Targeted capture probe and kit for detecting fusion genes of TFE3, TFEB and MITF in renal cell carcinoma and application of targeted capture probe and kit

Abstract

The invention discloses a targeted capture probe and kit for detecting TFE3, TFEB and MITF fusion genes in renal cell carcinoma and application of the targeted capture probe and kit, and relates to the technical field of biomedicine, and the sequence of the probe is shown as SEQ ID NO: 1-75. The target capture probe provided by the invention can accurately and effectively detect RNA level TFE3, TFEB and MITF genes and related fusion genes including known fusion genes and unknown fusion genes, determines the fusion gene panel targeting MiT family translocation renal cell carcinoma, overcomes the limitations of lower resolution and lower flux of a traditional detection method, and can be used for detecting the miT family translocation renal cell carcinoma. And a genetic basis can be provided for efficient accurate diagnosis and typing of MiT family translocation renal cell carcinoma through one-time detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a targeted capture probe, a kit and applications thereof for detecting TFE3, TFEB and MITF fusion genes in renal cell carcinoma. Background Art

[0002] MiT familial translocation renal cell carcinomas primarily include TFE3-rearranged renal cell carcinoma (TFE3-RCC) and TFEB fusion-associated renal cell carcinoma (TFE3-RCC). These molecularly driven malignancies are caused by the disruption of the TFE3 gene at region 11.2 of the short arm of chromosome X (Xp11.2) and the TFEB gene at region 21.1 of the short arm of chromosome 6 (6p21.1), respectively, and the subsequent specific translocations with other partner genes. The clinicopathological features of TFE3-RCC and TFEB-RCC are closely related to the functional heterogeneity of the fusion partner genes. Epidemiological data show that this subtype accounts for 1% to 4% of adult renal cancers but is predominant in adolescents and children (accounting for 15% and 20% to 50%, respectively), making it the most common renal cancer subtype in children. Notably, TFE3-RCC and TFEB-RCC exhibit significantly aggressive biological behavior, with approximately 40% of cases presenting with regional lymph node metastasis at initial diagnosis. This results in a five-year overall survival rate of less than 60% (approximately 30% lower than that of clear cell carcinoma). A recent multicenter study confirmed that the prognosis of localized TFE3-RCC and TFEB-RCC is similar to that of clear cell renal cell carcinoma (ccRCC) (5-year DFS 68% vs. 72%), but the survival of metastatic patients is significantly shortened (median OS 16 months vs. 28 months), highlighting the urgent need for precise molecular classification and treatment.

[0003] Currently, more than 20 TFE3 and TFEB partner genes (including SFPQ, ASPSCR1, NONO, PRCC, and MALAT1) have been identified internationally. Different fusion subtypes lead to significant differences in tumor phenotypes by regulating signaling pathways such as MAPK / mTOR and Wnt / β-catenin. Based on multi-omics analysis, our team found that ASPSCR1-TFE3 fusion subtypes exhibit highly invasive characteristics (ISUP nuclear grade ≥3 in 82%, lymph node metastasis rate in 64%, median OS of 31 months), while MED15-TFE3 fusion subtypes exhibit unique immune microenvironment characteristics (PD-L1 CPS ≥10 in 58%) (Nature Communications 2021). In terms of treatment response, targeted immunotherapy combination therapy can increase the objective response rate of patients with ASPSCR1-TFE3 to 46% (vs. 22% for other subtypes), while patients with PRCC-TFE3 fusions have demonstrated a 78% disease control rate with everolimus (Molecular Cancer 2024; Modern Pathology 2025). These findings suggest that accurate identification of fusion subtypes is crucial for prognostic stratification and treatment decisions.

[0004] However, the clinical diagnosis of MiT family translocation renal cell carcinoma faces two technical bottlenecks: First, extensive overlap between histomorphology and conventional immunophenotyping (e.g., CAIX, CD10) results in a misdiagnosis rate as high as 35%. Second, existing molecular diagnostic techniques have inherent limitations. Immunohistochemical staining for TFE3 and TFEB can provide initial screening (sensitivity 89%, specificity 76%), but the FISH technique, which confirms the diagnosis, has a false-negative rate exceeding 25% for short-segment translocations and paracentric translocations due to probe design limitations (resolution >50 kb), and is unable to resolve fusion partner gene information. Although next-generation sequencing (NGS) can overcome these limitations (sensitivity >99% for fusion gene detection), whole-genome / transcriptome sequencing is hindered by high costs (>$2000 per sample) and lengthy turnaround times (>14 days), resulting in clinical adoption rates of less than 15%.

[0005] In view of this, this application is hereby filed. Summary of the Invention

[0006] The purpose of the present invention is to provide a targeted capture probe, a kit and its application for detecting TFE3, TFEB and MITF fusion genes in renal cell carcinoma, so as to solve the problems of misdiagnosis and missed diagnosis in the prior art.

[0007] First, an embodiment of the present invention provides a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, characterized in that the sequence of the probe is shown in SEQ ID NOs: 1-75.

[0008] As an optional embodiment, the probe covers the mRNA coding region and non-coding region of the three main genes TFE3, TFEB, MITF and their partner genes of MiT family translocation renal cell carcinoma; The partner genes include SFPQ, ASPSCR1, NONO, MED15, PRCC, RBM10, PARP14, GRIPAP1, EWSR1, CLTC, FUBP1, KHSRP, LUC7L3, MATR3, RBMX, SETD1B, ZC3H4, VCP, PTPN12, ZNF627, YAP1, PHF1, EIF4A2, CADM2, COL21A1, ACTB, CLTC, ACTG1, KATA6A, AR1D1B, NEAT1, MALAT1, and DVL2.

[0009] As an optional embodiment, the probe is 120 bp in length, the flanking length is 10 bp, the interval between adjacent probes is 10 to 15 bp, and a 3X tiling design is used to reduce coverage omission areas.

[0010] As an optional embodiment, the sequence design of the probe is based on the GRCh37 gencode v19 CTATlib Mar012021.source.targz.md5sum genome. The probe can specifically bind to the fusion site region of TFE3, TFEB, MITF and their partner genes, and can distinguish normal gene sequences from fusion gene sequences to avoid false positive results.

[0011] Secondly, an embodiment of the present invention also provides a kit for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, comprising an RNA extraction kit, a library construction kit, a hybridization capture kit, and the above-mentioned targeted capture probe.

[0012] As an optional implementation method, the RNA extraction kit is suitable for extracting total RNA from FFPE samples, the library construction kit can break the extracted RNA fragments and construct a chain-specific RNA library, and the hybridization capture kit can be combined with a targeted capture probe to achieve specific capture of TFE3, TFEB, and MITF fusion genes.

[0013] Finally, the present invention also provides an application of a targeted capture probe for detecting the TFE3 fusion gene in renal cell carcinoma, including a method for detecting the TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, the method comprising the following steps: S1: RNA extraction from FFPE samples; S2: Construction of pre-library for sequencing analysis; S3: Using the targeted capture probe to perform hybridization capture on the pre-library to obtain a cDNA capture final library; S4: Perform high-throughput sequencing on the captured cDNA fragments.

[0014] As an optional embodiment, in S1, the specific steps of extracting RNA from FFPE samples include: FFPE samples were deparaffinized using xylene; Ethanol was used for precipitation; Total RNA was extracted using a column-based extraction method to ensure RNA purity.

[0015] As an optional implementation, in S2, constructing a pre-library for sequencing analysis includes: Total RNA was collected for library construction; RNA fragments were randomly broken into 300-400 bp using enzyme digestion technology; Perform first-strand cDNA reverse transcription to construct a strand-specific RNA library; The end-repaired product is tailed with dA and connected to a specific Illumina adapter; The ligation product was purified and UDI Primer was added for library amplification to obtain a prelibrary.

[0016] As an optional embodiment, in S4, the high-throughput sequencing includes: Sequencing was performed using the Illumina second-generation sequencing platform; Single-end sequencing or double-end sequencing mode is used, and the sequencing length is 100~150bp; Perform quality control on sequencing data to remove low-quality reads and adapter sequences; The high-quality sequencing data were compared with the reference genome to determine the presence or absence of TFE3, TFEB, and MITF fusion genes and their specific fusion sites.

[0017] Compared with the prior art, the embodiments of the present invention have the following advantages and beneficial effects: 1. The targeted capture probe provided by the embodiments of the present invention can accurately and effectively detect TFE3, TFEB, MITF genes and their related fusion genes at the RNA level, including known fusion genes and unknown fusion genes, and has identified a fusion gene panel targeting TFE3 rearrangement renal cell carcinoma as the main target. This overcomes the limitations of low resolution and low throughput of traditional detection methods, and provides a genetic basis for the accurate diagnosis and typing of TFE3 rearrangement renal cell carcinoma with a single test.

[0018] 2. The embodiment of the present invention adopts a shingled probe design, which is based on 3 main genes and 33 fusion genes to capture fusion gene probes mainly for TFE3 rearrangement renal cell carcinoma, with extremely high targeting. The length of the two wings of the probe is extended, and the amount of sequencing data is increased, which is conducive to the discovery of new fusion sites and new fusion genes around the target gene, and can provide more accurate companion diagnostic medical information for clinical treatment.

[0019] 3. The present invention uses transcriptome-targeted hybridization capture technology for gene fusion detection, which has the following advantages compared to traditional DNA-based detection methods: Traditional DNA-based gene fusion detection methods often reduce detection sensitivity because they need to cover long intron regions containing a large number of repetitive sequences. However, the embodiments of the present invention target transcribed RNA, which does not contain introns. Therefore, the impact of intron regions on detection sensitivity is avoided, significantly improving the detection sensitivity of fusion genes. When performing gene fusion detection at the DNA level, existing technologies place high demands on bioinformatics algorithms due to the complexity of intron regions, increasing the difficulty and cost of data analysis. The embodiments of the present invention avoid interference from intron regions, simplify the bioinformatics analysis process, reduce algorithm requirements, and improve the accuracy and efficiency of data analysis. In the prior art, the breakpoints of some genes are located in introns, which are long. This makes it difficult to completely cover these regions when designing probes at the DNA level, which may lead to failure in fusion gene detection. However, the embodiments of the present invention use RNA for detection, which does not contain introns. Therefore, there is no problem of covering intron regions, and fusion genes can be detected more comprehensively. Complex structural variations may occur in the DNA of tumor patients. These variations may affect subsequent capture sequencing, resulting in failure of fusion gene detection. Since the embodiments of the present invention are based on RNA for detection, and RNA does not contain introns, it can avoid the impact of these complex structural variations on detection and improve the success rate of fusion gene detection.

[0020] 4. The embodiments of the present invention are aimed at low-quality FFPE samples. Targeted capture sequencing can reduce false results caused by sample quality problems and improve detection accuracy.

[0021] 5. In this embodiment, the library is constructed by reverse transcription of RNA into cDNA. Enzymatic digestion randomly breaks the fragments into 300-400 bp, followed by immediate reverse transcription of the first-strand cDNA to construct a strand-specific RNA library. This eliminates the need for second-strand cDNA synthesis and degradation or template switching, reduces the generation of dimers and repeats, and provides higher alignment rates and transcript detection. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Flowchart for constructing a pre-library for sequencing analysis according to an embodiment of the present invention; Figure 2 Flowchart of hybridization capture in an embodiment of the present invention.

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0024] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0025] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0026] In the description of the present invention, it should be noted that the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0027] Example Note: The ideal detection system should meet the following technical indicators: (1) Covering all reported TFE3, TFEB, and MITF partner genes (including PRCC, NONO, ASPSCR1, etc.); (2) Detection sensitivity ≥ 98%, specificity ≥ 99%; (3) It can identify new fusion events; (4) The testing period is ≤ 5 working days; (5) The cost of testing a single sample is controlled within $500.

[0028] Therefore, by optimizing the probe design strategy (such as using double-ended anchor probes), introducing a digital PCR quantitative quality control system, and establishing an intelligent bioinformatics analysis process, the construction of a clinical-grade detection system can be achieved.

[0029] Traditional FISH technology is limited by probe resolution (>50kb) and detection principles. The false-negative rate for intrachromosomal rearrangements and microtranslocations is as high as 25%-30%, and it cannot identify partner gene types. Conventional immunohistochemistry (TFE3 staining) suffers from nonspecific nuclear staining interference and has a specificity of only 76%, leading to misdiagnosis and missed diagnoses.

[0030] Although NGS can theoretically cover all fusion subtypes (sensitivity > 99%), the cost of whole genome / transcriptome sequencing exceeds $2,000 per sample, the detection cycle is as long as 14 to 21 days, and the sample quality requirements are strict (FFPE sample DV200 needs to be >50%). As a result, the clinical penetration rate is less than 15%, which makes it difficult to meet real-time diagnosis and treatment needs.

[0031] The existing detection system lacks the ability to capture TFE3, TFEB, and MITF partner genes in a targeted manner. In approximately 10% to 25% of cases, the specific fusion subtype cannot be clearly identified (especially those involving low-frequency / rare partner genes), resulting in the inability to effectively link molecular typing with targeted treatment options.

[0032] Therefore, the present invention provides a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma. The main purpose is to accurately detect the transcriptional level of TFE3, TFEB, and MITF as the main genes and their fusion partner genes, improve the detection rate and sensitivity, and have the advantages of short detection cycle and low detection cost. The partner genes include the following 33: SFPQ, ASPSCR1, NONO, MED15, PRCC, RBM10, PARP14, GRIPAP1, EWSR1, CLTC, FUBP1, KHSRP, LUC7L3, MATR3, RBMX, SETD1B, ZC3H4, VCP, PTPN12, ZNF627, YAP1, PHF1, EIF4A2, CADM2, COL21A1, ACTB, CLTC, ACTG1, KATA6A, AR1D1B, NEAT1, MALAT1, and DVL2.

[0033] Specifically, the embodiment of the present invention provides a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, the nucleotide sequence of which is shown in Table 1 below:

[0034] Probes were designed for the coding and noncoding regions of the mRNAs of the main genes TFE3, TFEB, and MITF and their fusion partner genes, respectively, using the GRCh37 gencode v19 CTAT lib Mar012021.source.targz.md5sum genome. The transcript numbers of the main genes and their corresponding genomic consensus coding sequence numbers are shown in Table 2 below: Table 2

[0035] The targeted capture probe is 120 bp long, with a 10 bp flank length, and a 10-15 bp interval between adjacent probes. The 3X tiling design reduces coverage omissions. The genomic coding sequence number is the distribution combination sequence number of the transcript RNA sequence on the genomic DNA. Experimental verification has shown that the probe combination of the present invention can accurately and effectively detect RNA levels of TFE3, TFEB, MITF genes and their related fusion genes, including known fusion genes and unknown fusion genes.

[0036] The embodiment of the present invention also provides a kit for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, wherein the kit includes a targeted capture probe, an RNA extraction kit, a library construction kit, and a hybridization capture kit.

[0037] In order to better verify the effect of the targeted capture probe in the embodiment of the present invention, the embodiment of the present invention also provides an application of the targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, which is also a method for constructing the above-mentioned kit, comprising the following steps: S1: RNA extraction from FFPE samples Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples to ensure RNA purity. The extraction process included: a. Deparaffinize FFPE samples using xylene. b. Dehydration with ethanol; c. Extract total RNA using column extraction or magnetic bead extraction to remove protein and genomic DNA contamination.

[0038] S2: Construction of pre-library for sequencing analysis Library construction is performed on the extracted RNA, including the following sub-steps: a. Take an appropriate amount of total RNA (e.g., 500 ng) and randomly fragment the RNA into 300-400 bp using enzyme digestion technology; b. Perform first-strand cDNA reverse transcription to construct a strand-specific RNA library; c. Add dA tails to the end-repaired products and connect to specific Illumina adapters; d. Purify the ligation product and add UDI Primer for library amplification to obtain a pre-library.

[0039] S3: Use the targeted capture probe to perform hybridization capture on the pre-library to obtain the cDNA capture final library The constructed prelibrary is hybridized with the target capture probe, including the following sub-steps: a. Hybridize the prelibrary with the target capture probe under appropriate conditions (e.g., 65°C for 12-16 hours); b. Use magnetic beads or column purification to enrich the cDNA fragments bound to the probe and remove unbound nonspecific fragments; c. Elute the enriched cDNA fragments to obtain the captured final library.

[0040] S4: High-throughput sequencing of captured cDNA fragments High-throughput sequencing of captured cDNA fragments includes the following sub-steps: a. Sequencing using an Illumina next-generation sequencing platform (e.g., NovaSeq or NextSeq); b. Use paired-end sequencing mode with a sequencing length of 150bp; c. Perform quality control on sequencing data and remove low-quality reads and adapter sequences; d. Compare the high-quality sequencing data to the reference genome to determine the presence and specific fusion sites of TFE3, TFEB, and MITF genes.

[0041] The present invention will be further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] Example 1: Detection of SFPQ-TFE3 fusion gene in 1 / 200 positive standards Specimen information: Total RNA extracted from FFPE tissue specimens adjacent to the cancer was diluted at a ratio of 1:200 for gene detection.

[0043] The experimental steps are as follows: Step 1: FFPE RNA extraction: 1. Place 8 (typically 5-10) paraffin samples into a 1.5 mL centrifuge tube. Add 1 mL of xylene (in other examples, equivalent substitutes may be used). Vortex to mix. Incubate in a 56°C metal bath / water bath for 3 minutes. Centrifuge at 13,000 rpm for 1 minute and discard the supernatant.

[0044] 2. Add 1 mL of anhydrous ethanol, shake to mix, let stand at room temperature for 2 minutes, centrifuge at 13000 rpm for 1 minute, and discard the supernatant.

[0045] 3. Add 240 μL of lysis buffer and 20 μL of proteinase K and vortex to mix. Place the centrifuge tube in a 56°C metal bath / water bath and incubate for 20 minutes.

[0046] 4. Transfer to a 90°C metal bath / water bath and incubate for 30 minutes.

[0047] 5. Remove the centrifuge tube, place it in an ice bath for 3 minutes, centrifuge at 13,000 rpm for 5 minutes, and carefully transfer the supernatant to a new 1.5 mL centrifuge tube.

[0048] 6. Add 250 μL of binding solution and 800 μL of anhydrous ethanol and shake to mix.

[0049] 7. Add the mixed solution to the centrifuge column, centrifuge at 10,000 rpm for 1 minute, discard the filtrate, and repeat this step until the mixed solution has passed through the column.

[0050] 8. Add 500 μl of cleaning solution and centrifuge at 10,000 rpm for 1 minute. Discard the filtrate and repeat once. Centrifuge the empty column at 13,000 rpm for 2 minutes. Transfer the adsorption column to a new 1.5 ml centrifuge tube and add 80 μl (50-100 μl can be selected) of enzyme-free water. Let it stand at room temperature for 2 minutes. Centrifuge at 13,000 rpm for 1 minute and collect the filtrate.

[0051] 9. Perform quality control on total RNA concentration and fragments, requiring the total RNA amount to be greater than or equal to 500 ng and DV200 ≥ 20%.

[0052] Step 2: Total RNA library construction (refer to Figure 1 shown) 1. Take 500ng of total RNA and fragment it at 65℃ for 5 minutes. The required target fragment is 350~400bp.

[0053] 2. Reverse transcription, using random primers to synthesize the first-strand cDNA. 3. Using Adaptase technology, tailing and ligation are performed simultaneously to integrate the R2 Stubby Adapter into the 3' end of the first-strand cDNA molecule.

[0054] 4. Extension reaction: This step extends one strand into two strands to form dsDNA.

[0055] 5. Ligation reaction: Add R1 Stubby Adapter to the 3' end of the primer-extended double-stranded cDNA molecule.

[0056] 6. Library amplification: Add specific UDI Primers to both ends of the library for PCR amplification and enrichment.

[0057] 7. After library purification, perform concentration quantification and fragment quality control.

[0058] Step 3: Hybridization capture (refer to Figure 2 shown) 1. Take equal amounts of library and hybridize with fusion probe overnight.

[0059] 2. Post-hybridization capture: capture the hybridized library using streptavidin magnetic beads and elute with elution buffer.

[0060] 3. After capture, library amplification is performed and the number of PCR cycles is optimized based on the amount of library to be hybridized.

[0061] 4. After amplification, the library was purified and quantified and quality checked, and the library was sequenced using the Illumina NovaSeqXPlus sequencing platform.

[0062] 5. Sequencing Analysis: Fusion gene results were analyzed using Ariba and STAR FUSION software. The instrument used was the Illumina NovaSeqX Plus. The STAR FUSION software analysis results are shown in Table 3.

[0063] Table 3

[0064] As shown in Table 3, the SFPQ-TFE3 fusion gene transcript was detected, indicating that a fusion event between the SFPQ gene and the TFE3 gene occurred in the sample. JunctionReadCount (673) indicates that 673 reads spanned the fusion junction, while SpanningFragCount (14) indicates that 14 fragments spanned the fusion junction, indicating that the fusion event was sufficiently supported by sequencing data and had a high degree of confidence. The FFPM data indicated that the number of fusion fragments per million fragments was 15.5585, indicating that the expression level of the fusion gene in the sample was relatively high, further supporting the existence of the fusion gene.

[0065] Example 2: Example 2 Clinical Detection of SFPQ-TFE3 Fusion Gene Specimen information: Tissue specimens from patients positive for the SFPQ-TFE3 fusion gene.

[0066] Experimental steps: Same as Example 1.

[0067] Detection instrument: illumina NovaSeqXPlus.

[0068] Detection results: SFPQ-TFE3 fusion gene transcripts were detected. The results of STAR FUSION software analysis are shown in Table 4: Table 4

[0069] The sample was also subjected to conventional transcriptome sequencing, which detected the SFPQ-TFE3 fusion gene transcript. The number of fusion reads detected in this sample using the TFE3 rearrangement renal cell carcinoma fusion gene was approximately 14 times that of full-length transcriptome sequencing. The results of STARFUSION software analysis are shown in Table 5: Table 5

[0070] Combining Tables 4 and 5, we can see that in the TFE3 fusion probe assay, the JunctionReadCount was 783, the SpanningFragCount was 22, the total fusion reads were 805, and the FFPM was 13.5629. In contrast, in the full-length transcriptome sequencing assay, the JunctionReadCount was 54, the SpanningFragCount was 2, the total fusion reads were 56, and the FFPM was 0.775. This indicates that the sensitivity of the TFE3 fusion probe assay is significantly higher than that of the full-length transcriptome sequencing assay. Furthermore, in this sample, the number of fusion reads detected by the fusion probe assay was approximately 14 times that of the full-length transcriptome sequencing assay (805 vs. 56), indicating that the fusion probe assay is more effective in enriching and detecting fusion genes, especially with higher sensitivity in detecting low-abundance fusion genes.

[0071] Therefore, TFE3 fusion probe detection has high sensitivity and specificity, and can more accurately detect the presence of SFPQ-TFE3 fusion gene, which is of great significance for the clinical diagnosis and treatment of TFE3 rearrangement renal cell carcinoma.

[0072] Example 3: Clinical Detection of SFPQ-TFE3 Fusion Gene Specimen information: Tissue specimens from patients positive for the SFPQ-TFE3 fusion gene.

[0073] Experimental steps: Same as Example 1.

[0074] Detection instrument: Illumina NovaSeqXPlus, STAR FUSION software analysis results are shown in Table 6: Table 6

[0075] As shown in Table 6, the SFPQ-TFE3 fusion gene transcript was detected, indicating that a fusion event between the SFPQ gene and the TFE3 gene existed in this sample. The JunctionReadCount was 30, indicating that 30 sequencing reads spanned the fusion junction of the SFPQ and TFE3 genes. The number of fusion reads was 30, indicating that a total of 30 reads supported the existence of the SFPQ-TFE3 fusion gene. The FFPM was 0.6258, indicating that 0.6258 fragments per million sequenced fragments supported the existence of the SFPQ-TFE3 fusion gene. The LeftBreakpoint was chr1:35652602:-, indicating that the breakpoint of the SFPQ gene was on chromosome 1 at position 35,652,602, and the direction was the negative strand. The SpanningFragCount was 0, indicating that no fragments spanned the fusion junction. This shows that despite its low abundance (FFPM is 0.6258), the existence of the fusion gene is supported by sufficient sequencing data, indicating that the SFPQ-TFE3 fusion gene does exist in this sample.

[0076] Example 4: Clinical Detection of SFPQ-TFE3 Fusion Gene Specimen information: Tissue specimens from patients positive for the SFPQ-TFE3 fusion gene.

[0077] Experimental steps: Same as Example 1.

[0078] Detection instrument: Illumina NovaSeqXPlus, STAR FUSION software analysis results are shown in Table 7: Table 7

[0079] As shown in Table 7, the SFPQ-TFE3 fusion gene transcript was detected, indicating that a fusion event between the SFPQ gene and the TFE3 gene existed in this sample. The JunctionReadCount was 34, indicating that 34 sequencing reads spanned the fusion junction of the SFPQ and TFE3 genes. The number of combined reads was 35, indicating that a total of 35 reads supported the existence of the SFPQ-TFE3 fusion gene. The FFPM was 0.5639, indicating that 0.5639 fragments per million sequenced fragments supported the existence of the SFPQ-TFE3 fusion gene, indicating that although the abundance of the fusion gene was relatively low, it could still be detected.

[0080] LeftBreakpoint is chr1:35654604:-, indicating that the breakpoint of the SFPQ gene is on chromosome 1 at position 35,654,604, and the direction is the negative strand. RightBreakpoint is chrX:48898095:-, indicating that the breakpoint of the TFE3 gene is on chromosome X at position 48,898,095, and the direction is the negative strand. SpanningFragCount is 1, indicating that one fragment spans the fusion junction.

[0081] Example 5: Clinical Detection of ASPSCR1-TFE3 Fusion Gene Specimen information: Tissue specimens from patients positive for the ASPSCR1-TFE3 fusion gene.

[0082] Experimental steps: Same as Example 1.

[0083] Detection instrument: Detection instrument: Illumina NovaSeqXPlus, STAR FUSION software analysis results are shown in Table 8: Table 8

[0084] As shown in Table 8, the ASPSCR1-TFE3 fusion gene transcript was successfully detected in this sample. Although its abundance was low (FFPM was 0.1155), the presence of the fusion gene was sufficiently supported by sequencing data (JunctionReadCount was 9, and the number of fusion reads was 9). The detection of the ASPSCR1-TFE3 fusion gene transcript can serve as a molecular marker for TFE3-rearranged renal cell carcinoma, helping to confirm this type of renal cell carcinoma. The abundance of the fusion gene (such as the FFPM value) can be used as one of the indicators for prognostic assessment, helping to predict the patient's disease progression and treatment response. Although the fusion gene abundance was low, it was still detectable, indicating that the detection method of the embodiment of the present invention has high sensitivity.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, improvements, and the like made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention. It should be noted that the structures or components illustrated in the accompanying drawings are not necessarily drawn to scale, and that descriptions of known components, processing techniques, and processes are omitted to avoid unnecessarily limiting the present invention.

Claims

1. A targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, characterized in that: The sequences of the probes are shown in SEQ ID NOs: 1-75.

2. A targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 1, characterized in that: The probe covers the mRNA coding and non-coding regions of the main genes TFE3, TFEB, MITF and their partner genes in MiT family translocation renal cell carcinoma; The partner genes include SFPQ, ASPSCR1, NONO, MED15, PRCC, RBM10, PARP14, GRIPAP1, EWSR1, CLTC, FUBP1, KHSRP, LUC7L3, MATR3, RBMX, SETD1B, ZC3H4, VCP, PTPN12, ZNF627, YAP1, PHF1, EIF4A2, CADM2, COL21A1, ACTB, CLTC, ACTG1, KATA6A, AR1D1B, NEAT1, MALAT1, and DVL2.

3. A targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 2, characterized in that: The probe is 120 bp in length, with a flank length of 10 bp. The interval between adjacent probes is 10 to 15 bp. A 3X tiling design is used to reduce coverage omission areas.

4. A targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 2, characterized in that: The sequence design of the probe is based on the GRCh37 gencode v19 CTAT libMar012021.source.targz.md5sum genome. The probe can specifically bind to the fusion site region of TFE3, TFEB, MITF and their partner genes, and can distinguish normal gene sequences from fusion gene sequences, avoiding false positive results.

5. A kit for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma, characterized in that: The invention comprises an RNA extraction kit, a library construction kit, a hybridization capture kit and the targeted capture probe according to any one of claims 1 to 4.

6. A kit for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 5, characterized in that: The RNA extraction kit is suitable for extracting total RNA from FFPE samples, the library construction kit can break the extracted RNA fragments and construct a chain-specific RNA library, and the hybridization capture kit can be combined with a targeted capture probe to achieve specific capture of TFE3, TFEB, and MITF fusion genes.

7. Use of a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to any one of claims 1 to 4, characterized in that: A method for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma comprises the following steps: S1: RNA extraction from FFPE samples; S2: Construction of pre-library for sequencing analysis; S3: Using the targeted capture probe to perform hybridization capture on the pre-library to obtain a cDNA capture final library; S4: Perform high-throughput sequencing on the captured cDNA fragments.

8. The use of a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 7, characterized in that: In S1, the specific steps for extracting RNA from FFPE samples include: FFPE samples were deparaffinized using xylene; Ethanol was used for precipitation; Total RNA was extracted using a column-based extraction method to ensure RNA purity.

9. The use of a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 7, characterized in that: In S2, constructing a pre-library for sequencing analysis includes: Total RNA was collected for library construction; RNA fragments were randomly broken into 300-400 bp using enzyme digestion technology; Perform first-strand cDNA reverse transcription to construct a strand-specific RNA library; The end-repaired product is tailed with dA and connected to a specific Illumina adapter; The ligation product was purified and UDI Primer was added for library amplification to obtain a prelibrary.

10. The use of a targeted capture probe for detecting TFE3, TFEB, and MITF fusion genes in renal cell carcinoma according to claim 7, characterized in that: In S4, high-throughput sequencing includes: Sequencing was performed using the Illumina second-generation sequencing platform; Single-end sequencing or paired-end sequencing mode is used, and the sequencing length is 100-150bp; Perform quality control on sequencing data to remove low-quality reads and adapter sequences; The high-quality sequencing data were compared with the reference genome to determine the presence or absence of TFE3, TFEB, and MITF fusion genes and their specific fusion sites.

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