene chip” or a “nucleic acid chip”) for analyzing the sequence and difference of genes in the tissue. Expression analysis, as well as genetic diagnosis.
  • the corresponding transcripts can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using specific primers for the SPOP gene, EZH1 gene, and ZNF148 gene.
  • RT-PCR RNA-polymerase chain reaction
  • Detection can be directed to cDNA as well as to genomic DNA.
  • Mutated forms of the SPOP gene, EZH1 gene, and ZNF148 gene include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type DNA sequences. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of related proteins, so the presence or absence of mutations can be indirectly determined by Northern blotting and Western blotting.
  • the most convenient method for detecting the mutation site of the present invention is to obtain an amplification product by separately amplifying the SPOP gene, the EZH1 gene, and the ZNF148 gene with specific primers of the SPOP gene, the EZH1 gene, and the ZNF148 gene; Whether the single nucleotide mutation (SNV) of the present invention exists in the amplification product.
  • SNV single nucleotide mutation
  • primer sequences that can be used for detection are as follows:
  • R AACCAGTGGAAAGAGAATGC (SEQ ID NO.: 10)
  • ZNF148 last exon primer sequence:
  • the primers are 15 to 50 bp in length, preferably 20 to 30 bp.
  • the primer is fully complementary to the template sequence, those skilled in the art will recognize that in the case of a certain non-complement of the primer and the template (especially the 5' end of the primer), it is also capable of specific amplification (ie only Amplify the desired fragment). Kits containing these primers and methods of using the same are within the scope of the present invention as long as the amplified product amplified by the primer contains the corresponding position of the gene mutation site of the present invention.
  • the length of the amplification product is not particularly limited, the length of the amplification product is usually 100 to 2000 bp, preferably 150 to 1500 bp, more preferably 200 to 1000 bp. These amplification products should all contain a single nucleotide mutation (SNV) site of the invention.
  • SNV single nucleotide mutation
  • tissue samples were obtained from surgical specimens of 28 patients, including 21 patients with cancerous nodules (also with benign thyroid knots) Section and papillary thyroid carcinoma) and 8 patients with simple benign nodules.
  • a simple benign nodule is defined as having at least one thyroid nodule and is present for more than 2 years without malignant histological signs. All patients were not treated (radiotherapy or chemotherapy) prior to specimen collection. Patient blood samples were used as germ cell line controls (to identify somatic variations). All tissues were quickly stored in liquid nitrogen for collection and analyzed independently to minimize contamination and interference.
  • the BSA software (version 0.7) was used to sequence the entire exon-sequenced paired sequence with the human reference genome (hg19) using its default parameter settings. Repetitive products resulting from PCR amplification were removed using the Picard tool (version 1.1). In a localized region with an insertion or deletion mutation, the sequence alignment is re-sequenced and the base quality score is corrected. After these analyses, the BAM file (binary alignment file) was finally obtained, and the mutation site was identified using the UnifiedGenotyper module in the GATK software package. In order to compare the mutations of specific patient-matched tissues, a single normal tissue-multiple tumor sample strategy was used based on the GATK combined recognition of somatic mutation sites.
  • tissue and control blood samples must have complete, sufficient sequence coverage (at least 10x depth); 2) at least one sequence covering the site in the tissue, at least 10% support there are mutated bases (if the local depth is >50 times, it is set to 5%); 3) In the tissue, the mutation data is found to be at least 3 times more. 4) For each possible somatic mutation site, use chi-square test to detect multiple The allelic depth and frequency of the tissue and control blood samples; 5) Exclude sites in the control blood sample where mutations also occur (more than 2 sequences support mutations in the blood sample).
  • the mutation density is calculated.
  • the somatically mutated base uses the aforementioned SNV analysis results.
  • the matched mutant sequences of tumors and benign nodules were compared to find important mutations unique to benign tumors.
  • the mutation sites found in the whole exome sequencing were further verified by PCR using a 96-well plate (GeneAmp PCR System 9700, supplied by Biosystems, France), using 20 ng of DNA template per reaction.
  • the PCR product was sequenced by a 3730xl DNA Analyzer (Applied Biosystems, Courtaboeuf, France) and analyzed using sequencing analysis software (version 5.2, Applied Biosystems, Courtaboeuf, France). All positive mutations were confirmed by an artificial check based on the original sequenced trace file.
  • SPOP detected in 4 patients, 14.3%
  • EZH1 detected in 3 patients, 10.7%
  • ZNF148 detected in 6 patients, 21.4%
  • Both SPOP and EZH1 are hot-spot mutations (P94R) and (Q571R); the mutations of ZNF148 are located in the last exon, which is a nonsense mutation or a frameshift mutation.
  • Thyroid nodule formation is a primary early stimulator of goiter.
  • causes of nodule formation include iodine deficiency, nutritional goiter or autoimmune diseases.
  • thyroid nodules resulting from local proliferation of follicular epithelial cells form monoclonal proliferation and are caused by somatic mutations.
  • TSHR, GNAS, or RAS family genes have somatic mutations.
  • SPOP, EZH1, and ZNF148 are mutually dissociated gene mutations that occur only in 29.2% of benign nodules and do not occur in paired PTC tumor tissues.
  • the expanded sample was validated in 259 benign nodules, and 25.8% of the nodules contained these three gene mutations.
  • these three genes are involved in tumor-associated cell biological behavior, the inventors performed functional experiments in thyroid cell lines, and found that these three genes only promote proliferation, but do not affect the invasion function. The above findings suggest that these three gene mutations are involved in the formation of benign thyroid nodules, but do not lead to their transformation into tumors.
  • the gene mutation detection of thyroid nodules only contains thyroid cancer conversion-related genes, which is the "inclusion” test; and the inventors found three benign nodule-related genes such as SPOP, EZH1 and ZNF148, providing "excluded” information in gene mutations. There is an important diagnostic significance in the test.

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Abstract

本发明公开了三种良性甲状腺结节特异基因SPOP、EZH1和ZNF148及其在良性甲状腺结节检测中的应用。本发明还提供了一种检测良性甲状腺结节的方法和相应的检测试剂盒。

Description

良性甲状腺结节特异基因 技术领域
本发明涉及医学检测领域，更具体地涉及三种良性甲状腺结节特异基因。
背景技术
随着常规甲状腺超声检查的普及，甲状腺结节的检出率明显上升。大规模的人群调查研究发现，女性和老年人群中甲状腺结节的患病率最高，达到19-68％。大多数新发结节都为良性结节，明确诊断为恶性的结节不到5％。虽然高分辨率超声联合细针穿刺细胞学使得甲状腺良恶性结节诊断准确率达到85％，患者与临床医生始终焦虑于良性结节是否具有恶性化潜能。因此，2009年美国甲状腺协会(ATA)推荐对于良性结节每12-18个月进行定期随访，从而造成了巨大的医疗资源与社会心理负担。
数年来，对于甲状腺结节的分子机制研究主要集中于恶性结节，甲状腺癌分子机制的研究。2014年The Cancer Genome Atlas(TCGA)甲状腺癌研究组详细描述了乳头状癌的基因组特点，发现96.5％的甲状腺乳头状癌存在明确的驱动基因变异。然而，甲状腺良性结节的遗传特征极少有研究报道。本领域迫切需要对良性甲状腺结节的遗传特征进行研究。
发明内容
本发明的目的在于提供良性甲状腺结节的特异基因。
在本发明的第一方面，提供了一种用于检测良性甲状腺结节的试剂盒，所述试剂盒包括选自下组的一对或多对引物：
(i)特异性扩增SPOP基因或转录本的引物，所述的引物扩增出长度为80-2000bp且含有SEQ ID NO.:1中第281位的扩增产物；
(ii)特异性扩增EZH1基因或转录本的引物，所述的引物扩增出长度为80-2000bp且含有SEQ ID NO.:3中第1712位的扩增产物；
(iii)特异性扩增ZNF148基因或转录本的引物，所述的引物扩增出长度为1000-3000bp且含有SEQ ID NO.:5中第1273-2871位的扩增产物。
在另一优选例中，所述特异性扩增SPOP基因或转录本的引物的核苷酸序列如SEQ ID NO.:7和8所示。
在另一优选例中，所述特异性扩增EZH1基因或转录本的引物的核苷酸序列如SEQ ID NO.:9和10所示。
在另一优选例中，所述特异性扩增ZNF148基因或转录本的引物选自下组：
(i)核苷酸序列如SEQ ID NO.:11和12所示的引物对；
(ii)核苷酸序列如SEQ ID NO.:13和14所示的引物对；
(iii)核苷酸序列如SEQ ID NO.:15和16所示的引物对。
在另一优选例中，所述试剂盒还含有选自下组的试剂：
(a)与SEQ ID NO.:1中第281位的C→G突变结合的探针或芯片；
(b)识别SEQ ID NO.:1中第281位C→G突变的限制性内切酶；
(c)与SEQ ID NO.:3中第1712位的A→G突变结合的探针或芯片；
(d)识别SEQ ID NO.:3中第1712位A→G突变的限制性内切酶。
在另一优选例中，所述的突变包括单链突变和双链突变。
在另一优选例中，所述试剂盒还含有选自下组的试剂：
(I)检测SEQ ID NO.:2中第94位P→R突变的特异性抗体；
(II)检测SEQ ID NO.:4中第571位Q→R突变的特异性抗体。
在另一优选例中，所述试剂盒用于良性甲状腺结节的辅助判断。
在另一优选例中，所述试剂盒用于甲状腺结节组织样本和/或血液样本的检测。
在另一优选例中，所述检测为预先检测。
在另一优选例中，所述的血液样品包括血清和血浆。
在另一优选例中，所述的检测是对亚洲人群进行检测。
在另一优选例中，所述的检测是对中国人群进行检测。
在另一优选例中，所述的检测是用于判断甲状腺结节是否为良性。
在另一优选例中，所述的检测是用于判断甲状腺结节不为恶性甲状腺结节，较佳地，用于判断甲状腺结节不为乳头状甲状腺癌。
在本发明的第二方面，提供了一种多核苷酸分子的用途，用于制备检测良性甲状腺结节的试剂盒；其中，所述的多核苷酸分子包括：
(i)SPOP基因、特异性扩增SPOP基因或转录本的引物、与SPOP基因的核苷酸序列即SEQ ID NO.:1中第281位C→G突变特异性结合的探针或芯片、和/或检测SEQ ID NO.:2中第94位P→R突变的特异性抗体；
(ii)EZH1基因、特异性扩增EZH1基因或转录本的引物、与EZH1基因的核苷酸序列即SEQ ID NO.:3中第1712位A→G突变特异性结合的探针或芯片、和/或检测SEQ ID NO.:4中第571位Q→R突变的特异性抗体；和/或
(iii)、特异性扩增ZNF148基因或转录本的引物、与ZNF148基因的核苷酸序列即SEQ ID NO.:5中第1273-2871位突变特异性结合的探针。
在另一优选例中，所述试剂盒用于良性甲状腺结节的辅助判断。
在另一优选例中，所述试剂盒还包括一说明书，说明书中记载以下内容：
当检测对象具有一种或多种所述突变，则提示检测对象的甲状腺结节为良性。
在本发明的第三方面，提供了一种良性甲状腺结节相关基因的用途，用于制备检测良性甲状腺结节的试剂或试剂盒，并且所述的良性甲状腺结节相关基因包括SPOP基因、EZH1基因、和/或ZNF148基因。
在另一优选例中，所述的试剂或试剂盒用于检测以下单核苷酸突变：
SPOP基因的核苷酸序列：即SEQ ID NO.:1中第281位C→G。
在另一优选例中，所述的试剂包括特异性扩增SPOP基因或转录本的引物、含有所述突变位点的扩增产物、与所述突变位点特异性结合的探针、特异性检测所述突变位点的核酸芯片。
在另一优选例中，所述的试剂盒包括使用说明书以及一种或多种以下试剂：
容器(a)以及位于所述容器内的特异性扩增SPOP基因或转录本的引物；
容器(b)以及位于所述容器内的与所述突变位点特异性结合的探针；
容器(c)以及位于所述容器内的特异性检测所述突变位点的核酸芯片。
在另一优选例中，所述的SPOP基因用作标准品或对照品。
在另一优选例中，所述的试剂或试剂盒用于检测以下单核苷酸突变：
EZH1基因的核苷酸序列：即SEQ ID NO.:3中第1712位A→G。
在另一优选例中，所述的试剂包括特异性扩增EZH1基因或转录本的引物、含有所述突变位点的扩增产物、与所述突变位点特异性结合的探针、特异性检测所述突变位点的核酸芯片。
在另一优选例中，所述的试剂盒包括使用说明书以及一种或多种以下试剂：
容器(a)以及位于所述容器内的特异性扩增EZH1基因或转录本的引物；
容器(b)以及位于所述容器内的与所述突变位点特异性结合的探针；
容器(c)以及位于所述容器内的特异性检测所述突变位点的核酸芯片。
在另一优选例中，所述的EZH1基因用作标准品或对照品。
在另一优选例中，所述的试剂或试剂盒用于检测以下突变：
ZNF148基因的核苷酸序列：即SEQ ID NO.:5中第1273-2871位突变。
在另一优选例中，所述的ZNF148基因用作标准品或对照品。
在本发明的第四方面，提供了一种体外非诊断性检测样品是否存在良性甲状腺结节相关基因突变的方法，包括步骤：
(a)用特异性引物扩增样品的SPOP基因、EZH1基因、和/或ZNF148基因，得到扩增产物；和
(b)检测扩增产物中是否存在以下突变位点：
SPOP基因的核苷酸序列：即SEQ ID NO.:1中第281位C→G；
EZH1基因的核苷酸序列：即SEQ ID NO.:3中第1712位A→G；
ZNF148基因的核苷酸序列：即SEQ ID NO.:5中第1273-2871位突变。
在另一优选例中，所述的扩增产物的长度为80-2000bp且含有SEQ ID NO:1中第281位、SEQ ID NO.:3中第1712位、和/或SEQ ID NO.:5中第1273-2871位。
在另一优选例中，所述的扩增样本是甲状腺结节组织样本。
在本发明的第五方面，提供了一种检测个体良性甲状腺结节的方法，所述方法包括步骤：
检测该个体的下述基因、转录本和/或蛋白：
SPOP基因、转录本和/或蛋白，并与正常的SPOP基因、转录本和/或蛋白相比较，
EZH1基因、转录本和/或蛋白，并与正常的EZH1基因、转录本和/或蛋白相比较，
ZNF148基因、转录本和/或蛋白，并与正常的ZNF148基因、转录本和/或蛋白相比较，
其中，存在差异就提示该个体的甲状腺结节为良性。
在另一优选例中，检测待测个体结节样本中的基因、转录本和/或蛋白，并与该个体血液样本中的基因、转录本和/或蛋白相比较。
在另一优选例中，所述的差异是存在以下突变：
SPOP基因的核苷酸序列即SEQ ID NO.:1中第281位C→G；
EZH1基因的核苷酸序列即SEQ ID NO.:3中第1712位A→G；
ZNF148基因的核苷酸序列即SEQ ID NO.:5中第1273-2871位发生突变。
在另一优选例中，对个体的甲状腺结节组织样本进行检测，从而检测个体甲状腺结节是否为良性。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现与三种性结节相关的基因，分别是SPOP基因、EZH1基因和ZNF148基因。实验表明，SPOP,EZH1和ZNF148为互不相交的基因突变，在29.2％的良性结节中出现，在配对的PTC(乳头状甲 状腺癌，papillary thyroid carcinoma)肿瘤组织中不发生。上述3个良性结节相关基因，提供了恶性甲状腺结节的“排除”信息，在基因突变检测中有重要诊断意义。
SPOP基因
SPOP基因(NM_001007226)编码的蛋白调节死亡相关蛋白(DAXX)的转录抑制活性，DAXX与组蛋白去乙酰化酶、核心组蛋白和其他组蛋白相关蛋白相互作用。在小鼠中，SPOP编码的蛋白结合到macroH2A1.2的亮氨酸拉链结构域，后者是H2A组蛋白的异构体，富集在失活的X染色体上。此蛋白的BTB/POZ结构域与其他蛋白作用，调节转录抑制活性，与组蛋白去乙酰化酶的共抑制复合物的组件相互作用。SPOP基因的选择性剪切会产生许多转录变异体，编码相同的蛋白。
EZH1基因
EZH1基因(NM_001991)编码的蛋白是一个非经典的多梳抑制复合物2(PRC-2)的组成部分，调节组蛋白H3第27位赖氨酸(H3K27)的甲基化作用，在维持胚胎干细胞的多能性和可塑性中起重要作用。
ZNF148基因
ZNF148基因(NM_021964)编码的蛋白(锌指蛋白148)属于Krüppel-like转录因子的一类，对其目标蛋白既有转录激活作用也可抑制转录。ZNF148的低表达与结直肠癌的预后不良相关，在肝细胞癌细胞系中，ZNF148过表达的克隆形成明显减少。
甲状腺结节
甲状腺结节是指在甲状腺内的肿块，可随吞咽动作随甲状腺而上下移动，是临床常见的病症，可由多种病因引起。临床上有多种甲状腺疾病，如甲状腺退行性变、炎症、自身免疫以及新生物等都可以表现为结节。甲状腺结节可以单发，也可以多发，多发结节比单发结节的发病率高，但单发结节甲状腺癌的发生率较高。
甲状腺结节分为良性甲状腺结节和恶性甲状腺结节，大多数新发结节都为良性结节，明确诊断为恶性的结节不到5％。
检测方法、检测试剂及试剂盒
本发明提供了一种检测个体良性甲状腺结节的方法，通过检测甲状腺结节中的SPOP基因、EZH1基因、和ZNF148基因，并将其与血液样本中的相应基因进行比 较，提前预测甲状腺结节是否为良性。本发明的方法可以用于辅助诊断分型，尤其是早期辅助性诊断。
具体地，本发明的方法、试剂及试剂盒检测如下突变：
Figure PCTCN2017114889-appb-000001
本领域的技术人员知道，有大量的分析技术可用于检测基因中所述位点是否存在突变。这些技术包括(但并不限于)：DNA测序、杂交测序；酶促错配切割、异源双链分析、点杂交、寡核苷酸阵列(芯片)、焦磷酸测序、Taqman探针检测技术、分子信标等。
用于本发明的测试样品没有特别限制，对于检测突变位点而言，可以是从细胞或组织等样品中抽提出的DNA或mRNA。由于本发明的突变主要存在于甲状腺结节细胞中，通常而不存在于外周血细胞中。因此，优选的测试样品是甲状腺结节细胞，外周血细胞可以用作对照。
本发明的基因序列检测的一部分或全部可作为探针固定在微阵列(microarray)或DNA芯片(又称为“基因芯片”或“核酸芯片”)上，用于分析组织中基因的序列和差异表达分析，以及基因诊断。用SPOP基因、EZH1基因、和ZNF148基因的特异性引物进行RNA-聚合酶链反应(RT-PCR)体外扩增也可检测相应的转录产物。
检测可以针对cDNA，也可针对基因组DNA。SPOP基因、EZH1基因、和ZNF148基因的突变的形式包括与正常野生型DNA序列相比的点突变、易位、缺失、重组和其它任何异常等。可用已有的技术如Southern印迹法、DNA序列分析、PCR和原位杂交检测突变。另外，突变有可能影响相关蛋白的表达，因此用Northern印迹法、Western印迹法可间接判断基因有无突变。
最方便的检测本发明突变位点的方法，是通过用SPOP基因、EZH1基因、和ZNF148基因的特异性引物分别扩增样品的SPOP基因、EZH1基因、和ZNF148基因，得到扩增产物；然后检测扩增产物中是否存本发明的单核苷酸突变(SNV)。
具体地，可用于检测的代表性引物序列如下所示：
SPOP引物序列:
F:CCAGATCAAAGCCACAAC(SEQ ID NO.:7)
R:CTGGACGATAGAGTAAGACC(SEQ ID NO.:8)
EZH1引物序列：
F:ACACCTGCTTTTTTGACTCG(SEQ ID NO.:9)
R:AACCAGTGGAAAGAGAATGC(SEQ ID NO.:10)
ZNF148最后一个外显子引物序列：
1)F:TCTTGGTTGACCAAAACCAC(SEQ ID NO.:11)
R:GGCCCCTCCTGCAAATTATC(SEQ ID NO.:12)
2)F:TTTGGGAGGGTCTGGTTATC(SEQ ID NO.:13)
R:CCACATATGAAGAGAGCAAAG(SEQ ID NO.:14)
3)F:CAGGCTTTGGACAGAACTAG(SEQ ID NO.:15)
R:TACACAGAGTAACCCCACTC(SEQ ID NO.:16)
应理解，在本发明首次揭示了SPOP基因、EZH1基因、和ZNF148基因的突变位点与良性甲状腺结节的相关性之后，本领域技术人员可以方便地设计出可特异性扩增出含该突变位点位置的扩增产物，然后通过测序等方法确定是否存在本发明的突变。通常，引物的长度为15-50bp，较佳地为20-30bp。虽然引物与模板序列完全互补是优选的，但是本领域技术人员知道，在引物与模板存在一定的不互补(尤其是引物的5'端)的情况下，也能够特异性地扩增(即仅扩增出所需的片段)。含有这些引物的试剂盒和使用这些引物的方法都在本发明范围之内，只要该引物扩增出的扩增产物含有本发明基因突变位点的对应位置。
虽然扩增产物的长度没有特别限制，但是通常扩增产物的长度为100-2000bp，较佳地为150-1500bp，更佳地为200-1000bp。这些扩增产物都应含有本发明的单核苷酸突变(SNV)位点。
本发明的主要优点包括：
(a)发现SPOP,EZH1和ZNF148这3个良性结节相关基因，提供了甲状腺乳头状癌的“排除”信息；
(b)强有力地证实了大部分良性结节并非癌前状态，与乳头状癌的发生无关；
(c)存在SPOP,ZNF148,或EZH1突变时，可能无需对良性甲状腺结节进行常规监测，节省大量医疗资源。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则 百分比和份数按重量计算。
通用材料和方法
样本准备和DNA提取
经上海交通大学医学院附属瑞金医院伦理委员会批准，并在患者知情同意后，从28位患者的手术标本中共获得127例组织样本，其中包括21位伴癌结节患者(同时患有甲状腺良性结节和甲状腺乳头状癌)与8位单纯良性结节患者。单纯良性结节定义为至少患有一个甲状腺结节并存在时间大于2年，且无恶性组织学征象。所有患者在标本采集前均未接受治疗(放疗或化疗)。使用患者血液标本作为生殖细胞系对照(以识别体细胞变异)。所有组织收集时迅速放入液氮中储存，并独立分析，以最大程度减少污染和干扰。经有经验的病理科医师检阅核对HE染色切片后，对病理证实的区域提取DNA(甲状腺乳头状癌组织的细胞密度＞80％)。使用数字病理切片扫描仪nanozoomer 2.0-RS(Hamamatsu)扫描病理切片，并使用QIAGEN DNeasy Blood & Tissue Kit提取组织DNA。对于伴癌结节患者，同时收集良性结节、乳头状癌和正常组织；对于单纯结节患者，收集良性结节和配对的正常甲状腺组织。
全外显子组测序
通过超声组织匀浆仪将28例患者共127个组织DNA随机打碎成小片段，构建测序文库，平均插入片段大小为300bp。使用SureSelect Human All Exon 50Mb kit(AgilentTechnologies,Santa Clara,CA)进行全外显子组捕捉，再用Illumina HiSeq2500测序系统进行深度测序，生成100bp的配对序列。所有样本测序数据经过严格过滤后，得到平均为13.26GB数据量的高质量原始测序数据(全外显子测序的平均有效覆盖为161x，最低为130倍覆盖，最高为180倍)。
变异位点的注释和命名
使用BWA软件(第0.7版)，采用其默认参数设置，将获得的全外显子测序的配对序列与人类参考基因组(hg19)进行序列比对。PCR扩增造成的重复产物使用Picard工具(1.1版)进行去除。在有插入或缺失突变的局部区域，重新进行序列比对，并矫正碱基质量打分。经过这些分析，最后获得BAM文件(二进制比对文件)，使用GATK软件包中的UnifiedGenotyper模块识别突变位点。为了比较具体每个患者配对组织的突变情况，在GATK联合识别体细胞突变位点的基础上，使用单个正常组织-多个肿瘤样本的策略。为避免测序或比对过程中的误差，采用以下标准：1)组织和对照血样都要有完整、足够的序列覆盖(至少10x深度)；2)组织中覆盖某个位点的序列中，至少10％支持此处有突变碱基(如果局部深度＞50倍，则设为5％)；3)组织中，测序数据中发现突变至少要超过3次。4)对每个可能的体细胞突变位点，使用卡方检验检测多个 组织和对照血样的等位基因深度和频率；5)排除对照血样品中也出现突变的位点(大于2个序列支持血样中也发生突变)。后续分析中，排除dbSNP数据库中的常见突变(build 142)、千人基因组中(最小等位基因频率MAF＞5％)、exome aggregation consortium数据库中(MAF＞1％)、内含子区域和基因间隔区的突变。使用ANNOVAR软件对体细胞突变的单碱基突变(SNV)和插入缺失突变进行注释，找出位于的基因及可能受影响的蛋白编码。
突变分析
假设人类外显子的蛋白编码基因共30Mb并完全覆盖，计算突变密度。体细胞突变的碱基使用前述的SNV分析结果。分析良性肿瘤的共有突变时，比较配对肿瘤和良性结节的突变序列，以找到良性肿瘤特有的重要突变。
使用PCR和Sanger一代测序验证突变
对全外显子组测序发现的变异位点，进一步使用96孔板进行PCR检测验证(GeneAmp PCR系统9700，由法国的Biosystems提供)，每个反应使用20ng的DNA模板。PCR产物由3730xl DNA Analyzer(Applied Biosystems,Courtaboeuf,France)测序，并使用测序分析软件(5.2版本Applied Biosystems,Courtaboeuf,France)进行分析。所有阳性的突变均根据原始测序的trace文件通过了人工核对得到了确认。
扩大群体评估良性结节中重要突变的频率
进一步收集了259例患者共328例液氮冰冻的甲状腺良性结节组织。按上述方法抽提基因组DNA，对SPOPP94R和EZH1Q571R热点突变位点，设计引物对位点所在外显子区段进行PCR扩增，并使用Sanger测序方法对PCR产物进行测序，根据原始测序的trace文件人工核对确认变异，计算变异频率。对于ZNF148基因(NM_021964)，由于全外显子组发现多个位于最后一个外显子的变异，因此设计PCR产物，对所有编码区域和内含子-外显子连接区域的侧翼逐一测序，统计整个ZNF148编码区的变异。
实施例1甲状腺结节的突变分析
对手术收集的28例患者的127例组织样本进行全外显子测序和突变分析，来自21位伴癌结节患者(同时患有甲状腺良性结节和甲状腺乳头状癌PTC)的样本为PTC组样本，来自8位单纯良性结节患者的样本为非PTC组样本。
在28位患者的样本库中，共发现了535个基因的734个体细胞突变。良性结节中的突变频率(每Mb有0.36个突变)实际上高于乳头状癌(每Mb有0.33个突变)(P＝0.58)。经比较，来源于PTC组和非PTC组的良性结节的突变频率无明显差异(分别为每Mb有0.34和每Mb有0.38个突变，P＝0.70，t检验)。
28位患者的良性结节中，最常见的频发突变为SPOP(有4位患者中检出， 14.3％)，EZH1(有3位患者中检出，10.7％)和ZNF148(有6位患者中检出，21.4％)。SPOP和EZH1均为热点突变，分别为(P94R)和(Q571R)；ZNF148的突变均位于最后一个外显子，为无义突变或移码突变。
实施例2良性甲状腺结节特异基因的扩大分析
为扩大样本验证这三个基因与甲状腺良性结节的特异关系，对231例额外的良性甲状腺结节患者进行检测，结果发现，231例患者中，有29例患者(11.2％)存在SPOPP94R突变，24例患者存在EZH1Q571R突变，14例患者存在ZNF148突变(5.4％)，每个突变互不相交。
对甲状腺癌的TCGA数据库中的信息进行分析，结果显示上述三个基因的发生率极低(几千份样本中，只发现有1个SPOP，2个EZH1，2个ZNF148)，且均同时伴有已知的PTC驱动突变。
讨论
甲状腺结节形成是甲状腺肿大的原发早期刺激因素。结节形成的原因包括碘缺乏、营养性甲状腺肿或自身免疫疾病。相比之下，滤泡上皮细胞的局部增殖导致的甲状腺结节形成为单克隆增殖，且由体细胞突变引起。甲功正常的甲状腺结节中，仅有一小部分发生TSHR，GNAS，或RAS家族基因的体细胞突变。此外，多结节疾病中尚不清楚是否有特异的亚群为癌前病变。利用全外显子测序技术首次描述了良性甲状腺结节的基因突变情况。有趣的是，发明人发现虽然良性结节和乳头状癌的基因突变频率相似，但特异的基因却各不相同。SPOP、EZH1和ZNF148为互不相交的基因突变，仅在29.2％的良性结节中出现，在配对的PTC肿瘤组织中不发生。扩大样本到259个良性结节中验证，25.8％的结节含有这3种基因突变。虽然这3个基因参与肿瘤相关的细胞生物学行为，发明人在甲状腺细胞系中做了功能实验，结果发现这3个基因仅促进增殖，但不影响侵袭功能。以上发现提示，这3个基因突变参与良性甲状腺结节形成，但并不导致其向肿瘤转化。目前甲状腺结节的基因突变检测仅包含甲状腺癌转换相关基因，为“纳入”检测；而发明人发现了SPOP,EZH1和ZNF148这3个良性结节相关基因，提供“排除”信息，在基因突变检测中有重要诊断意义。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于检测良性甲状腺结节的试剂盒，其特征在于，所述试剂盒包括选自下组的一对或多对引物：
    (i)特异性扩增SPOP基因或转录本的引物，所述的引物扩增出长度为80-2000bp且含有SEQ ID NO.:1中第281位的扩增产物；
    (ii)特异性扩增EZH1基因或转录本的引物，所述的引物扩增出长度为80-2000bp且含有SEQ ID NO.:3中第1712位的扩增产物；
    (iii)特异性扩增ZNF148基因或转录本的引物，所述的引物扩增出长度为1000-3000bp且含有SEQ ID NO.:5中第1273-2871位的扩增产物。
  2. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒还含有选自下组的试剂：
    (a)与SEQ ID NO.:1中第281位的C→G突变结合的探针或芯片；
    (b)识别SEQ ID NO.:1中第281位C→G突变的限制性内切酶；
    (c)与SEQ ID NO.:3中第1712位的A→G突变结合的探针或芯片；
    (d)识别SEQ ID NO.:3中第1712位A→G突变的限制性内切酶。
  3. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒还含有选自下组的试剂：
    (I)检测SEQ ID NO.:2中第94位P→R突变的特异性抗体；
    (II)检测SEQ ID NO.:4中第571位Q→R突变的特异性抗体。
  4. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒用于良性甲状腺结节的辅助判断。
  5. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒还包括一说明书，说明书中记载以下内容：
    当检测对象具有一种或多种所述突变，则提示检测对象的甲状腺结节为良性。
  6. 一种多核苷酸分子的用途，其特征在于，用于制备检测良性甲状腺结节的试剂盒；其中，所述的多核苷酸分子包括：
    (i)SPOP基因、特异性扩增SPOP基因或转录本的引物、与SPOP基因的核苷酸序列即SEQ ID NO.:1中第281位C→G突变特异性结合的探针或芯片、和/或检测SEQ ID NO.:2中第94位P→R突变的特异性抗体；
    (ii)EZH1基因、特异性扩增EZH1基因或转录本的引物、与EZH1基因的核苷酸序列即SEQ ID NO.:3中第1712位A→G突变特异性结合的探针或芯片、和/或检测SEQ ID NO.:4中第571位Q→R突变的特异性抗体；和/或
    (iii)、特异性扩增ZNF148基因或转录本的引物、与ZNF148基因的核苷酸序列即SEQ ID NO.:5中第1273-2871位突变特异性结合的探针。
  7. 一种良性甲状腺结节相关基因的用途，其特征在于，用于制备检测良性甲状腺结节的试剂或试剂盒，并且所述的良性甲状腺结节相关基因包括SPOP基因、EZH1基因、和/或ZNF148基因。
  8. 如权利要求7所述的用途，其特征在于，所述的试剂或试剂盒用于检测以下单核苷酸突变：
    SPOP基因的核苷酸序列：即SEQ ID NO.:1中第281位C→G。
  9. 如权利要求7所述的用途，其特征在于，所述的试剂或试剂盒用于检测以下单核苷酸突变：
    EZH1基因的核苷酸序列：即SEQ ID NO.:3中第1712位A→G。
  10. 一种体外非诊断性检测样品是否存在良性甲状腺结节相关基因突变的方法，其特征在于，包括步骤：
    (a)用特异性引物扩增样品的SPOP基因、EZH1基因、和/或ZNF148基因，得到扩增产物；和
    (b)检测扩增产物中是否存在以下突变位点：
    SPOP基因的核苷酸序列：即SEQ ID NO.:1中第281位C→G；
    EZH1基因的核苷酸序列：即SEQ ID NO.:3中第1712位A→G；
    ZNF148基因的核苷酸序列：即SEQ ID NO.:5中第1273-2871位突变。
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