Primers, kits and methods for detecting KMT2D gene mutations
By designing a kit for KMT2D gene, combined with PCR and Sanger sequencing technology, the problem of difficult to quickly detect polymorphic loci mutations in the KMT2D gene in the prior art is solved, and rapid and accurate detection is achieved, providing a reliable diagnostic basis.
Patent Information
- Application Number
- CN202411898817.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art is difficult to quickly and effectively detect polymorphic locus mutations in the KMT2D gene in patients with follicular lymphoma, affecting disease diagnosis and treatment decisions.
A kit is designed to detect hot spots of polymorphic mutations of KMT2D genes. Using PCR technology and Sanger sequencing, all 45 exon sequences of the KMT2D gene are amplified and sequenced through specific primer sequences to ensure that various mutations in the gene can be detected.
The rapid and accurate detection of polymorphic mutation sites of the KMT2D gene is achieved, reducing the detection cost and difficulty, and providing reliable experimental basis to guide clinical treatment and diagnosis.
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Figure CN119753142A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of molecular detection, and particularly relates to a primer, a kit and a method for detecting KMT2D gene mutation. Background Art
[0002] Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma after diffuse large B-cell lymphoma. In most cases, FL is a systemic tumor of lymphoid tissue that occurs in adults. FL accounts for 5% of all hematological neoplasms, about 35% of all non-Hodgkin lymphomas, and 70% of indolent lymphomas. In most patients, FL is a chronic, incurable disease with a long natural course and survival typically measured in decades. FL is a tumor derived from follicular center B cells, usually including centrocytes and centroblasts, with varying proportions depending on the lymphoma grade. The growth pattern may be completely follicular, follicular and diffuse, and rarely completely diffuse. FL is characterized by diffuse lymphadenopathy, bone marrow involvement, and splenomegaly, with extranodal involvement being less common and cytopenias being relatively common. Systemic symptoms such as fever, night sweats, and weight loss are uncommon in the absence of transformation to diffuse large B-cell lymphoma.
[0003] Approximately 85% of FL patients have t(14;18) leading to overexpression of BCL-2 protein, a member of a family of proteins that block apoptosis. Mutations in chromatin-modifying genes (KMT2D, CREBBP, EZH6) are a common feature of FL, and among these genes, KMT2D (also known as MLL2 / ALR / MLL4) is the most commonly mutated gene, present in 70% to 90% of FL patients. The gene KMT2D has a total of 54 exons and encodes a highly conserved protein that belongs to the SET1 family of histone lysine methyltransferases (KMTs), a group of enzymes that catalyze the methylation of lysine 4 on histone H3 (H3K4) and are associated with transcriptionally active chromatin. The enzymatic function of KMT2D depends on a set of C-terminal conserved domains, including a PHD domain, two FY-rich motifs (FYRC and FYRN), and a catalytic SET domain. In FL, KMT2D mutations predominantly manifest as premature stop codons, frameshift insertions / deletions, and splice site mutations, which are predicted to produce truncated proteins lacking part or all of the C-terminal protein domain. In addition, multiple missense mutations have been identified in the KMT2D protein, but their functional consequences remain unexplored.
[0004] FL is diagnosed by identifying nodular B-cell proliferation in lymph node biopsy. After PET-CT staging and bone marrow biopsy, a large proportion of patients do not require immediate treatment. When treatment is required, common regimens include anti-CD20 immunotherapy with or without chemotherapy. This experiment used Sanger sequencing to obtain the mutation of the KMT2D gene, which effectively helped to reveal the mechanism of enhancer activation related to the destruction of key B-cell networks by KMT2D gene mutations, and further helped to understand the etiology and pathogenesis of the disease. In addition, although the overall survival of most patients is prolonged, relapses are very frequent, and early relapses and transformation to aggressive lymphomas indicate a worse prognosis. Therefore, this project can provide a reliable experimental basis for clinical treatment and diagnosis, and provide guidance for its treatment plan. Gene therapy has become a fundamental treatment method, thereby promoting research progress in related fields and providing a reference for basic research. Summary of the invention
[0005] The purpose of the present invention is to provide a kit for detecting KMT2D gene polymorphic mutation hotspots, which adopts PCR technology and Sanger sequencing and can be used to quickly detect the mutation of KMT2D gene polymorphic sites in patients.
[0006] Primers for detecting KMT2D gene mutations are characterized by comprising primers for amplifying KMT2D genes, the base sequence of which is:
[0007]
[0008]
[0009] Furthermore, the primers also include sequencing primers for detecting the KMT2D gene, and the base sequence thereof is: M13 F: TGTAAAACGACGGCCAGT
[0010] M13 R:AACAGCTATGACCATG.
[0011] Further, primer sequences K1-F and K1-R are primers for amplifying exon sequences of KMT2D gene 1, 2, 3, 4, and 5, primer sequences K2-F and K2-R are primers for amplifying exon sequences of KMT2D gene 6, 7, 8, and 9, primer sequences K3-F and K3-R are primers for amplifying exon sequences of KMT2D gene 10, primer sequences K4-F and K4-R are primers for amplifying exon sequences of KMT2D gene 11, primer sequences K5-F and K5-R are primers for amplifying exon sequences of KMT2D gene 12 and 13, primer sequences K6-F and K6-R are primers for amplifying exon sequences of KMT2D gene 14, primer sequences K7-F and K7-R are primers for amplifying exon sequences of KMT2D gene 15 Primers K8-F and K8-R are primers for amplifying exon sequences of KMT2D gene 15, 16, 17, 18, primers K9-F and K10-R are primers for amplifying exon sequences of KMT2D gene 22, 23, 24, primers K10-F and K10-R are primers for amplifying exon sequences of KMT2D gene 25, 26, 27, primers K11-F and K11-R are primers for amplifying exon sequences of KMT2D gene 28, 29, 30, primers K12-F and K12-R as well as K13-F and K13-R are primers for amplifying exon sequences of KMT2D gene 31 Primers of the sequence, primer sequences K14-F and K14-R are primers for amplifying exon sequences of KMT2D gene 32 and 33, primer sequences K15-F and K15-R and K16-F and K16-R are primers for amplifying exon sequences of KMT2D gene 34, primer sequences K17-F and K17-R are primers for amplifying exon sequences of KMT2D gene 35, 36, 37, 38, primer sequences K18-F and K18-R, K19-F and K19-R and K20-F and K20-R are primers for amplifying exon sequences of KMT2D gene 39, primer sequences K21-F and K21-R are primers for amplifying exon sequences of KMT2D gene 40, 41, 42 Primers, primer sequences K22-F and K22-R are primers for amplifying exon sequences 43, 44, and 45 of the KMT2D gene, primer sequences K23-F and K23-R are primers for amplifying exon sequences 46 and 47 of the KMT2D gene, primer sequences K24-F and K24-R are primers for amplifying exon sequences 48 of the KMT2D gene, primer sequences K25-F and K25-R are primers for amplifying exon sequences 49 and 50 of the KMT2D gene, primer sequences K26-F and K26-R are primers for amplifying exon sequences 51, 52, and 53 of the KMT2D gene, and primer sequences K27-F and K27-R are primers for amplifying exon sequences 54 of the KMT2D gene.
[0012] The present invention also provides a method for detecting KMT2D gene polymorphism hotspot mutation, comprising the following steps:
[0013] (1) Extract tissue DNA from blood;
[0014] (2) Amplify the DNA extracted in step 1 by PCR; wherein the PCR amplification primers are:
[0015]
[0016]
[0017]
[0018] (3) Sequencing the amplified product in step 2; the base sequence of the sequencing primer is:
[0019] M13 F:TGTAAAACGACGGCCAGT
[0020] M13 R:AACAGCTATGACCATG;
[0021] (4) Judging the sequencing results, determining whether the KMT2D gene has mutated.
[0022] Finally, the present invention provides a kit for detecting polymorphic mutation sites of the KMT2D gene, comprising:
[0023] (i) Blood DNA extraction reagents;
[0024] (ii) Detection system PCR amplification reaction solution; including primers for amplifying the KMT2D gene, whose base sequence is:
[0025]
[0026]
[0027] (iii) Sequencing system reagents; including sequencing primers for detecting the KMT2D gene, whose base sequence is:
[0028] M13 F:TGTAAAACGACGGCCAGT
[0029] M13 R:AACAGCTATGACCATG.
[0030] Beneficial effects: The present invention designs primers for amplifying all 45 exon sequences of KMT2D. By adding a linker, the PCR products of all 27 pairs of primers can be sequenced with one sequencing primer. A stable amplification system is constructed using PCR technology. By adjusting reaction conditions such as primer concentration and annealing temperature, the amplification efficiency can be optimized. The mutation types of the KMT2D gene are various and spread throughout the entire gene. Therefore, the primers of the present invention can not only amplify the KMT2D full exon sequence, but also ensure that no matter where these exons mutate, there will be no missed detection. Compared with the fluorescent quantitative PCR method, the cost and difficulty of detection are reduced. The fluorescent quantitative PCR method requires the design of multiple probes for different mutation types, which is costly and difficult to detect. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is the chromosome location map of the KMT2D gene.
[0032] Figure 2 The figure shows the mutation sites of KMT2D gene that are currently reported to be associated with the disease FL.
[0033] Figure 3 The sequencing map of the clinical sample detection found that the 10th exon of the third pair of primers of KMT2D contained a c.1938C>G mutation.
[0034] Figure 4 The sequencing map of the clinical sample detection found that the 11th exon of the 4th pair of primers of KMT2D contained c.2826C>CT mutation.
[0035] Figure 5 The sequencing map of the clinical sample detection found that the 18th intron of the 7th pair of primers of KMT2D contained the c.4741+44G>A mutation.
[0036] Figure 6 The sequencing map of the clinical sample detection found that the 41st exon of the 21st pair of primers of KMT2D contained the c.13689C>T mutation. DETAILED DESCRIPTION
[0037] The present invention is further described below in conjunction with specific examples and drawings. It should be noted that conventional conditions and methods not described in the examples are usually in accordance with conventional methods used by experimenters in the relevant field: for example, the fourth edition of the "Compendium of Molecular Biology Experiment Guide" edited by Osborne and Kingston, or the steps and conditions recommended by the manufacturer.
[0038] Example 1
[0039] Primers for detecting polymorphic mutation sites of KMT2D gene are designed for amplification primers designed for the entire exon of KMT2D, including:
[0040] The primers for amplifying the full exon sequence of the KMT2D gene have the following base sequences:
[0041]
[0042]
[0043] The kit for detecting polymorphic mutation sites of KMT2D gene includes:
[0044] (i) Blood DNA extraction reagents;
[0045] (ii) PCR reaction solution of the detection system;
[0046] (iii) sequencing system reagents;
[0047] Among them, tissue DNA extraction reagents can be purchased from commercial reagents such as Tiangen DNA Extraction Kit.
[0048] The PCR amplification reaction solution of the detection system includes: 2×PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U / μl); the concentration of the upstream and downstream primers of the 45 exon sequences of the KMT2D gene are both 10μM.
[0049] The sequencing system reagents include: sequencing purification solution (ExoI: 0.6U, CIP: 1.2U), EDTA (125mmol), anhydrous ethanol, 75% ethanol, HIDI (highly deionized formamide), sequencing primers: the upstream and downstream primers for detecting the full exon sequence of the KMT2D gene are M13-F (3.2μm), M13-R (3.2μm), and Bigdye Terminator V3.1 (purchased from Applied Biosystems, USA).
[0050] Example 2
[0051] The operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Bio):
[0052] (1) Extracting tissue DNA from blood:
[0053] 1) Draw 300 μl of blood and add 900 μl of red blood cell lysis buffer, mix by inversion, and leave at room temperature for 5 minutes, and mix by inversion several times. Centrifuge at 12000 rpm for 1 min, remove the supernatant, keep the white blood cell pellet, add 200 μl of buffer GA, and oscillate until thoroughly mixed.
[0054] 2) Add 20 μl of proteinase K solution and mix well.
[0055] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. The solution should become clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.
[0056] 4) Add 200 μl of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.
[0057] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.
[0058] 6) Add 500 μl of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.
[0059] 7) Add 700 μl of rinse solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid, and place the adsorption column CB3 in a collection tube.
[0060] 8) Add 500 μl of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, and discard the waste solution.
[0061] 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.
[0062] 10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 100 μl of elution buffer TE to the middle part of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 12000 rpm for 2 minutes, and collect the solution into the centrifuge tube.
[0063] (2) Reagent preparation: Prepare X μl of the PCR reaction solution of the detection system according to the number of people to be tested, and dispense 19 μl per person:
[0064] X = 19 μl reaction solution × (n samples + 1 blank control)
[0065] n is the number of samples tested.
[0066] (3) Sample addition: Add 1 μl of DNA to the PCR reaction solution of the detection system; for the blank control, add 1 μl of saline or add nothing.
[0067] (4) Amplification: The test is performed on a conventional PCR instrument, such as ABI veriti (Applied Biosystems, USA). The reaction conditions are as follows:
[0068]
[0069] The preparation method of PCR amplification system reagents is as follows:
[0070]
[0071] The primer sequences are:
[0072]
[0073]
[0074] (5) Electrophoresis: 1.5% agarose gel electrophoresis, 110 V, 25 min, observed by gel imaging system.
[0075] (6) Sanger sequencing:
[0076] Take 9 μl of PCR product and 2 μl of purification system. Purify according to the following procedure:
[0077]
[0078] Mix 1 μl of the purified product with the upper and lower sequencing primers according to the following system:
[0079]
[0080] Reaction procedure:
[0081]
[0082] Sedimentation stage:
[0083] Add 2 μl of 125 mmol EDTA to the product of the completed sequencing reaction, let it stand for 5 minutes; add 15 μl of anhydrous ethanol and vortex mix; centrifuge at 3700 rpm for 30 minutes; inverted centrifuge for 15 seconds, add 50 μl of 70% ethanol, vortex mix; centrifuge at 3700 rpm for 15 minutes; inverted centrifuge for 15 seconds, place in a 95°C metal bath; add 10 μl Hi-Di and then conduct a denaturation test.
[0084] After the denaturation process, the DNA was sequenced on a sequencer (ABI3730).
[0085] Results judgment: Sequencing results were compared with the KMT2D wild-type reference sequence (Genbank:
[0086] NG_027827) and report the results based on the actual mutation situation.
[0087] Example 3
[0088] A sample of FL that has been clinically confirmed was taken for genome extraction, reagent preparation, amplification and sequencing according to the reagents and methods of Examples 1 and 2. 1 μl of sample DNA was added to each PCR reaction solution of the detection system.
[0089] The detection found that c.1938C>G, c.2826C>CT, c.4741+44G>A, c.13689C>T existed in exon 10, exon 11, intron 18, and exon 41, respectively. For specific mutations, see Figure 3 , 4 , 5, and 6. And according to Figure 2 In summary, c.13689C>T has been reported in cases and affects the splicing function of the gene; c.2826C>CT is a non-pathogenic site because the incidence of T in the population is over 80%; there are no detailed case reports on the other two sites, which require further experimental verification.
[0090] From the test results, it can be found that the series of primer designs and detection methods of the present invention can simply and quickly discover the mutations of all 45 exons of the KMT2D gene, and the display results are simple, fast and intuitive, providing a basis for the diagnosis of clinical blood diseases.
Claims
1. Primers for detecting KMT2D gene mutation, characterized in that: The primers for amplifying the KMT2D gene are included, and the base sequence thereof is:
2. The primer according to claim 1, characterized in that It also includes a sequencing primer for detecting the KMT2D gene, the base sequence of which is: M13 F:TGTAAAACGACGGCCAGT M13 R:AACAGCTATGACCATG.
3. The primer according to claim 1, characterized in that Primer sequences K1-F and K1-R are primers for amplifying exon sequences of KMT2D gene 1, 2, 3, 4, and 5, primer sequences K2-F and K2-R are primers for amplifying exon sequences of KMT2D gene 6, 7, 8, and 9, primer sequences K3-F and K3-R are primers for amplifying exon sequences of KMT2D gene 10, primer sequences K4-F and K4-R are primers for amplifying exon sequences of KMT2D gene 11, primer sequences K5-F and K5-R are primers for amplifying exon sequences of KMT2D gene 12 and 13, primer sequences K6-F and K6-R are primers for amplifying exon sequences of KMT2D gene 14, primer sequences K7-F and K7-R are primers for amplifying exon sequences of KMT2D gene Primers for amplifying exon sequences of MT2D gene 15, 16, 17, and 18, primer sequences K8-F and K8-R are primers for amplifying exon sequences of KMT2D gene 19, 20, and 21, primer sequences K9-F and K9-R are primers for amplifying exon sequences of KMT2D gene 22, 23, and 24, primer sequences K10-F and K10-R are primers for amplifying exon sequences of KMT2D gene 25, 26, and 27, primer sequences K11-F and K11-R are primers for amplifying exon sequences of KMT2D gene 28.29.30, primer sequences K12-F and K12-R as well as K13-F and K13-R are primers for amplifying exon sequences of KMT2D gene 31 Primers, primer sequences K14-F and K14-R are primers for amplifying exon sequences of KMT2D gene 32 and 33, primer sequences K15-F and K15-R and K16-F and K16-R are primers for amplifying exon sequences of KMT2D gene 34, primer sequences K17-F and K17-R are primers for amplifying exon sequences of KMT2D gene 35, 36, 37, 38, primer sequences K18-F and K18-R, K19-F and K19-R and K20-F and K20-R are primers for amplifying exon sequences of KMT2D gene 39, primer sequences K21-F and K21-R are primers for amplifying exon sequences of KMT2D gene 40, 41, 42 Primers, primer sequences K22-F and K22-R are primers for amplifying exon sequences 43, 44, and 45 of the KMT2D gene, primer sequences K23-F and K23-R are primers for amplifying exon sequences 46 and 47 of the KMT2D gene, primer sequences K24-F and k24-R are primers for amplifying exon sequences 48 of the KMT2D gene, primer sequences K25-F and k25-R are primers for amplifying exon sequences 49 and 50 of the KMT2D gene, primer sequences K26-F and K26-R are primers for amplifying exon sequences 51, 52, and 53 of the KMT2D gene, and primer sequences K27-F and K27-R are primers for amplifying exon sequences 54 of the KMT2D gene.
4. A method for detecting polymorphic hotspot mutations of the KMT2D gene, characterized in that: The following steps are involved: (1) Extract tissue DNA from blood; (2) amplifying the DNA extracted in step 1 by PCR; The PCR amplification primers are: (3) Sequencing the amplified product in step 2; the sequencing primer base sequence is: M13 F: TGTAAAACGACGGCCAGT M13 R:AACAGCTATGACCATG; (4) Judging the sequencing results, determining whether the KMT2D gene has mutated.
5. A kit for detecting polymorphic mutation sites of KMT2D gene, characterized in that: The kit comprises: (i) Blood DNA extraction reagents; (ii) Detection system PCR amplification reaction solution; including primers for amplifying the KMT2D gene, whose base sequence is: (iii) Sequencing system reagents; including sequencing primers for detecting the KMT2D gene, whose base sequence is: M13 F:TGTAAAACGACGGCCAGT M13 R:AACAGCTATGACCATG.