CN114774554A - Molecular marker of epithelial-like vascular endothelioma and detection kit thereof - Google Patents
Molecular marker of epithelial-like vascular endothelioma and detection kit thereof Download PDFInfo
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- CN114774554A CN114774554A CN202210679461.0A CN202210679461A CN114774554A CN 114774554 A CN114774554 A CN 114774554A CN 202210679461 A CN202210679461 A CN 202210679461A CN 114774554 A CN114774554 A CN 114774554A
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Abstract
The invention relates to the technical field of biomedical detection, in particular to a PDGFRA-USP46 fusion gene as a molecular marker of an epithelioid vascular endothelial tumor and a kit, wherein the molecular marker is the PDGFRA-USP46 fusion gene, and the nucleotide sequence of the PDGFRA-USP46 fusion gene is shown as SEQ ID No. 1. The fusion gene is formed by fusing the tail end of PDGFRA gene Exon16 positioned on chromosome 4q12 and the front end of USP46 gene Exon2 positioned on 4q12, and belongs to a splice-site fusion site in the version type. The kit for detecting the epithelial angioendothelioma based on the one-step method qRT-PCR technology can realize absolute quantitative detection of PDGFRA-USP46 mRNA in paraffin-embedded tissues, and has high sensitivity and specificity and strong operability and repeatability.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a PDGFRA-USP46 fusion gene serving as a molecular marker of an epithelial angioid endothelioma and a one-step method qRT-PCR detection kit thereof.
Background
Epithelial-like intravascular endothelioma (EHE) is a rare malignant angiogenic tumor that was first reported and described by Weiss and Enzinger in 1982 as a group of vascular tumors occurring in the surrounding soft tissues. The sarcoma typing published by WHO in 2020 considers EHE to be divided into two types, one of which is the common classical gene t (1; 3) (p36.3; q25) translocation forming the WWTR1-CAMTA1 fusion gene and the other being the uncommon YAP1-TFE3 fusion gene. About 100 EHE documents at home and abroad are reported so far, and the clinical pathology and the molecular genetics characteristics of the EHE documents are not sufficiently known. About 90% of EHEs are associated with the WWTR1-CAMTA1 fusion gene, a typical pathology is represented by epithelial-like endothelial cell nests and cord composition within the mucohyaline stroma, lacking vascularization; a small fraction (10%) of EHE has a unique morphology, including marked angiogenesis, endothelial cells lined with abundant eosinophilic cytoplasm, and YAP1-TFE3 fusions. EHE tumor cells often express the vascular associated markers CD31, CD34, ERG, FLI-1, FVIII, etc., CAMTA1(+) in CAMTA1 fused tumors and TFE3 core (+) in YAP1-TFE3 fused tumors.
In recent years, more than 90% of EHEs have been found to have reproducible chromosomal translocations, forming the WWTR1-CAMTA1 fusion gene. In 2013, Cristina R and the like detect the chromosomal translocation and the fusion gene thereof existing in EHE reported in the literature at present in YAP1-TFE3 gene fusion in a WWTR1-CAMTA1 fusion negative EHE subset, and the chromosomal translocation and the fusion gene thereof are shown in Table 1. In addition to WWTR1-CAMTA1 and YAP1-TFE3, Suurmeijer AJH et al detected different WWTR1 gene fusions in 6 cases in 2020, including WWTR1-MAML2 and WWTR1-ACTL6A, 4 of which were located in the heart. Song et al detected low abundance of PTPRM-CCDC120 fusion in EHE in 2020. The chromosome translocation or fusion genes are detected by means of RT-PCR, FISH or second-generation sequencing and the like, and diagnosis, typing and differential diagnosis of EHE are facilitated.
TABLE 1 chromosome translocation and fusion gene thereof in the current literature reporting the existence of epithelioid vascular endothelioma
Recently, applicants have discovered and validated a new fusion gene, PDGFRA-USP46, formed by fusion of PDGFRA gene Exon16 on chromosome 4q12 at its terminus to USP46 gene Exon2 on chromosome 4q12 at its terminus, as a splice-site in inversion type, in1 human epithelioid angioma paraffin-embedded tissue using transcriptome sequencing, RT-PCR and FISH techniques. At present, no research report of PDGFRA-USP46 fusion gene on epithelioid vascular endothelial tumor or other tumors is found at home and abroad.
The invention aims to provide a novel fusion gene PDGFRA-USP46 for diagnosis and typing of an epithelial-like vascular endothelial tumor and a kit for detecting the fusion gene PDGFRA-USP46 in a paraffin embedded tissue of the epithelial-like vascular endothelial tumor based on a one-step method qRT-PCR technology, so that absolute quantitative detection of PDGFRA-USP46 mRNA in a paraffin tissue is realized, and diagnosis and molecular typing of the epithelial-like vascular endothelial tumor are facilitated.
Disclosure of Invention
The first purpose of the invention is to provide a molecular marker of epithelioid vascular endothelial tumor, which is beneficial to diagnosis and molecular typing of the epithelioid vascular endothelial tumor;
the second purpose of the invention is to provide a detection kit for detecting human epithelial-like vascular endothelioma, which can realize absolute quantitative detection of PDGFRA-USP46 mRNA in paraffin-embedded tissues, and has the advantages of higher sensitivity and specificity, and strong operability and repeatability.
The invention provides a molecular marker of epithelioid vascular endothelial tumor, wherein the molecular marker is PDGFRA-USP46 fusion gene, and the nucleotide sequence of the PDGFRA-USP46 fusion gene is shown as SEQ ID No. 1.
Preferably, the PDGFRA-USP46 fusion gene is formed by fusing the PDGFRA gene Exon16 terminal positioned on chromosome 4q12 and the USP46 gene Exon2 front terminal positioned on 4q 12.
Preferably, the fusion sites of the PDGFRA-USP46 fusion gene are PDGFRA-chr4:55146649 and USP46-chr4: 53497311.
The invention also provides a detection kit for detecting human epithelioid vascular endothelioma aiming at the PDGFRA-USP46 fusion gene, and the detection kit also belongs to the protection scope of the invention.
Preferably, the technical scheme also comprises a primer composition of the PDGFRA-USP46 fusion gene.
Preferably, the primer composition comprises a forward primer and a reverse primer, wherein the sequence of the forward primer is 5'-ATCGTCCAGCCTCATATAAGAAG-3' as shown in SEQ ID No.2, and the sequence of the reverse primer is 5'-AAGCACGGAGTTACAGTAGC-3' as shown in SEQ ID No. 3.
Preferably, the technical scheme also comprises a probe of the PDGFRA-USP46 fusion gene, wherein the sequence of the probe is FAM-5'-ATCTATGTTAGGGCACCAATGCCTCTGCT-3' -BHQ1, and is shown as SEQ ID No. 4.
Preferably, the kit further comprises a positive quantitative reference substance, wherein the positive quantitative reference substance is PESI-T plasmid containing PDGFRA-USP46 fusion gene junction site fragment.
Preferably, the technical scheme also comprises a negative control, wherein the negative control is soft tissue angiofibroma paraffin embedded tissue with negative fusion gene PDGFRA-USP 46.
The molecular marker for the epithelioid hemangioma has at least the following technical effects:
the molecular marker of epithelioid vascular endothelial tumor, namely PDGFRA-USP46 fusion gene, is formed by fusing the tail end of PDGFRA gene Exon16 positioned on chromosome 4q12 with the front end of USP46 gene Exon2 positioned on 4q12, and belongs to a splice-site in inversion type.
The PDGFRA can be combined with a corresponding ligand PDGF to activate, and then activates phosphorylation pathways of phosphatidylinositol, cAMP and various proteins, and regulates division and proliferation of cells. PDGFRA mutations are closely related to gastrointestinal stromal tumors GIST, with mutations occurring in approximately 5-7% of gastrointestinal stromal tumors (GIST), especially alterations in exon 12/14/18, the most common mutation being exon 18D 842V, which is associated with specific clinical pathological features, such as primary imatinib resistance and higher inertia. In addition, various PDGFRA gene fusions are seen in eosinophilic acute myeloid leukemia.
While USP46 is totally called ubiquitin-specific peptidase and located on chromosome 4, no related fusion gene is reported at present. The modification of cellular proteins by ubiquitin is an important regulatory mechanism controlled by the synergistic effect of multiple ubiquitin-binding and deubiquitinating enzymes. USP46 belongs to a large class of cysteine proteases that are deubiquitinases and function in a variety of tumors. USP46 is important for the proliferation and tumor growth of HPV-transformed cancers, whereas USP46 is low expressed in hepatocellular, ovarian, lung, and renal cell cancers and is associated with a poor prognosis.
According to the detection kit for the epithelial-like vascular endothelioma, PDGFRA-USP46 fusion genes are used as markers, a one-step method qRT-PCR technology is utilized, a specific probe is designed through the sequence of primers, complementary hybridization is carried out on the specific probe and an amplification product, and the type and the amount of fluorescence can specifically represent the type and the amount of a target product. Therefore, when the kit for detecting the epithelial-like vascular endothelioma based on the one-step method qRT-PCR technology is used for detecting the fusion gene in the paraffin-embedded tissue of the epithelial-like vascular endothelioma, the kit has the advantages of strong operability and repeatability, higher sensitivity and specificity, enrichment of molecular typing of the epithelial-like vascular endothelioma, and improvement of understanding and comprehension of clinical pathology and molecular characteristics of the rare soft tissue tumors.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the results of PDGFRA-USP46 fusion gene formation, expression and sequencing; specifically, fig. 1A is a schematic diagram of the PDGFRA-USP46 fusion gene formation provided by the present invention; FIG. 1B shows a one-step RT-PCR method for detecting PDGFRA-USP46 mRNA expression in paraffin-embedded tissues of epithelioid vascular endothelial tumors; FIG. 1C is the PDGFRA-USP46 mRNA sequencing result;
FIG. 2 shows that one-step RT-PCR is adopted to detect the expression of 4 transcripts of WWTR1-CAMTA1 and YAP1-TFE3 fusion genes in paraffin-embedded tissues of epithelioid vascular endothelial tumors;
FIG. 3 is a standard curve of PDGFRA-USP46 standard substance provided by the invention, the slope of the standard curve is-3.861, and the efficiency value is 1.816;
FIG. 4 is a one-step qRT-PCR amplification curve of PDGFRA-USP46 standard and PDGFRA-USP46 mRNA in epithelioid vascular endothelial tumor tissue provided by the present invention;
FIG. 5 shows the result of FISH detection of CAMTA1 gene rearrangement in epithelioid angioendothelioma;
FIG. 6 shows the result of FISH detection of TFE3 gene rearrangement in epithelioid angioendothelioma;
FIG. 7 is a diagram showing the analysis of PDGFRA-USP46 gene rearrangement and fusion sites in the result of RNA sequencing of an epithelioid endothelioma.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
The invention provides a PDGFRA-USP46 fusion gene as a molecular marker of epithelial angioid tumors and a quantitative detection kit thereof.
The technical idea is as follows: after 1 case of human epithelioid vascular endothelial paraffin embedded tissues are subjected to common fusion gene WWTR1-CAMTA1/YAP1-TFE3 and CAMTA1/TFE3 gene rearrangement detection without fruits by using RT-PCR and FISH technologies, a new fusion gene is detected in the sample by a high-throughput sequencing (RNA-sq) technology: PDGFRA-USP46, and analysis shows that the fusion gene is formed by fusing the PDGFRA gene Exon16 end located on chromosome 4q12 with the USP46 gene Exon2 front end located on 4q12 (FIG. 1A), and belongs to the splice-site fusion site in the version type, wherein the fusion site is PDGFRA-chr4:55146649 and USP46-chr4: 53497311.
Since the PDGFRA-USP46 fusion gene is discovered for the first time at home and abroad, the applicant further designs and preferably selects an amplification primer of PDGFRA-USP46, successfully amplifies a positive target fragment of 150bp in the paraffin-embedded tissue of the epithelial angioma in the example by using a one-step RT-PCR technology, and verifies that the fusion gene PDGFRA-USP46 is obtained by performing Sanger sequencing on a PCR product and comparing the PCR product by using an NCBI database, wherein the nucleotide sequence of the fusion gene PDGFRA-USP46 is shown as SEQ ID NO. 1.
Meanwhile, the applicant provides an absolute quantitative detection kit for PDGFRA-USP46 mRNA in paraffin tissue based on one-step qRT-PCR technology, the kit is high in operability and repeatability, high in sensitivity and specificity and beneficial to diagnosis and molecular typing of EHE.
First, experimental material
1. Epithelial-like intravascular endothelioma and tissue samples of control cases
In this group, 1 example of epithelial angioendothelioma (EHE) and 1 example of soft tissue Angiofibroma (AFST) in the control case were formalin-fixed paraffin-embedded tissues, and the clinical and pathological information thereof is shown in table 2.
TABLE 2 clinical pathological information table of epithelioid endothelioma and control case
2. Various PCR related primer and probe sequences in experiment
2.1 PDGFRA-USP46 fusion gene primer and Probe design
As the PDGFRA-USP46 fusion gene is formed by fusing the end of PDGFRA gene Exon16 on chromosome 4q12 and the front end of USP46 gene Exon2 on 4q12, the fusion sites are PDGFRA-chr4:55146649 and USP46-chr4:53497311 respectively. Therefore, the inventors designed forward and reverse primers of the PDGFRA-USP46 fusion gene by knowing the PDGFRA gene sequence and the USP46 gene sequence when designing primers, the sequences are shown in table 3, and the amplified fragment size of the fusion gene is 150 bp. Similarly, based on the PDGFRA-USP46 fusion gene sequence, the fusion gene specific probe is designed, the sequence is shown in Table 3, and the PDGFRA-USP46 fusion gene is quantitatively detected by qRT-PCR.
TABLE 3 PCR primers and Probe sequence Listing for the experiments
2.2 common fusion Gene primer sequence WWTR1-CAMTA1/YAP1-TFE3
4 pairs of primers of a common fusion gene WWTR1-CAMTA1/YAP1-TFE3 in the epithelioid vascular endotheliomas are respectively designed and synthesized, and the sequence and the size of an amplified fragment are shown in a table 3.
2.3 preparation of Positive quantitative reference
Amplifying total RNA extracted from an epithelial hemangioma paraffin embedded tissue by using forward primers and reverse primers of SEQ ID No.2 and SEQ ID No.3 to obtain a 150bp fragment product containing a PDGFRA-USP46 fusion gene locus, introducing the fragment product into a PESI-T plasmid, transferring the PESI-T plasmid into escherichia coli for mass culture, extracting purified plasmid, determining plasmid concentration, calculating copy number of unit volume, diluting according to proportion to obtain a positive quantitative plasmid reference product series: 1X 106 copies/ml、1×105 copies/ml、1×104 copies/ml、1×103copies/ml and 1X 102 copies/ml。
3. FISH probe
TABLE 4 FISH Probe used for the experiment
4. Other main reagents
1) Trizol reagent (Invitrogen)
2）Proteinase K (PK) Solution（Promega）
3）QIAGEN One Step RT-PCR Kit（Qiagen）
4）RNeasy FFPE Kit (Qiagen)
5）TruSeq RNA Exome Kit (Illumina Inc)
6) CAMTA1 separation probe (Anbiping)
7) TFE3 separation probe (anbiping);
8) pepsin working solution (Anbiping)
9) DAPI staining solution (Anbiping)
10) 50X EDTA (Anbiping)
11) 2 XSSC (Anbiping)
12) 0.1% NP-40/2 XSSC (Anbitong)
Second, Experimental methods and procedures
1. One-step RT-PCR and qRT-PCR
1.1 extraction of Paraffin-Embedded tumor tissue RNA
Continuously cutting 10 pieces of 10-micron wax film by a new cutter, and putting into a 1.5 ml disinfection centrifuge tube; adding 1.0ml of xylene and incubating for 10 minutes at 55 ℃; centrifuging at 13000rpm/4 ℃ for 2 minutes; repeating for 1 time; adding 100% alcohol 1.0ml, incubating at room temperature for 10 min, centrifuging at 13000rpm/4 ℃ for 2 min, and discarding the supernatant; repeating for 1 time; adding 250ul of prepared cell lysate in advance, centrifuging at 13000rpm/4 ℃ for 10 seconds, and mixing uniformly; protease K (protease K) (20 mg/ml) was added 50ul overnight (12 hours) in a dry thermostat at 55 ℃.
Inactivating the proteinase K at 95 ℃ for 5 minutes in a dry thermostat, centrifuging at 13000rpm/4 ℃ for 2 minutes, removing the precipitate, and collecting the supernatant to a 2ml centrifuge tube; add 1.0ml Trizol solution (Invitrogen) into the centrifuge tube and incubate for 5 minutes at room temperature; adding chloroform to 200ul, violently mixing for 15 seconds by using vortex, and incubating for 5 minutes at room temperature; then, the mixture is centrifuged at 13000rpm and 4 ℃ for 10 minutes; transferring the water phase to another 2.0 ml centrifuge tube (about 700 ul), adding isopropanol according to the volume of 1:1 (namely 700 ul), blowing and beating, and precipitating for 30 minutes at room temperature and minus 25 ℃; 13000rpm/4 ℃ for 10 minutes, and discarding the supernatant; adding 1.0ml of 75% alcohol, and turning the centrifuge tube upside down for several times; centrifuging at 13000rpm at 4 ℃ for 5 minutes, discarding the supernatant, and drying; 50ul of enzyme-free water was added to dissolve the RNA (30-60 min), and the RNA concentration and the A260/A280 and A260/A230 ratios were measured by Nanodrop and recorded.
1.2 one-step RT-PCR
1.2.1 sample RNA one-step RT-PCR amplification reaction
Preparing a PCR amplification reaction system according to the table 5 by using a marked 200 mu L enzyme-free EP tube, uniformly mixing and centrifuging;
TABLE 5 one-step RT-PCR amplification reaction System
And (3) putting the EP tube added into the reaction system into a PCR instrument, and starting PCR amplification according to the following set reaction conditions:
1.2.2 agarose gel electrophoresis of PCR amplification products
Preparing glue: agarose gel was prepared according to the recipe 2.5g agarose, 1 XTAE 100mL, Gelstain 10. mu.L agarose gel.
Electrophoresis: the agarose gel was placed in an electrophoresis tank, filled with 1 × TAE, 8 μ L of PCR product was added to each gel well, 2 μ L of 50bp DNA marker was added to a standard well, and the voltage was adjusted to 90V for 30 min.
Exposure: and (4) placing the agarose gel into an exposure instrument for automatic exposure and photographing for storage.
1.3 one-step method qRT-PCR
1.3.1 taking a marked 96-well plate, preparing a qRT-PCR amplification reaction system according to the table 6, uniformly mixing and centrifuging;
TABLE 6 one-step method qRT-PCR amplification reaction system
1.3.2 putting the 96-well plate added to the reaction System into a Light Cycler 480 PCR apparatus, starting PCR amplification according to the set reaction conditions (annealing temperature, time, etc. are set depending on the length, etc.)
1.3.3 data reading, analysis and result calculation
LightCycler 480 PCR instrument according to standard solution series concentration 1X 106、1×105、1×104、l×103、1×102 copies/mL, standard curve was plotted.
The LightCycler 480 PCR instrument system software reads and analyzes expression data of the PDGFRA-USP46 fusion gene, and calculates the content (copies/mL) of the PDGFRA-USP46 fusion gene in the tissue sample after comparing with a standard curve.
2. Second generation sequencing
1 case of epithelioid vascular endothelioma paraffin-embedded tissue Sendai corporation for transcriptome sequencing and sarcoma panel sequencing (BSR), respectively.
3. Tissue Fluorescence In Situ Hybridization (FISH)
3.1 flaking and dewaxing
3.1.1 tableting
5 pieces of white slices of 3 μm are cut continuously and baked in an oven at 60 ℃ for 2 hours.
3.1.2 dewaxing
Placing the glass slide in xylene at room temperature for 10 minutes and three times; taking out the slide, and putting the slide into 100% ethanol for 3 minutes and three times; the slide was removed and washed once in deionized water at room temperature.
3.2 pretreatment of the slices
Putting the glass slide into 1 multiplied by EDTA working solution, putting the glass slide into a water bath kettle at 99 ℃ for 25 minutes, taking the glass slide out, cooling the glass slide to room temperature, and washing the glass slide once by using deionized water; putting the glass slide into 2 XSSC at room temperature for soaking for 3 minutes, and airing at room temperature; 200ul of preheated pepsin digestion solution (preheated for 15 minutes in a water bath kettle at 37 ℃) is dripped into the slide sample area, and the slide sample area is put into a hybridization instrument to be incubated for 7 minutes at 37 ℃; throwing off the redundant liquid on the slide, and putting the slide into room temperature 2 XSSC for 3 minutes; taking out the glass slide, and respectively putting the glass slide into 70%, 90% and 100% ethanol for 1 minute; the slide is taken out and dried at room temperature.
3.3 denaturing hybridization
Taking out the CAMTA1 separation probe and the TFE3 separation probe from a refrigerator at the temperature of-20 ℃, shaking, uniformly mixing, and centrifuging for 15 s; respectively dripping 10ul of CAMTA1/TFE3 separation probe into the sample area of the slide, quickly placing a cover glass, and uniformly distributing probe liquid by light pressure; then, mounting the cover glass along the edge of the cover glass by using mounting glue, and completely covering the contact part of the cover glass and the glass slide; the slide was placed into a hybridization apparatus (Thermo Fisher, usa), the hybridization apparatus humidity strip was wetted and inserted into the hybridization apparatus, the hybridization apparatus upper cover was closed, and the procedure was set: denaturation at 85 ℃ for 5 min, followed by hybridization overnight at 37 ℃ in the absence of light for 16 h.
3.4 washing and counterdyeing
Firstly, putting 2 XSSC and 0.1 percent NP-40/2 XSSC into a constant-temperature water bath kettle at 37 ℃ and preheating for 30 minutes; closing the power supply of the hybridization instrument, removing the rubber sealing glue after taking out the slide, and removing the cover glass; placing the slide in 2 XSSC preheated to 37 ℃ for 10 minutes; the slide was removed and placed in 0.1% NP-40/2 XSSC preheated to 37 ℃ for 5 minutes; taking out the glass slide, and sequentially placing the glass slide into 70 percent of room temperature, 90 percent of room temperature and 100 percent of ethanol for 1 minute; taking out the slide, and airing in a dark place; at room temperature, 10ul of DAPI staining solution was dropped onto a dry slide, the slide was covered lightly to avoid the generation of bubbles, stored in the dark, and observed under a fluorescence microscope.
3.5 interpretation of results
The slide glass is observed under a high power microscope and an oil microscope by a fluorescence microscope, and cells with consistent cell nucleus size, complete cell nucleus boundary, uniform DAPI staining, no overlapping of the cell nucleus and clear signals are selected.
3.5.1 CAMTA1 isolation Probe assay
Two-color signals in 100 cancer cell nuclei were counted randomly. When the distance between the red and green signals is greater than twice the length of the signal, it is considered that the CAMTA1 separates positive cells.
Interpretation criteria: positive cells greater than 15% (15 cells) were positive for CAMTA1 isolation.
3.5.2 TFE3 separation Probe assay
Two-color signals in 100 tumor nuclei were counted randomly. When the distance between the red and green signals is greater than twice the length of the signal, it is considered that TFE3 separates positive cells.
Interpretation criteria: if the positive cells are greater than 15% (15 cells), the case is positive for isolation of TFE 3.
Three, result in
1. One-step RT-PCR and qRT-PCR detection of expression of fusion gene
1.1 expression of PDGFRA-USP46 fusion gene in epithelioid vascular endothelioma
Paraffin embedded tissues of 1 Epithelioid Hemangioma (EHE) were selected and 1 soft tissue Angiofibroma (AFST) was used as a negative control for one-step RT-PCR using PDGFRA-USP46 primers SEQ ID No.2 and SEQ ID No. 3.
Gel electrophoresis showed that 150bp PDGFRA-USP46 mesh band was amplified from epithelial-like intravascular endothelioma (EHE) tissue, whereas negative controls were negative for soft tissue Angiofibroma (AFST) tissue and blank controls. Sanger sequencing results were consistent with sequences in the NCBI database (fig. 1B, fig. 1C). The nucleotide sequence of PDGFRA-USP46 is shown in the attached table SEQ ID No. 1.
The results of detecting the four transcripts of WWTR1-CAMTA1/YAP1-TFE3, which are common fusion genes in epithelioid vascular endotheliomas, by using one-step RT-PCR technology, show that the expression of WWTR1-CAMTA1/YAP1-TFE3, which are four transcripts, is not detected in the 2 cases and the negative control (FIG. 2, the four transcripts from left to right in FIG. 2 are WWTR1(ex4) -CAMTA1(ex9), WWTR1(ex3) -CAMTA1(ex9), YAP1(ex1) -TFE3(ex4) and YAP1(ex1) -TFE3(ex 6)), which is also consistent with the RNA-Seq high-throughput sequencing results (FIG. 7).
1.2qRT-PCR detection of PDGFRA-USP46 mRNA expression in EHE
The tissue is embedded by paraffin of the epithelioid hemangioma, RNA is successfully extracted, the expression of PDGFRA-USP46 mRNA in the epithelioid hemangioma sample is detected by one-step qRT-PCR with the negative control of the soft tissue hemangiofibroma, the slope of the experimental standard curve is-3.861, and the efficiency value is 1.816 (figure 3). The results of the quantitation of PDGFRA-USP46 mRNA after comparison with the standard curve are shown in Table 7 and FIG. 4.
TABLE 7 quantitative detection results of PDGFRA-USP46 mRNA in EHE tissues
2. Second generation sequencing results
The analysis of the 1 st example of the transcriptome sequencing result of the epithelioid hemangioma does not find the existence of the four transcripts of the common fusion gene WWTR1-CAMTA1/YAP1-TFE3 reported in the literature at present.
However, in this example, a new PDGFRA-USP46 gene fusion was detected in the epithelioid vascular endothelial tumor sample at the following specific fusion sites: PDGFRA-chr4: 55146649; USP46- -chr4:53497311, PDGFRA gene exon16 end (chr 4: 55146649) fused with USP46 gene exon2 front end (chr 4: 53497311), belonging to the splice-site fusion site in inversion type (FIG. 7).
Applicants further spliced out the sequence Seq ID No.1 of the PDGFRA-USP46 gene by the known sequence of the PDGFRA gene and the sequence of the USP46 gene, as well as the predicted break binding site of RNA-Seq.
3. FISH test results
3.1 detection result of CAMTA1 Gene rearrangement in epithelioid intravascular endothelioma
The CAMTA1 gene rearrangement assay was performed on EHE samples using CAMTA1 dual color separation probe, and FISH results showed: 100 tumor cells were counted, 0 positive cells, negative for CAMTA1 gene rearrangement (fig. 5). The result is consistent with the RT-PCR detection result of the prior fusion gene WWTR1-CAMTA 1.
3.2 detection of TFE3 Gene rearrangement in epithelioid intravascular endothelioma
The TFE3 gene rearrangement assay was performed on EHE samples using a TFE3 two-color separation probe, and FISH results showed 100 cells counted, 0 positive cells, and negative for TFE3 gene rearrangement (fig. 6). The results were consistent with the RT-PCR assay results of the previously fused gene YAP1-TFE 3.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Sequence listing
<110> Beijing Chaoyang Hospital affiliated to capital medical university
<120> epithelial-like vascular endothelial tumor molecular marker and detection kit thereof
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tacacagtat gtccccatgc tagaaaggaa agaggtttct aaatattccg acatccagag 120
atcactctat gatcgtccag cctcatataa gaagaaatct atgttagggc accaatgcct 180
ctgctctgga aaaagacatt ggtccagagc agtttccaat caatgaacac tatttcggat 240
tggtcaat 248
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Claims (9)
1. The molecular marker of the epithelioid vascular endothelial tumor is a PDGFRA-USP46 fusion gene, and the nucleotide sequence of the PDGFRA-USP46 fusion gene is shown as SEQ ID No. 1.
2. The molecular marker of epithelioid vascular endothelial tumor according to claim 1, wherein the PDGFRA-USP46 fusion gene is formed by fusion of the PDGFRA gene Exon16 terminus located on chromosome 4q12 and the USP46 gene Exon2 terminus located on 4q 12.
3. The molecular marker of an epithelioid vascular endothelial tumor according to claim 1, wherein the fusion site of the PDGFRA-USP46 fusion gene is PDGFRA-chr4 and USP46-chr4, respectively.
4. A test kit for detecting an epithelioid endothelioma, comprising the reagent for the PDGFRA-USP46 fusion gene according to any one of claims 1 to 3.
5. The detection kit for detecting an epithelioid endovascular endothelioma according to claim 4, further comprising a primer composition for the PDGFRA-USP46 fusion gene.
6. The detection kit for detecting the epithelioid hemangioma according to claim 5, wherein the primer composition comprises a forward primer and a reverse primer, the sequence of the forward primer is shown as SEQ ID No.2, and the sequence of the reverse primer is shown as SEQ ID No. 3.
7. The detection kit for detecting the epithelioid hemangioendothelioma according to claim 4, further comprising a probe of the PDGFRA-USP46 fusion gene, wherein the sequence of the probe is shown in SEQ ID No. 4.
8. The detection kit for detecting an epithelioid endothelioma according to claim 4, further comprising a positive quantitative reference, wherein the positive quantitative reference is PESI-T plasmid containing PDGFRA-USP46 fusion gene junction site fragment.
9. The detection kit for detecting an epithelioid intravascular endothelioma according to claim 4, further comprising a negative control, wherein the negative control is soft tissue angiofibroma paraffin embedded tissue negative for the fusion gene PDGFRA-USP 46.
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