CN107267608A - The new transposition companion of Xp11.2 a kind of and its detection primer and application - Google Patents

Abstract

The invention discloses a new translocation partner of Xp11.2, detection primers and applications thereof. A new translocation partner of Xp11.2 is the translocation of MATR3‑TFE3 gene, which is located on chromosome 5 5q31.2, and the gene fusion occurs between exon 15 of MATR3 and exon 4 of TFE3 gene. A PCR primer for detecting MATR3-TFE3 translocation tumors is a PCR primer for detecting the MATR3-TFE3 gene translocation described in the present invention. Application of the PCR primers of the present invention in the preparation of MATR3-TFE3 translocation tumor diagnostic reagents. The present invention designs specific PCR primers for the new fusion sites discovered by high-throughput sequencing, expands the detection range of the original primer combination, and is applied in clinical practice to improve the detection rate and accuracy rate of diagnosing such tumors.

Description

A new translocation partner of Xp11.2 and its detection primer and application

technical field

The invention belongs to the field of medical testing, and relates to a new translocation partner of Xp11.2, detection primers and applications thereof.

Background technique

In 2004, WHO revised the histopathological classification of RCC in 1997, adding Xp11.2 translocation/TFE3 gene fusion-related RCC (Xp11.2 translocation RCC). The TFE3 gene is located at XP11.2, and it is the only driving gene of Xp11.2 translocation RCC. The fusion gene can highly express the TFE3 fusion protein through the transformation of the promoter, and TFE3, as a transcription factor, binds to a specific DNA structure, transcriptionally regulates the expression of various genes in the body, and eventually causes disease. At least 10 different translocation partners and fusion genes have been reported, including ASPL-TFE3, PRCC-TFE3, SFPQ-TFE3, NONO-TFE3, CLTC-TFE3, LUC7L3-TFE3, KHSRP-TFE3, PARP14-TFE3, DVL2 -TFE3 and RBM10-TFE3, etc., there is only a single translocation form in each tumor. In addition to renal cell carcinoma, some soft tissue mesenchymal tumors also have pathogenic gene translocation of TFE3 gene, including alveolar soft tissue sarcoma, some epithelioid vascular pericytic tumors, etc. These mesenchymal tumors and Xp11.2 translocation Renal carcinomas are collectively referred to as Xp11.2 translocation tumors.

Xp11.2 translocation tumor is a rare type of tumor. Although its overall incidence rate is very low, about 1/3 of children with renal cancer are of this type, and retrospective studies have shown that such tumors are rare in my country. rare. The disease is characterized by a young age of onset, which causes an extremely heavy burden on families and society. And studies have confirmed that the prognosis of Xp11.2 translocation RCC is worse than that of non-Xp11.2 translocation RCC, and the prognosis of Xp11.2 translocation RCC is different among different genotypes. In addition, there is clear evidence that patients with Xp11.2 translocation tumors are sensitive to vascular endothelial growth factor receptor (VEGFR) or mammalian target of rapamycin (mTOR) molecularly targeted therapy. Other studies have shown that MET tyrosine kinase is the target gene of the ASPL-TFE3 fusion gene, and it is expected to become a therapeutic target for Xp11.2 translocation tumors. Therefore, it is very important to accurately diagnose such tumors based on genotype.

Currently, the commonly used detection methods for the diagnosis of Xp11.2 translocation tumors mainly include immunohistochemistry and fluorescence in situ hybridization. Immunohistochemical detection of nuclear TFE3 fusion protein is intuitive, fast, and low in price, but its disadvantage is that this method is easily affected by many factors, such as tissue fixation time, tissue repair method, antibody clone number, and artificial interpretation factors, etc., making the result False positives or false negatives may occur. Chromosomal fluorescence in situ hybridization (FISH) starts from the combination of traditional cytogenetics and DNA technology, which is fast, sensitive, and specific, and can detect hidden or small chromosomal aberrations and complex karyotypes; a variety of fluorescent markers can also be used to display The relative position and direction between DNA fragments and genes, the spatial positioning is precise. However, these two detection methods can only indicate the high expression of TFE3 protein or the translocation of TFE3 gene, and neither can clarify the specific translocation changes in the tumor. Accurate detection of translocation partners and fusion gene sites in Xp11.2 translocation tumors is not only the basis for predicting the prognosis of patients, but also the guidance for future targeted therapy. Therefore, it is necessary to clarify the gene translocation of Xp11.2 translocation tumors point, which has important clinical significance.

Xp11.2 translocation tumor is a tumor type with large heterogeneity among individuals. Currently known fusion gene forms include ASPL-TFE3, PRCC-TFE3, SFPQ-TFE3, NONO-TFE3, CLTC- More than ten kinds of TFE3, LUC7L3-TFE3, KHSRP-TFE3, PARP14-TFE3, DVL2-TFE3 and RBM10-TFE3.

At present, high-throughput sequencing is the only detection method that can clarify unknown translocation sites. However, high-throughput sequencing is expensive, the detection cycle is long, the detection platform is scarce, and the requirements for sample quality are high, which is not conducive to popularization and promotion. For most patients It is not the preferred detection method. Relying on past experience, designing a specific PCR primer combination for known fusion sites, performing reverse transcription on the RNA extracted from tumor tissues, performing PCR amplification and sequencing, and detecting specific translocation sites of fusion genes is the most accurate and effective method. Convenient and economical method. Therefore, the discovery of new pathogenic fusion sites and the design of PCR primers will help to further improve the detection accuracy of Xp11.2 translocation tumors.

Contents of the invention

The purpose of the present invention is to provide a new translocation partner of Xp11.2 aiming at the above-mentioned deficiencies of the prior art.

Another object of the present invention is to provide detection primers for this new translocation partner.

Another object of the present invention is to provide the application of primers.

The purpose of the present invention can be achieved through the following technical solutions:

A new translocation partner of Xp11.2, which is MATR3-TFE3 gene translocation, MATR3 gene is located on 5q31.2 (chromosome 5, position 139273752-139331677, sequence from GeneBank, sequence version number GRCh38.p7), a total of 19 Exon; gene fusion occurred between exon 15 of MATR3 and exon 4 of TFE3 gene. We used high-throughput sequencing to detect samples from Xp11.2 tumor patients with unknown fusion partners, and found the new translocation partner of Xp11.2: MATR3-TFE3 gene translocation, which has not been reported in domestic and foreign literature However, the original TFE3 fusion gene amplification primers cannot detect this fusion gene, which will lead to missed diagnosis.

The new translocation partner of Xp11.2 preferably comprises the nucleotide sequence shown in SEQ ID NO.5.

The application of the new translocation partner of Xp11.2 in the preparation of MATR3-TFE3 translocation tumor diagnostic reagent.

A PCR primer for detecting MATR3-TFE3 translocation tumors is a PCR primer for detecting the MATR3-TFE3 gene translocation described in the present invention. All primer pairs capable of detecting the MATR3-TFE3 gene translocation of the present invention are within the protection scope of the present invention.

For the new translocation partner found in the present invention, we designed primers respectively in exon 15 of MATR3 and exon 4 of TFE3. Follow the principle of primer design, the primers should be designed in the conserved region of the template cDNA, the primer length should be between 15bp and 30bp, the GC content of the primer should be between 40% and 60%, the annealing temperature should be close to 72°C, the primer itself and the primer There should be no complementary sequence between them, and the amplified band is single and specific. Since the work often needs to be carried out in paraffin-fixed specimens, the RNA fragmentation of paraffin specimens is severe, so the amplified product should not be too long and needs to be controlled below 300bp.

After many times of debugging and verification, the final designed primer sequence is: MATR3-E15-F1: CAGTTCAGTGGGAGACGAGA (SEQ ID NO.1); TFE3-E4-R1: GCGTTGGGTTTCCAGAT (SEQ ID NO.2), the theoretical amplification product is 161bp in length , the entire sequence of the amplified product (fusion gene) is as follows: CAGTTCAGTGGGAGACGAGACCGATCTTGCTAATTTAGGTGATGTGGCTTCTGATGGGAAAAAGGAACCATCAGATAAAGCTGTGAAAAAAGATGGAAGTGCTTCAGCAGCAGCAAAGAAAAAGCTTAAAAAGGTGCAGACCCATCTGGAGAACCCAACGC (SEQ ID NO.5). In order to ensure the detection success rate, we also designed another pair of substitute primers: the primer sequence is: MATR3-E15-F2: GGTGATGTGGCTTCTGATG (SEQ ID NO.3); TFE3-E4-R2: CGAGTGTGGTGGACAGGT (SEQ ID NO.4), The theoretical amplified product is 184 bp in length, and the entire sequence of the amplified product (fusion gene) is as follows: GGTGATGTGGCTTCTGATGGGAAAAAGGAACCATCAGATAAAGCTGTGAAAAAAGATGGAAGTGCTTCAGCAGCAGCAAAGAAAAAGCTTAAAAAGGTGCAGACCCATCTGGAGAACCCAACGCGCTACCACCTGCAGCAGGCGCGCCGGCAGCAGGTGAAACAGTACCTNOGTCCACCACACTCG.6 (SEQ ID). It was verified by experiments that both pairs of primers could successfully amplify the target band, and the band was single and specific.

The application of the PCR primers of the present invention in the preparation of MATR3-TFE3 translocation tumor diagnostic reagents.

A diagnostic kit for MATR3-TFE3 translocation tumors, comprising the PCR primers of the present invention.

Beneficial effect:

The present invention designs specific PCR primers for the new fusion sites discovered by high-throughput sequencing, expands the detection range of the original primer combination, and is applied in clinical practice to improve the detection rate and accuracy rate of diagnosing such tumors. Provide basis for diagnosis and typing and molecular targeted therapy. According to our experimental results, the specificity and sensitivity of the primer combination diagnosis have reached 100%, and the operation object only needs to be performed on paraffin-embedded tissue sections, and the time is only three working days. Using the probe combination provided by the present invention to detect MATR3-TFE3 translocation tumors is not only convenient, fast and reliable, but also has a high detection rate, and can be used to prepare a MATR3-TFE3 translocation tumor diagnostic kit, which is MATR3-TFE3 translocation Provides new tools for rapid and accurate diagnosis of malignant neoplasms.

Description of drawings

Figure 1: The sequence diagram of the MATR3-TFE3 fusion gene successfully detected by applying the primer combination of the present invention (MATR3-E15-F1; TFE3-E4-R1) in known MATR3-TFE3 fusion tumors for verification.

Figure 2: The sequence map of the successfully detected MATR3-TFE3 fusion gene in known MATR3-TFE3 fusion tumors using the substitute primer combination (MATR3-E15-F2; TFE3-E4-R2) of the present invention for verification.

Fig. 3: The sequence diagram of the successfully detected MATR3-TFE3 fusion gene by applying the primer combination of the present invention (MATR3-E15-F1; TFE3-E4-R1) in Xp11.2 tumor cases with unknown fusion partners.

Fig. 4: The sequence diagram of the successfully detected MATR3-TFE3 fusion gene in Xp11.2 tumor cases with unknown fusion partner by applying the substitute primer combination (MATR3-E15-F2; TFE3-E4-R2) of the present invention.

detailed description

The present invention is described further below by embodiment.

Embodiment 1 is verified for the case of definite diagnosis:

For cases in which new fusion sites of MATR3exon15-TFE3exon4 were detected by high-throughput sequencing RNA-seq, the primers designed by us were used for verification.

1. Extraction of RNA:

Strictly follow the RNeasy FFPE Kit operating instructions for extraction. ① Dewaxing: dewax the collected slides with xylene, rinse with absolute ethanol, air-dry and scrape off with a scalpel blade and put them into a 1.5ml EP tube; Proteinase K, mix well, enzymatically digest at 56°C for 15min, then at 80°C for 15min, cool on ice; ③add 16μl DNase buffer, then add 10μl DNase I, mix well, let stand at room temperature for 15min, centrifuge at 12000rpm for 15min, and take the supernatant;④ Add 320μl of binding solution and 720μl of absolute ethanol, mix well, transfer to the adsorption column twice, centrifuge at 8000rpm for 1min, discard the waste liquid; ⑤Wash: add 500μl of washing solution, centrifuge at 8000rpm for 1min; For waste liquid, transfer the adsorption column to a new 2ml collection tube, centrifuge at 12000rpm for 5min; ⑥Elute: transfer the adsorption column to a 1.5ml EP tube, add 100μl eluent, let stand at room temperature for 1min, and centrifuge at 12000rpm After 1 min, the concentration and purity of the collected eluate (ie, the DNA extract) was determined, and stored at -80°C until use.

2. Reverse transcription PCR RT-PCR

RNA was reverse-transcribed using a kit (K1622, RevertAid First Strand cDNA Synthesis Kit, MBI), and the method is detailed in the kit instructions. The PCR amplification primers are the MATR3-TFE3 fusion gene primer combination of this patent. The reaction system includes: 0.125 μl TaKaRa Ex Taq ^TM HS solution, 2.5 μl 10×Taq Buffer (Mg ²⁺ plus), 2 μl ldNTP (both purchased from Japan Takara Company), the primer concentration is 20 μmol/l, the cDNA template is 100 ng, sterile Deionized water was added to 25 μl. PCR amplification conditions were denaturation at 94°C for 3 minutes, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with a total of 35 cycles, and finally extension at 72°C for 5 min. The PCR product was electrophoresed with 3% agarose at a voltage of 100V, stained with ethidium bromide, observed under ultraviolet light, and sent for sequencing.

Result: after PCR with two pairs of primers (MATR3-E15-F1/TFE3-E4-R1; and MATR3-E15-F2/TFE3-E4-R2) of the present invention, a single specific electrophoresis band can be seen, and the amplified product Sequencing was carried out to obtain the fusion gene sequence of MATR3exon15-TFE3exon4 (Figure 1, Figure 2), which proved that the primer combination designed in this project was reliable and sensitive.

Example 2 Detection of Xp11.2 tumors with unknown translocation partners

We used the primer combination of the present invention to detect 35 Xp11.2 tumors with unknown translocation partners, and the RNA extraction, reverse transcription PCR and sequencing methods were the same as above.

Results: A total of 1 case detected MATR3-TFE3 fusion gene (Fig. 3, Fig. 4) by using the primer combination designed by the present invention. The results were verified by immunohistochemistry and fluorescence in situ hybridization, and the results were true and reliable.

Evaluation: The primer combination of the present invention is a supplement to the original Xp11.2 fusion gene primers, covers the unreported sites of the TFF3 fusion gene, and increases the detection rate of the RT-PCR method for diagnosing the tumor.

<110> Nanjing General Hospital of Nanjing Military Region of the Chinese People's Liberation Army

<120> A new translocation partner of Xp11.2 and its detection primers and application

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<212>DNA

<213> Artificial sequence

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<223> Primer MATR3-E15-F1

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cagttcagtg ggagacgaga 20

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<223> Primer TFE3-E4-R1

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gcgttgggtt ctccagat 18

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ggtgatgtgg cttctgatg 19

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<223> Primer TFE3-E4-R2

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cgagtgtggt ggacaggt 18

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cagttcagtg ggagacgaga ccgatcttgc taatttaggt gatgtggctt ctgatgggaa 60

aaaggaacca tcagataaag ctgtgaaaaa agatggaagt gcttcagcag cagcaaagaa 120

aaagcttaaa aaggtgcaga cccatctgga gaacccaacg c 161

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ggtgatgtgg cttctgatgg gaaaaaggaa ccatcagata aagctgtgaa aaaagatgga 60

agtgcttcag cagcagcaaa gaaaaagctt aaaaaggtgc agacccatct ggagaaccca 120

acgcgctacc acctgcagca ggcgcgccgg cagcaggtga aacagtacct gtccaccaca 180

ctcg 184

Claims (7)

1. A new translocation partner of Xp11.2, characterized in that it is MATR3-TFE3 gene translocation, the gene is located on chromosome 5, position 139273752-139331677, the sequence version number in GeneBank is GRCh38.p7, a total of 19 exons; gene fusion occurred between exon 15 of MATR3 and exon 4 of TFE3 gene. 2. The new translocation partner of Xp11.2 according to claim 1, characterized in that it comprises the nucleotide sequence shown in SEQ ID NO.5 or the nucleotide sequence shown in SEQ ID NO.6. 3. The application of the new translocation partner of Xp11.2 according to claim 1 in the preparation of MATR3-TFE3 translocation tumor diagnostic reagents. 4. A PCR primer for detecting MATR3-TFE3 translocation tumors, characterized in that it is a PCR primer for detecting the MATR3-TFE3 gene translocation according to claim 1. 5. The PCR primer for detecting MATR3-TFE3 translocation tumors according to claim 4, characterized in that it is selected from the following any pair of primers: MATR3-E15-F1 shown in SEQ ID NO.1 and SEQ ID NO. TFE3-E4-R1 shown in 2; or MATR3-E15-F2 shown in SEQ ID NO.3 and TFE3-E4-R2 shown in SEQ ID NO.4. 6. The application of the PCR primers described in claim 4 or 5 in the preparation of MATR3-TFE3 translocation tumor diagnostic reagents. 7. A diagnostic kit for MATR3-TFE3 translocation tumors, characterized in that it comprises the PCR primers according to claim 4 or 5.