Acute myelogenous leukemia gene detection capture probe, kit and application

Abstract

The invention discloses a group of capture probes for detecting related genes of acute myelogenous leukemia, a kit and application. The kit comprises related probes and corresponding reagents, is based on a next-generation sequencing technology, can detect 20 gene probes highly related to acute myelogenous leukemia at one time by utilizing a hybrid capture method, is wide in coverage and high in sequencing depth, and can detect somatic variation of all exon regions of designed genes. All related acute myelogenous leukemia subtypes of 20 related genes of acute myelogenous leukemia and corresponding targeted drugs and prognosis evaluation can be reported.

Description

Technical Field

[0001] The present invention relates to the technical field of multi-gene detection, and in particular to a method for detecting acute myeloid leukemia-related genes, a kit and an application thereof. Background Art

[0002] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. According to the main affected cell series, acute leukemia can be divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

[0003] The annual incidence of acute myeloid leukemia is about 3.6 / 100,000, and the median age of onset is 66 years old. The incidence increases with age. The incidence of 10-25 years old is about 1 / 100,000. AML accounts for about 70% of acute leukemia, with slightly more men than women. The annual mortality rate of acute myeloid leukemia in the United States is about 2.2 / 100,000; my country lacks relevant statistical data, but it is estimated to be higher than that in Western developed countries.

[0004] Molecular biological examinations include: PML-RARα, RUNX1-RUNX1T1, CBFβ-MYH11, MLL rearrangement, BCR-ABL1, KIT, FLT3-ITD, NPM1, CEBPA, TP53, RUNX1, ASXL1, IDH1, IDH2, and DNMT3A gene mutations. These examinations are the basis for acute myeloid leukemia classification, risk stratification, and guidance of treatment plans.

[0005] The prognostic factors of acute myeloid leukemia patients include many aspects. In the field of molecular biology, the current domestic genetic prognostic grouping of acute myeloid leukemia is mainly based on the changes in acute myeloid leukemia cell genetics and molecular genetics at the time of initial diagnosis.

[0006] In the fifth edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissue (WHO-HAEM5) released in 2022, the classification of acute myeloid leukemia further emphasized the importance of genetic characteristics. The new classification system requires clinical testing of gene mutations in patients with acute myeloid leukemia for the following reasons:

[0007] 1. Strengthening of genetic definition: The fifth edition of the WHO classification divides acute myeloid leukemia into two categories: genetic definition and cell differentiation definition, highlighting the core position of gene mutation in the diagnosis of acute myeloid leukemia.

[0008] 2. Cancellation of the original cell ratio limit: In the genetically defined acute myeloid leukemia types, except for a few types, the 20% base requirement of original cells has been cancelled. This means that even if the original cell ratio is less than 20%, as long as there is a specific gene mutation, it can be diagnosed as acute myeloid leukemia. This change further highlights the importance of NGS testing in the diagnosis of acute myeloid leukemia.

[0009] 3. Prognosis and treatment decisions: The new classification system emphasizes the impact of gene mutations on prognosis and treatment decisions. For example, the detection results of gene mutations such as NPM1, FLT3, and TP53 directly affect the patient's prognosis assessment and treatment plan selection. NGS technology can comprehensively and accurately detect these gene mutations, thus providing an important basis for clinical decision-making.

[0010] The existing detection of acute myeloid leukemia-related genes is mostly based on the first generation sequencing technology, which can only detect some hot spot mutations of the selected genes, but cannot detect unknown mutations, and the minimum reportable mutation abundance (AF) is usually not less than 10%. Therefore, the present invention is based on the second generation sequencing and adopts the hybridization capture method, which can realize the one-time detection of the mutation of the whole exon region of 20 genes in the sample to be tested by the same system, and develops a kit for detecting acute myeloid leukemia-related genes. Summary of the invention

[0011] In response to the above-mentioned problems, the present invention provides, on the one hand, a multi-gene panel for detecting acute myeloid leukemia-related genes, which can detect 20 acute myeloid leukemia-related genes and has a specific chamber, so that the variation of the whole exon region of 20 genes in the test sample can be detected in the same system at one time, with a wide coverage area, and capable of detecting germline variation in all exon regions.

[0012] The technical solution adopted by the present invention is:

[0013] The present invention provides a group of capture probes, wherein the capture probes include at least one of the base sequences SEQ ID NOs 1-58.

[0014] As a further preferred embodiment of the present invention, a capture probe is used for manufacturing a library construction kit for detecting acute myeloid leukemia-related genes.

[0015] A library construction kit for detecting acute myeloid leukemia-related genes comprises at least one set of capture probes, wherein the capture probes comprise at least one of base sequences SEQ ID NOs 1-58.

[0016] The above-mentioned kit also includes at least one of the following: genomic DNA extraction reagents (such as Tiangen blood genomic extraction kit), DNA library construction reagents (such as Watchmaker's Equinox Library Amp Mix), capture DNA library construction reagents (such as Twist's liquid phase hybridization capture kit) and instruments or reagents required for sequencing (such as Illumina high-throughput sequencing platform and human genome sequence).

[0017] The present invention also provides the application of the above library construction kit in detecting genes related to acute myeloid leukemia. Further, the application is to detect somatic mutations in genes highly related to acute myeloid leukemia, and somatic mutations are divided into Class I mutations (including Class A or B evidence), Class II mutations (including Class C or D evidence), Class III mutations (clinical significance unknown), and Class IV mutations (harmless or potentially harmless).

[0018] Furthermore, the kit can also be used to provide related diseases and corresponding targeted drugs and prognosis evaluation.

[0019] A library construction method, characterized in that the above-mentioned capture probe or library construction kit is used to capture acute myeloid leukemia-related genes.

[0020] As a further preference of the present invention, the highly associated genes for acute myeloid leukemia include one or more of the following genes: CEBPA, DNMT3A, EZH2, FLT3, IDH1, IDH2, KIT, NPM1, NRAS, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, ZRSR2, ASXL1, BCOR, and WT1.

[0021] The included related genes were derived from multiple databases, relevant guidelines and expert consensus, covering 20 genes related to acute myeloid leukemia.

[0022] Related diseases include: acute myeloid leukemia, acute myeloid leukemia with defined genetic changes, acute promyelocytic leukemia with PML::RARA fusion, acute myeloid leukemia with RUNX1::RUNX1T1 fusion, acute myeloid leukemia with CBFB::MYH11 fusion, acute myeloid leukemia with DEK::NUP214 fusion, acute myeloid leukemia with RBM15::MRTFA fusion, acute myeloid leukemia with BCR::ABL1 fusion, acute myeloid leukemia with KMT2A rearrangement, acute myeloid leukemia with MECOM rearrangement, acute myeloid leukemia with NUP98 rearrangement, acute myeloid leukemia with NPM1 variant, acute myeloid leukemia with CEBPA variant, acute myeloid leukemia (myelodysplasia-related), acute myeloid leukemia with other specified genetic alterations, acute myeloid leukemia (defined by differentiation), acute myeloid leukemia, minimally differentiated, acute myeloid leukemia without mature cell type, acute myeloid leukemia with mature cell type, acute basophilic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia.

[0023] According to the evidence-based classification of somatic mutations in the 2020 "Guidelines for the Interpretation of Clinical Reports of Next-Generation Sequencing", the clinical significance of somatic mutations is classified into: Class I variants (including level A or B evidence), Class II variants (including level C or D evidence), Class III variants (clinical significance is unknown), and Class IV variants (harmless or potentially harmless).

[0024] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0025] The present invention is based on second-generation sequencing and adopts a hybridization capture method, which can realize one-time detection of the variation of the whole exon region of 20 genes in the sample to be tested by the same system. It has a wide coverage area and can detect somatic variation in all exon regions. The minimum reportable mutation abundance (AF) can reach 1%.

[0026] The test kit of this project is designed for acute myeloid leukemia. It can detect 20 genes with clear pathogenic effects that are highly related to acute myeloid leukemia at one time. It has strong specificity for patients with acute myeloid leukemia. Compared with the detection method of simultaneously detecting harmful gene mutations of multiple blood diseases, it can reduce costs and increase efficiency.

[0027] The determination of gene variation in this project is based on the "Guidelines for Interpretation of Clinical Reports of Next Generation Sequencing". There is a standard annotation process for variations not included in the clinvar database to effectively avoid missed detections. The annotation of gene-related diseases comes from a wide range of sources, not just relying on a single database. Multiple databases, relevant guidelines and expert consensus are manually collated to output all relevant acute myeloid leukemia subtypes, and report the corresponding targeted drugs and prognosis assessment (if any).

[0028] The capture probes designed in this project cover the entire coding region sequence of the genes involved, with a target region coverage of 99%, an average sequencing depth of up to 1500x, and a minimum reportable mutation abundance (AF) of 1%.

[0029] Reference numerals

[0030] Figure 1 This is the fragment analysis result of the hybridization capture library. DETAILED DESCRIPTION

[0031] 1. Probe design

[0032] 1. Determine the target capture region based on the target gene and generate the capture region bed file.

[0033] 2. According to the bed file and the human hg19 reference genome, the target region probe sequence was designed using the catch software (reference: Metsky HC, Siddle KJ, Gladden-Young A, et al. Capturing sequence diversity in metagenomes with comprehensive and scalable probe design[J]. Nat Biotechnol, 2019: 160-168.);

[0034] Among them, the catch software operation method is as follows:

[0035] nohup design.py-pl 120-l 60-m 5-e 5-o AML.fasta&&\

[0036] makeblastdb-in AML.fasta-out blast / AML.fasta-dbtype nucl&&\

[0037] echo -e

[0038] "qseqid\tsseqid\tpident\talignment_length\tmismatch\tgapopen\tqstart\tqend\tsstart\tsend\tevalue\tbitscore">blast / AML.probe.blastn.txt

[0039] blastn-num_threads 10-query probe / AML.probe.fasta-db blast / AML.fasta-evalue 1e-50-max_target_seqs 1000-outfmt 6>>blast / AML.probe.blastn.txt&&\

[0040] perl filter_mulialign_probe.pl blast / AML.probe.blastn.txt probe / AML.probe.fasta probe / AML.uniq.probe.fasta

[0041] The designed exploration sequence was aligned with hg19 using blastn software, and the sequences that were aligned to hg19 multiple times were removed to obtain the probe sequence as shown in the appendix.

[0042] 2. Nucleic Acid Extraction

[0043] Extract genomic DNA from whole blood or bone marrow samples.

[0044] 3. Library Construction

[0045] 1. Extract DNA, fragment it (enzyme digestion), end repair and dA tailing.

[0046] The concentration of gDNA was determined using the Qubit quantitative analysis kit. The reaction solution was prepared (on ice) and kept on ice for later use. The reaction solution was configured as follows:

[0047] Reagents Single reaction volume Frag / AT Buffer 4uL Frag / AT Enzyme 6uL Total volume 10uL

[0048] Set the following program on the PCR instrument, with the heated cover temperature at 70°C:

[0049]

[0050]

[0051] Add 40 μL of DNA sample to the above reaction solution, with a total amount of 50-200 ng. Seal the reaction tube or cover it with a lid, gently shake and mix, centrifuge for a moment, and run it on a PCR instrument with a preset program.

[0052] 2. Connect the universal connector

[0053] Universal adapter (Twist Bioscience) and DNA Ligation Mix (Twist Bioscience) were thawed on ice; the PCR program was set to 20°C hold with the heated lid closed.

[0054] In each sample plate well or single tube containing DNA fragments with dA tails, add 5μl of UniversalAdapter, mix gently by pipetting, and then place on ice. Add 20μl of Ligation Mix, mix gently by pipetting, seal the reaction tube or cover it with a lid, centrifuge for a moment, and run on a pre-programmed PCR instrument.

[0055] 3. First step magnetic bead purification

[0056] After connecting the universal adapter, add 60 μL of mixed DNA purification magnetic beads (0.8x) to each sample, vortex to mix, incubate at room temperature for 5 minutes, and then stand on the magnetic rack for 5 minutes after centrifugation.

[0057] Aspirate the supernatant, add 200 μl of freshly prepared 80% ethanol, and incubate for one minute. Repeat once.

[0058] Aspirate the supernatant and carefully remove the remaining ethanol using a 10μl pipette, avoiding touching the magnetic beads. Dry the magnetic beads on a magnetic rack until they are non-reflective and cracked. Add 18μl of ddH2O to each sample, pipette to mix, and incubate at room temperature for 25min; after a quick spin, place on a magnetic rack and let stand for 3min. Transfer 15μl of supernatant to a PCR tube and place on ice for later use.

[0059] 4. UDI Primer PCR Amplification

[0060] Thaw Equinox Library Amp Mix (2x) and UDI primers (Twist Bioscience) on ice; set the following program on the PCR instrument with a heated lid temperature of 105°C:

[0061]

[0062] Add 10 μl of UDI primers and 25 μl of Equinox Library Amp Mix (2x) after inversion to each of the libraries purified in the first step, and gently pipette to mix. After centrifugation, transfer to a PCR instrument and run on the PCR instrument according to the preset program.

[0063] 5. Second step magnetic bead purification

[0064] Vortex the pre-equilibrated DNA purification beads until mixed. Add 50μl (1x) of pre-mixed DNA purification beads to each ligated sample after connecting the UDI connector. Vortex to mix and incubate at room temperature for 5 minutes. After centrifugation, let it stand on the magnetic rack for 5 minutes, aspirate the supernatant, add 200μl of freshly prepared 80% ethanol, incubate for one minute, and discard the supernatant. Repeat once. Use a 10μl pipette to aspirate the remaining ethanol. Dry the beads on the magnetic rack until the beads are non-reflective and dry. Add 24μl of ddH2O to each sample, pipette to mix, incubate at room temperature for 5 minutes, centrifuge, place on the magnetic rack, and let it stand for 2 minutes.

[0065] Take 2 μl and dilute 10 times, then use Qubit to measure the library concentration and use fragment analyzer to measure the fragment size. The average fragment length should be between 350 and 425 bp.

[0066] 4. Hybridization Capture

[0067] 1. Library hybridization

[0068] The amount of library pool input is calculated according to the library DNA concentration, and the library pool is obtained by mixing. The total amount of library pool DNA is 1.5 to 4 ug.

[0069] Add pre-hybridization reagent to the mixed sample according to the table below, gently shake to mix, and centrifuge briefly:

[0070] Reagents Single reaction volume Universal Blockers 8uL Blocking Solution 5uL The probe designed by the present invention 4uL

[0071] Open the cap of the reaction tube and place it in a vacuum concentrator. After balancing, dry it at 45°C until the solution in the tube is completely dried. The drying process takes about 30 to 60 minutes.

[0072] Incubate Fast Hybridization Mix (Twist Bioscience) at 65°C for 10 min until the precipitate is completely dissolved; set the PCR instrument program according to the table below and heat the lid at 85°C.

[0073] temperature time 95℃ 5min 60℃ 2h

[0074] After drying, remove the centrifuge tube from the vacuum concentrator, tear off the sealing film, quickly vortex the preheated Fast Hybridization Mix (Twist Bioscience) and take 20μl of the resuspended sample, flick to mix, and let stand at room temperature for 5 minutes. Use a pipette to blow up and down 10 times. After the DNA is completely dissolved, transfer all the solution in the tube to a new PCR tube as much as possible, add 30μl of paraffin oil to the tube, and centrifuge to remove bubbles. Transfer the PCR tube to the preheated PCR instrument and run it on the preset program PCR instrument.

[0075] 2. Library capture and cleaning

[0076] Prepare DNA-binding magnetic beads Streptavidin Binding Beads (TwistBioscience) while running the hybridization reaction program, shake well to mix, and equilibrate at room temperature for at least 30 min; preheat 450 μl Fast Wash Buffer 1 at 66°C on a thermostatic metal bath; preheat 700 μl Wash Buffer 2 at 48°C; dispense the enrichment reagent into centrifuge tubes according to the table below.

[0077] Reagents Single reaction volume Fast Binding Buffer(Twist Bioscience) 900ul Fast Wash Buffer 1(Twist Bioscience) 450ul Wash Buffer 2(Twist Bioscience) 700ul

[0078] Vortex the pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, and take 100μl of magnetic beads to a 1.5mL centrifuge tube. Add 200μl Fast Binding Buffer to the centrifuge tube and mix with a pipette tip; place the centrifuge tube on the magnetic rack for 1 minute or until the solution is clear, discard the supernatant, and remove the centrifuge tube; repeat the above washing steps 2 times, a total of 3 times. After 3 washes, add 200μl Fast Binding Buffer again, and vortex to resuspend and mix thoroughly.

[0079] After the hybridization reaction is completed, open the PCR instrument cover and quickly transfer all the hybridization solution to the balanced magnetic bead solution, and immediately blow and mix. Close the centrifuge tube and seal the tube mouth with sealing film, then place it on the Biocate oscillator, 1200rpm, and mix thoroughly for 30 minutes at room temperature.

[0080] Centrifuge the tube quickly, place it on the magnetic rack for 1 minute, and remove the supernatant. Remove the tube from the magnetic rack, add 200 μl of preheated FW1, mix gently, and then quickly incubate at 66°C for 5 minutes. Repeat once.

[0081] Transfer all the liquid in the tube to a new 1.5mL centrifuge tube, place it on the magnetic rack for 1 minute, and remove the supernatant. Remove the centrifuge tube from the magnetic rack, add 200μl preheated W2, mix gently and quickly place it at 48℃ for 5 minutes, place it on the magnetic rack for 1 minute, remove the supernatant and repeat the washing with W2 twice, a total of three times. After a quick centrifugation, place it on the magnetic rack and use a 10μl pipette tip to absorb all the residual liquid; add 45μl ddH2O, mix it by pipetting, and incubate it on ice.

[0082] 3. PCR Amplification

[0083] Prepare DNA purification magnetic beads, shake and mix thoroughly, and equilibrate at room temperature for at least 30 minutes; prepare 80% ethanol; thaw Equinox Library Amp Mix (2x) and Amplification Primers (Twist Bioscience) on ice; set the following program on the PCR instrument, with the hot cover temperature at 105°C.

[0084]

[0085] Prepare PCR tubes by adding 25 μl of Equinox Library Amp Mix (2x) and 2.5 μl of Amplification Primers to each tube.

[0086] Take 22.5 μl of the magnetic bead suspension prepared in step 2 and add it to the PCR tube, gently shake and mix, and centrifuge it momentarily. Transfer the PCR tube to the preheated PCR instrument and run the above program.

[0087] 4. Magnetic Bead Purification

[0088] Vortex the pre-equilibrated DNA purification magnetic beads until mixed. Add 90μl (1.8x) of mixed DNA purification magnetic beads to each enriched sample prepared in 2.3. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge and place on the magnetic rack for 5 minutes, then discard the supernatant. Add 200μl of freshly prepared 80% ethanol, incubate for one minute, and discard the supernatant. Repeat once. Use a 10μl pipette to absorb the remaining ethanol. Dry the magnetic beads on the magnetic rack until the beads are non-reflective and dry. Add 32μl of ddH2O to each sample, mix by pipetting, incubate at room temperature for 5 minutes, centrifuge and place on the magnetic rack, then let it stand for 2 minutes.

[0089] Transfer 30 μl of supernatant to a clean 1.5 ml centrifuge tube and use 2 μl The library concentration was determined by dsDNA HS Assay Kit (Vazyme), which should be greater than 0.5 ng / μl. The length distribution of library fragments was detected by 2100 fragment analyzer, and the average fragment length should be around 300-400 bp.

[0090] The fragment analysis results of the hybridization library are as follows Figure 1 .

[0091] 5. Sequencing and Data Analysis

[0092] 1. Sequencing was performed using the NextSeq 2000 instrument on the Illumina platform with sequencing parameters of PE 2x150.

[0093] 2. The obtained original sample fq data is split according to normal samples and standard mutation samples.

[0094] 3. All original sample FQ data were preprocessed by fastp to remove low-quality reads and adapter-contaminated reads; the preprocessing method reference is: Chen S, Zhou Y, Chen Y, et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. 2018.

[0095] 4. The sample data obtained in step 3 was aligned to hg19 using bwa mem, the original bam file was deduplicated, and then the bam file was recalibrated for base quality using GATK best practice to obtain the sample data bam file for variation detection.

[0096] 5. Perform the following processing on each sample obtained in step 4:

[0097] a. Use the Mutect2 tool to detect somatic mutations (snv / indel): establish a data set of normal samples as background filtering (PoN, Panel of Normals), analyze and filter meaningless / benign germline mutations;

[0098] b. LUMPY software detection of fusion genes: normal samples are used as the baseline to remove background variations. The specific detection method reference is: Layer RM, Hall IM, Quinlan A R. LUMPY: A probabilistic framework for structural variant discovery [J]. 2012;

[0099] c. Finally, the copy number variation is detected by cnvkit software to obtain the vcf result files of variation types such as SNV / INDEL / SV / CNV and the filtered variation list.

[0100] (8) The MSI status of each sample was detected based on the normal sample data using the MSIsensor-pro software (reference: Jia P, Yang X, Guo L, et al. MSIsensor-pro: Fast, Accurate, and Matched-normal-sample-free Detection of Microsatellite Instability[J]. Journal of Genomics Proteomics and Bioinformatics: English version, 2020(1):7). ​​A result greater than or equal to 10% was MSI-H, and a result less than 10% was MSI-L.

[0101] Example

[0102] A female patient diagnosed with acute myeloid leukemia was tested for mutations in the 20 genes described in the present invention using bone marrow samples. The genomic DNA of the bone marrow sample was extracted using a Tianlong automated extractor (model: NP968-C) and tested according to the experimental procedures described in the implementation method. The fragment analysis results of the hybrid capture library are shown in Figure 1 As shown:

[0103] The obtained hybridization capture library was sequenced using the Nextseq6000 instrument of the Illumina platform, and the sequencing parameters were: PE 2x150. The data off the machine were analyzed and quality controlled as described above, and the quality control was qualified.

[0104] The sequencing data was aligned with the human reference genome hg38 / GRCh38, and quality data such as coverage of the target region and average sequencing depth were collected. Bioinformatics methods and various professional databases (such as gnomAD, ClinVar, OMIM and other databases) were used to identify, annotate and filter variant sites. The gene names were in accordance with the rules of the Human Genome Organization Gene Nomenclature Committee (HGNC). The naming of variants refers to the nomenclature provided by the Human Genome Variation Society (HGVS). The interpretation of variant sites refers to the ACMG / AMP genetic variation classification standards and guidelines, as well as the updated supplementary recommendations and consensus of ClinGen and others. The obtained variant site information is shown in the following table.

[0105]

[0106] The test results show that the detection kit of the present invention can accurately detect the mutation of acute myeloid leukemia-related genes and provide prompts for related results.

[0107] The capture probe sequence of the present invention is as follows:

[0108]

[0109]

[0110]

[0111]

Claims

1. A set of capture probes, characterized in that The capture probe comprises at least one of the base sequences SEQ ID NOs 1-58.

2. The use of the capture probe according to claim 1, characterized in that The capture probe is used to manufacture a library construction kit for detecting acute myeloid leukemia-related genes.

3. A library construction kit for detecting acute myeloid leukemia-related genes, characterized in that: It comprises at least one group of capture probes, wherein the capture probes comprise at least one of the base sequences of SEQ ID NOs 1-58.

4. Use of the library construction kit as claimed in claim 3 in detecting genes related to acute myeloid leukemia.

5. A library construction method, characterized in that: The capture probe according to claim 1 or the library construction kit according to claim 3 is used to capture acute myeloid leukemia-related genes.

6. A library construction method according to claim 5, characterized in that, The highly associated genes for acute myeloid leukemia include one or more of the following genes: CEBPA, DNMT3A, EZH2, FLT3, IDH1, IDH2, KIT, NPM1, NRAS, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, ZRSR2, ASXL1, BCOR, and WT1.

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