Two-color separation probe for screening EWSR1 fracture condition, kit and method thereof

Abstract

The invention provides a double-color separation probe for screening an EWSR1 gene rearrangement condition, a kit and a method thereof, relates to an EWSR1 centromere side probe carrying a first fluorophore and an EWSR1 telomere side probe carrying a second fluorophore, and judges whether an EWSR1 gene breakage condition exists or not according to a detected fluorescence signal so as to determine whether gene rearrangement exists or not. The method disclosed by the invention can be used for simply and quickly screening whether the soft tissue tumor has the EWSR1 rearrangement condition or not, and a quick and reliable basis is provided for definite diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the fields of epigenetics and nucleic acid detection, and in particular to a dual-color probe FISH method and a kit for screening EWSR1 breakage. Background Art

[0002] The EWS RNA binding protein 1 (EWSR1) gene is located on human chromosome 22. This gene encodes a multifunctional protein involved in a variety of cellular processes including gene expression, cell signaling, and RNA processing and transport. The protein encoded by this gene contains an N-terminal transcriptional activation domain and a C-terminal RNA binding domain. Chromosomal translocations of this gene and multiple other genes generate chimeric proteins that are involved in tumor initiation and progression. Such translocations, particularly the t(11;22)(q24;q12) translocation, are known to cause Ewing sarcoma as well as neuroectodermal abnormalities and various other tumors.

[0003] It is now known that many soft tissue tumors have specific molecular genetic alterations, and at least one-third of tumor types have frequent chromosomal translocations. Ewing sarcoma, the classic "small round blue cell tumor", is the first type of soft tissue tumor that has been confirmed to have characteristic molecular genetic alterations. Most cases have t(11;22)(q24;q12) translocation, resulting in the fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) and the Freund's leukemia virus integration 1 gene (FLI1). Since the discovery of the EWSR1-FLI1 gene fusion in Ewing sarcoma, a large number of studies have elucidated the molecular mechanism of Ewing sarcoma and discovered many types of molecular alterations, which have affected the classification of soft tissue tumors. EWSR1-FLI1 fusion has long been considered to be caused by balanced chromosomal translocations, but recent studies have shown that about 40% of the gene rearrangements in Ewing sarcoma are due to chromplexy (a complex rearrangement) and have a poor prognosis. In these Ewing sarcoma cases, there is almost always fusion of other genes (up to 18 fusion genes) in the form of chromoplexy and ring chromosome rearrangements on the basis of EWSR1-FLI1 fusion. It is currently believed that regardless of its mechanism of occurrence, EWSR1-FLI1 fusion is an essential factor in the pathogenicity and progression of Ewing sarcoma, and studies have shown that EWSR1-FLI1 fusion is a key factor in the transformation of mesenchymal stem cells into Ewing sarcoma tumor cells.

[0004] Although the function of wild-type EWSR1 protein remains unclear, in existing studies, taking Ewing sarcoma as an example, EWSR1-FLI1 fusion protein, as an abnormal transcription factor (EWSR1 provides a transcriptional activation domain and FLI1 provides a DNA binding domain), deregulates tumorigenesis-related genes through a complex transcriptional process, including gene activation and repression. In cell lines, EWSR1-FLI1 directly induces or inhibits enhancers through chromatin remodeling mechanisms; EWSR1-FLI1 induces activation by binding to GGAA repeat motifs to form open chromatin and chromatin remodeling complexes; while inhibition is achieved by replacing wild-type ETS transcription factors. EWSR1-FLI1 can bind to BRG1 / BRM-related factors to form a chromatin remodeling complex (an ATP-dependent chromatin remodeling complex). EWSR1-FLI1-mediated transcriptional dysregulation exists in Ewing sarcoma, making the tumor sensitive to genotoxic chemotherapy drugs (etoposide is the first-line chemotherapy drug); in Ewing sarcoma cell lines, EWSR1-FLI1 fusion gene regulation of transcription leads to frequent formation and accumulation of R loops, resulting in replication stress and DNA damage, which may cause functional damage to BRCA1. The study of EWSR1 in Ewing sarcoma has also enhanced human understanding of "fusion genes". FLI1 is a member of the ETS transcription factor family and participates in tumor activation through multiple mechanisms. Although most Ewing sarcoma cases (about 90%) have EWSR1-FLI1 fusion, a small number of EWSR1 fusion partner genes are other members of the ETS family. For example, EWSR1-ERG fusion protein is the second most common fusion type in Ewing sarcoma (about 5%), and other partner genes include FEV, ETV1, ETV4 and ZSG. In addition, in "Ewing sarcoma family tumors", EWSR1 is fused with genes of non-ETS family members (including NFATC2, PATZ1, SP3ER and SMARCA5). These tumors are morphologically difficult to distinguish from Ewing sarcoma and are currently treated according to the treatment plan for Ewing sarcoma.

[0005] EWSR1 rearrangements have been shown to be the most common fusion gene in the tumors, and not just in Ewing sarcoma. Many soft tissue tumors, some special types of carcinomas, and some mesotheliomas also harbor EWSR1 fusions. Desmoplastic small round cell tumors harbor EWSR1-WT1 fusions, and extraskeletal myxoid chondrosarcomas harbor EWSR1-NR4A3 fusions. EWSR1 fusions are present in at least half of myoepithelioma and myoepithelioma carcinomas, and partner genes include POU5F1, PBX1, ZNF444, KLF17, ATF1, and PBX3. Interestingly, tumors with the same fusion gene can have completely different histologic features and biological potentials, such as EWSR1-ATF1 and EWSR1-CREB fusions in clear cell sarcomas, gastrointestinal clear cell sarcoma-like tumors, primary pulmonary myxoid sarcomas, and angiomatoid fibrous histiocytomas. Given that EWSR1 and FUS can replace each other, in tumors with FUS fusion, such as myxoid liposarcoma, EWSR1-DDIT3 fusion can be present, and low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma can present EWSR1-CREB3L1 / 2 fusion. EWSR1 fusion is also present in non-mesenchymal tumors, including salivary gland clear cell carcinoma (EWSR1-ATF1), odontogenic clear cell tumor (EWSR1-ATF1), mesothelioma (EWSR1-YY1 and EWSR1-ATF1), etc.

[0006] Given that EWSR1 has multiple fusion types, simply using QPCR methods cannot detect all fusion types at the same time, which can easily lead to missed diagnoses. However, as long as the probe is on EWSR1, FISH can screen whether EWSR1 is broken, thereby accurately determining the fusion partner based on the tumor, providing a reliable basis for treatment. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a dual-color separation probe for screening EWSR1 gene rearrangement, wherein the dual-color separation probe comprises an EWSR1 centromere side probe and an EWSR1 telomere side probe; the EWSR1 centromere side probe is a BAC clone carrying a CTA-984G1 sequence labeled with a first fluorescent group, and the CTA-984G1 sequence is 29156252 to 29274024 bp of human chromosome 22; the EWSR1 telomere side probe is a BAC clone carrying a RP11-90D17 sequence labeled with a second fluorescent group, and the RP11-90D17 sequence is 29399255 to 29574421 bp of human chromosome 22.

[0008] In some embodiments of the present invention, the first fluorescent group is rhodamine and the second fluorescent group is FITC.

[0009] In some embodiments of the present invention, the fluorescent groups carried by the EWSR1 centromere probe and the EWSR1 telomeric probe are labeled using phi29 DNA polymerase and random primers.

[0010] In some embodiments of the present invention, the mass ratio of the EWSR1 centromere probe to the EWSR1 telomere probe during use is 1:1.

[0011] The dual-color separation probe of the present invention is used in the preparation of a kit for screening EWSR1 gene rearrangement in clinical samples using fluorescence in situ hybridization. The clinical samples can be selected from fresh tissues or tissue sections.

[0012] The present invention also provides a kit for screening the EWSR1 gene rearrangement in a sample, the kit comprising a dual-color separation probe, the dual-color separation probe comprising an EWSR1 centromere side probe and an EWSR1 telomere side probe; the EWSR1 centromere side probe is a BAC clone carrying a CTA-984G1 sequence labeled with a first fluorescent group, the CTA-984G1 sequence is 29156252 to 29274024 bp of human chromosome 22; the EWSR1 telomere side probe is a BAC clone carrying a RP11-90D17 sequence labeled with a second fluorescent group, the RP11-90D17 sequence is 29399255 to 29574421 bp of human chromosome 22. The two probes are based on the original sequence position of the EWSR1 gene NG_023240 of NCBI (see Figure 2 The upper mark is the specific position of the separation probe. The EWSR1 gene separation probe is set on different BAC clones.

[0013] In some embodiments of the present invention, the kit further comprises a pretreatment solution, an SSC solution, and a proteinase K mother solution, wherein 1L of the pretreatment solution contains 22.6g of Na2EDTA-2H2O, 6.34g of Tris, and a pH of 7.0; the SSC solution is selected from a 20×SSC solution and a 2×SSC solution, and 1L of a 20×SSC solution contains 88g of sodium citrate, 176g of NaCl, and a pH of 7.0; the 2×SSC solution is prepared by diluting the 20×SSC solution 10 times with deionized water, and has a pH of 7.0; the proteinase K mother solution is prepared by adding proteinase K to the 2×SSC solution so that the concentration of proteinase K is 20mg / mL.

[0014] In some embodiments of the present invention, the kit further comprises a slow wash solution and a fast wash solution, the slow wash solution being prepared by adding NP-40 to a 2×SSC solution, wherein the volume of the added NP-40 is 1 / 1000 of the volume of the 2×SSC solution; the fast wash solution being prepared by adding NP-40 to a 2×SSC solution, wherein the volume of the added NP-40 is 3 / 1000 of the volume of the 2×SSC solution.

[0015] The present invention also provides a method for screening soft tissue tumor-related EWSR1 gene rearrangement using fluorescence in situ hybridization, wherein the FFPE sections of the soft tissue tumor samples are routinely processed, and then the dual-color separation probe of the kit is used for in situ hybridization, and whether there is a break in the EWSR1 gene is determined based on the detected fluorescence signal.

[0016] The beneficial effects of the present invention are as follows: (1) The present invention screens the optimal BAC sequence to label the probe through the original sequence of EWSR1, and adopts the FISH (Fluorescence In-Situ Hybridization) method to detect gene breaks, and the signal counting is accurate and fast, and the result has good repeatability; (2) The present invention applies phi29 DNA polymerase to label the probe, and establishes a phi29 DNA random primer labeling method; combined with fine component adjustment, its labeling effect is better than the currently commonly used gap translation method; (3) The EWSR1 gene is related to many diseases, and different diseases have different ligand fusions. In the case of unclear which gene the ligand is, screening whether the gene is broken by using a dual-color separation probe is very helpful for rapid tumor typing, efficacy judgment, drug research, and pathogenesis research; (4) The preferred BAC clone detection of the present invention has good specificity and high sensitivity. Through intuitive detection of large fragments, it is not easy to miss the identification of complex break positions, and it has good identification of unknown break points; (5) The probe design of the present invention is comprehensive, which overcomes the defect that one probe of the QPCR method on the market can only determine one rearrangement mode. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the specific location of the EWSR1 gene on chromosome 22. The area marked by the vertical line is the location of EWSR1.

[0018] Figure 2 is the specific location of the separation probe.

[0019] Figure 3 This is the FISH image of sample N1.

[0020] Figure 4 This is the FISH image of sample 1Z-636.

[0021] Figure 5This is the FISH image of sample 6-118. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used for the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined or specified, the scientific and technical terms described in this patent have the same meaning as those understood by ordinary technicians in the field.

[0023] Example 1 Preparation of a dual-color separation probe for detecting EWSR1 gene breakage (1) Design of a dual-color separation probe: First, the location of the target gene on the chromosome must be understood, such as Figure 1 As shown, the EWSR1 gene is located between 29268268 and 29300521 bp on human chromosome 22q12

[0024] (The location of EWSR1 is:

[0025] Chr22:29268268-29300521, NC_000022.11, NM_005243), so the BAC clone on the centromere side and the BAC clone on the telomere side are determined according to the position of EWSR1; the probe design of the present invention is that the EWSR1 centromere side probe is marked by the BAC clone CTA-984G1 sequence

[0026] The CTA-984G1 insert fragment is 29156252 to 29274024 bp of human chromosome 22 (Chr22: 29156252-29274024, NC_000022.11);

[0027] The EWSR1 telomeric probe was prepared by labeling the BAC clone RP11-90D17 sequence with FITC, where the RP11-90D17 insert is 29399255 to 29574421 bp of human chromosome 22.

[0028] (Chr22:29399255~29574421,NC_000022.11);

[0029] Among them, 29156252~29274024bp and 29399255~29574421bp of human chromosome 22 are public gene sequences, which can be found at: www.ncbi.nlm.nih.gov Available on the website.

[0030] (2) Extraction of BAC DNA: streak inoculation; select clones and culture overnight; use QIAGEN's plasmid extraction kit to extract plasmid DNA and store at -20 degrees for later use;

[0031] (3) Probe labeling: Using BAC cloning as the basic template, phi29 DNA polymerase and random primers are used to label the probe at room temperature. Phy29 DNA polymerase is a mesophilic DNA polymerase cloned from bacteriophage phi29. It has 3'→5' exonuclease proofreading ability and has special multiple displacement and continuous synthesis characteristics; due to its isothermal amplification characteristics, it is more convenient to operate. Random primers are used for amplification during labeling, the amplification temperature is 30°C, and the amplification time is 12 hours. The specific reaction system is as follows:

[0032] system 20μl DNA Xμl(80-160ng) 10xBuffer 2μl <![CDATA[dH2O]]> 4-X C / T dNTP deficiency 2μl FITC-dUTP / rhodamine-dCTP 4μl Random Primers (10PM) 2μl phi29 DNA polymerase (10U / μl) 6μl

[0033] (4) Purification and storage of DNA labeled with fluorescein: After the above system is mixed, label at 30°C for 16 hours and inactivate the enzyme at 65°C for 10 minutes. Take 20 μL of the labeled product and add it to a 1.5 mL centrifuge tube, then add 2 μL of 3 mol / L sodium acetate and 50 μL of anhydrous ethanol in sequence, mix well, place in a -70°C refrigerator for at least 2 hours, centrifuge at 4°C 13000 rpm for 30 minutes, remove the supernatant; add 500 μL of 70% ethanol, centrifuge at 4°C 13000 rpm for 15 minutes, remove the supernatant, and dry in the dark. Use 100 μL of purified water to dissolve the precipitate. Place the dissolved product in a Covaris M220 ultrasonic DNA disruptor and treat at 20°C for 40 seconds. After DNA fragmentation is completed, take out the product, purify the product using Ampure XP purification beads at a ratio of 1:1.8, and finally dissolve it in 20 μL of purified water to obtain the labeled probe.

[0034] Example 2 Reagent preparation

[0035] (1) Preparation of pretreatment solution (pH 7.0): Weigh 22.6 g Na2EDTA-2H2O and 6.34 g Tris in 800 mL deionized water, adjust the pH to 7.0 after dissolving, and make up to 1 L. Store sealed at 2-8°C for 12 months. If the reagent becomes turbid or contaminated, discard it immediately.

[0036] (2) Preparation of 20×SSC solution (pH 7.0): Weigh 88 g sodium citrate and 176 g NaCl in 800 mL deionized water, adjust the pH to 7.0 after dissolving, and make up to 1 L. Store sealed at 2-8°C for 12 months. If the reagent becomes turbid or contaminated, discard it immediately.

[0037] (3) Preparation of 2×SSC solution (pH 7.0): Dilute 20×SSC solution 10 times with deionized water and adjust the pH to 7.0. Store sealed at 2-8°C for 12 months. If the reagent becomes turbid or contaminated, please discard it immediately. (4) Preparation of 20 mg / mL proteinase K stock solution: Weigh 100 mg proteinase K dry powder into 5 mL 2×SSC solution pH 7.0 and shake gently without vortexing until completely dissolved. Aliquot 400 μL per tube and store at -20°C to avoid repeated freezing and thawing.

[0038] (5) Preparation of slow wash solution: Take 40 μL NP-40 and dissolve it in 40 mL 2×SSC solution (pH 7.0). Mix thoroughly. Prepare and use immediately.

[0039] (6) Preparation of quick wash solution: Dissolve 120 μL NP-40 in 40 mL 2×SSC solution (pH 7.0) and mix thoroughly. Prepare and use immediately.

[0040] Example 3 Application of the detection method of the present invention

[0041] Ten clinical FFPE samples related to the clinical EWSR1 fusion gene were tested using the FISH detection method. The specific steps are as follows:

[0042] (1) Pretreatment of FFPE samples, specifically including the following steps: baking, xylene dewaxing, gradient ethanol hydration, pretreatment solution treatment for 20 min, proteinase K digestion for 5-15 min, and gradient ethanol dehydration;

[0043] (2) Denaturing and hybridizing the probe and the tissue to be tested on the glass slide under light-proof conditions, specifically including the following steps: Turn on the hybridizer, set or select a saved program: denaturation at 85°C for 5 minutes, hybridization at 37°C overnight (12 to 16 hours), soak the wet strip of the hybridizer with water, put it into the hybridizer, and allow hybridization to proceed under certain humidity conditions, take 10 μL of the probe and add it to the center area of ​​the sample, carefully cover it with a cover slip and press it tightly to avoid bubbles, then seal the cover slip with a sealing glue (RUBBER CEMENT), gently place the slide in the hybridizer, and start the hybridization program;

[0044] (3) Wash the slides under light-proof conditions, specifically including the following steps: after hybridization, remove the slides from the hybridizer, remove the sealing glue with tweezers, immerse the slides in 2×SSC solution at room temperature for 5 minutes, and remove the coverslip; in the slow wash step, immerse the slides in a slow wash solution preheated to 46°C for 7 minutes; in the fast wash step, immerse the slides in a fast wash solution preheated to 72°C for 2 minutes; dehydrate step by step: immerse the slides in 70% ethanol (1 minute), 85% ethanol (1 minute), and anhydrous ethanol (3 minutes) in sequence, take them out and lay them flat on a clean bench or fume hood to dry;

[0045] (4) Under light-proof conditions, the hybridization area of ​​the slide is observed using a fluorescence microscope to interpret the results, which specifically includes the following steps: Under an oil immersion lens (100×), the red and green fluorescence signals are counted, 30 cells are counted, and a conclusion is drawn based on the interpretation of the results;

[0046] (5) Result determination: When the distance between the red and green fluorescence signals is greater than one signal width, it is recorded as a separation signal. To exclude false positives and false negatives, 100 cells were counted for each sample: two yellow signals (or red and green fluorescence close together) were visible in normal cases; one yellow signal and one red and one green separation signal were visible in broken cases.

[0047] Example 4 Accuracy comparison of clinical samples

[0048] Eight tissue section samples with or without EWSR1 gene fusion confirmed by next generation sequencing and one normal control were taken and tested according to Example 3. The test results are shown in the following table:

[0049]

[0050] The intuitive image results are shown in Figure 3 , 4 , 5, by Figure 3 It can be seen that there are two yellow fluorescent spots in each cell, indicating that the two probes (red and green) are not separated, suggesting that there is no break in the EWSR1 gene; Figure 4 It can be seen that there are two yellow fluorescent spots in each cell, indicating that the two probes (red and green) are not separated, suggesting that there is no break in the EWSR1 gene; Figure 5 It can be seen that there is a yellow fluorescent spot and separated red and green spots in each cell, indicating that the two probes (red and green) are separated, suggesting that the EWSR1 gene is broken.

[0051] From the above results, it can be seen that the detection method of the present invention has a 100% coincidence rate, low cost, and intuitive and accurate result judgment. In the absence of EWSR1 fusion but with other fusions (such as SYT fusion), the detection results show no breakage, so the specificity is good.

Claims

1. A dual-color separation probe for screening EWSR1 gene rearrangement, characterized in that: The dual-color separation probe includes an EWSR1 centromere side probe and an EWSR1 telomere side probe; the EWSR1 centromere side probe is a BAC clone carrying a CTA-984G1 sequence labeled with a first fluorescent group, and the CTA-984G1 sequence is 29156252 to 29274024 bp of human chromosome 22; the EWSR1 telomere side probe is a BAC clone carrying an RP11-90D17 sequence labeled with a second fluorescent group, and the RP11-90D17 sequence is 29399255 to 29574421 bp of human chromosome 22.

2. The dual-color separation probe according to claim 1, characterized in that: The first fluorophore is rhodamine and the second fluorophore is FITC.

3. The dual-color separation probe according to claim 1, characterized in that: The fluorescent groups carried by the EWSR1 centromere probe and the EWSR1 telomeric probe were labeled using phi29 DNA polymerase and random primers.

4. Use of the dual-color separation probe according to any one of claims 1 to 3 in the preparation of a kit for screening EWSR1 gene rearrangement in clinical samples using fluorescence in situ hybridization.

5. The use according to claim 4, characterized in that Clinical samples were selected from fresh tissues or tissue sections.

6. A kit for screening EWSR1 gene rearrangement in a sample, characterized in that: The kit comprises the dual-color separation probe according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that The kit also includes a pretreatment solution, an SSC solution, and a proteinase K mother solution.

8. A method for screening soft tissue tumor-related EWSR1 gene rearrangement using fluorescence in situ hybridization, characterized in that: Obtain FFPE sections of soft tissue tumor samples, then use the dual-color separation probe described in any one of claims 1 to 4 to perform in situ hybridization, and determine whether the EWSR1 gene is broken based on the detected fluorescent signal.

9. A method for preparing a dual-color separation probe for screening EWSR1 gene rearrangement, characterized in that: The EWSR1 centromere probe was prepared by labeling the BAC clone CTA-984G1 sequence with rhodamine, and the EWSR1 telomere probe was prepared by labeling the BAC clone RP11-90D17 sequence with FITC. The probes were labeled at room temperature using BAC clones as basic templates, phi29 DNA polymerase, and random primers.

10. The preparation method according to claim 9, characterized in that: The CTA-984G1 insert is 29156252-29274024 bp of human chromosome 22, and the RP11-90D17 insert is 29399255-29574421 bp of human chromosome 22.

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