Combined gene kit for second-generation sequencing of hereditary thrombocytopenia, application of combined gene kit and sequencing method based on combined gene kit

Abstract

The invention discloses a combined gene kit for second-generation sequencing of hereditary thrombocytopenia, application of the combined gene kit and a sequencing method based on the combined gene kit, and belongs to the field of clinical application of a second-generation gene sequencing technology. The combined gene detection kit covers all exons and untranslated regions of 51 main pathogenic genes and 21 potential pathogenic genes, so that the mutation detection rate and the diagnosis accuracy are remarkably improved; detection of various thrombocytopenia related mechanism genes is newly added, and mutation in a non-hotspot region of the gene, especially pathogenic sites in a 5 '-UTR region, can be found. By detecting specific genes such as GATAl, MYH9, RUNX1 and the like, the hereditary thrombocytopenia can be accurately diagnosed, misdiagnosis is effectively avoided, early diagnosis of diseases is realized, and a basis is provided for formulating a personalized treatment scheme; in addition, the method can also identify new genetic variation or prognosis related gene mutation, detect carriers of recessive genetic disease virulence genes, contribute to reduction of genetic disease risks and have remarkable clinical application value and technical advantages.

Description

Technical Field

[0001] The present invention belongs to the field of clinical application of second-generation gene sequencing technology, and specifically relates to a combined gene kit for second-generation sequencing of hereditary thrombocytopenia and its application and a sequencing method based thereon. Background Art

[0002] Hereditary thrombocytopenia ( Inherited thrombocytopenia , IT) is a group of rare diseases characterized by decreased platelet count. The main clinical symptoms of the disease include petechiae, ecchymoses, epistaxis, menorrhagia and gastrointestinal bleeding. Some patients have symptoms other than the hematopoietic system, such as skeletal dysplasia, cardiac malformations, renal failure, hearing loss and cataracts. At the same time, such patients have a significantly increased risk of developing hematological malignancies. Due to the high clinical heterogeneity of the disease, it is often misdiagnosed as immune thrombocytopenia, causing patients to receive unnecessary or even harmful treatments such as corticosteroids, immunosuppressants and splenectomy. Some patients do not experience spontaneous bleeding, and clinical manifestations such as bleeding will only occur after exposure to other risk factors. In addition, the diagnostic criteria for special groups such as children are somewhat different from those for adults, and further evaluation is needed. At present, the disease is mainly diagnosed clinically through the patient's medical history, physical examination, family history and laboratory tests. These methods cannot clarify the pathogenesis of the disease, the classification of the disease, and cannot detect whether the offspring have genetic risks.

[0003] Conventional PCR methods cannot intuitively obtain specific mutation sequences; first-generation sequencing has significant limitations in sequencing throughput and cannot detect all exon regions of multiple genes at one time.

[0004] Next Generation Sequencing (NGS), a new high-resolution molecular detection method, is a high-throughput detection method that can accurately measure the exons and related sequences of multiple genes at one time, making it possible to accurately diagnose hereditary thrombocytopenia. With the introduction of sequencing technology, studies have continuously discovered pathogenic genes related to hereditary thrombocytopenia, and significant results have been achieved in the basic research on the pathogenesis of hereditary thrombocytopenia. The Chinese patent (CN107916290A) discloses a detection kit for detecting hereditary thrombocytopenia-related gene groups, including 18 genes such as ACTN1. The kit has the advantages of high detection efficiency, wide coverage of mutant genes, and high detection rate. However, with the in-depth study of the pathogenesis of hereditary thrombocytopenia, more related genes need to be covered. At the same time, how to further optimize the detection performance, reduce costs and improve user experience are technical problems that need to be solved in this field. Summary of the invention

[0005] In view of the technical problems that existing test kits cover few genes related to hereditary thrombocytopenia, are time-consuming, cumbersome to operate, costly and have a low user experience, the present invention aims to provide a combined gene detection kit for second-generation sequencing of hereditary thrombocytopenia and its application and a sequencing method based thereon, so as to solve the relatively limited diagnosis and prediction methods of hereditary thrombocytopenia in the prior art, and provide a molecular basis for clinical diagnosis and treatment and judgment of genetic risks.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The present invention provides a combined gene detection kit for second-generation sequencing of hereditary thrombocytopenia, wherein the combined gene detection kit comprises 72 related genes, wherein the 72 related genes consist of 51 major pathogenic genes and 21 potential pathogenic genes; The 51 major pathogenic genes include: ABCG5, ABCG8, ACTB, ACTN1, ANKRD26, ARPC1B, CDC42, CYCS, DIAPH1, ETV6, FLI1, FLNA, FYB, GATA1, GFI1B, GNE, GP1BA, GP1BB, GP9, HOXA11, IKZF5, ITGA2B, ITGB3, MECOM, MPIG6B, MPL, MYH9, NBEAL2, ORAI1, PRKACG, PTPN11, RBM8A, RUNX1, SLFN14, SRC, STIM1, THPO, TPM4, TUBB1, WAS, WIPF1, GALE, KDSR, MASTL, PTPRJ, RNU4ATAC, SLC35A1, TRPM7, ADAMTS13, TUBA8 and VWF.

[0007] The 21 potential pathogenic genes include: THBD, GBA, LYST, SOCS1, TPP2, SMAD4, PROS1, ERCC6L2, RAP1B, AP3B1, BLOC1S6, CD36, WDR1, CFH, CFHR1, CFHR3, MCP, CFI, CFB, C3 and DGKE.

[0008] The application of the above-mentioned combination gene for second-generation sequencing of hereditary thrombocytopenia in the preparation of products related to the diagnosis, treatment prediction or prognosis evaluation of hereditary thrombocytopenia.

[0009] The present invention provides a detection kit for second-generation sequencing of hereditary thrombocytopenia, wherein the detection kit comprises the above-mentioned combination gene for second-generation sequencing of hereditary thrombocytopenia.

[0010] The detection kit detects all exons and untranslated regions of the 72 related genes.

[0011] The detection kit includes reagents for amplifying all exons and untranslated regions of the 72 related genes and for second-generation sequencing.

[0012] The detection kit includes reagents for preparing genomic DNA extracted from the detection object into a library for sequencing.

[0013] The detection kit also includes bioinformatics software or system for processing and analyzing sequencing data, and the software or system can determine the gene mutation status and generate a corresponding report.

[0014] The present invention provides a method for detecting hereditary thrombocytopenia gene mutations using the above-mentioned detection kit, comprising the steps of extracting genomic DNA from a detection object, preparing a sequencing library, performing second-generation sequencing, and applying bioinformatics software or a system to analyze sequencing data to determine the gene mutation status and identify new gene mutations.

[0015] The detection of hereditary thrombocytopenia gene mutations includes any one of hereditary thrombocytopenia pathogenic gene mutations, treatment prediction gene mutations and prognosis-related gene mutations.

[0016] The identified new gene mutations include: GP1BA: c. 1856-1857del, GP1BB: c. 53dup, RUNX1: c. 508+2del, SLFN14: c. 277C>T, SLFN14: c. 1120C>T, TUBB1: c. 22C>T, ERCC6L2: c. 3438dup and TPP2: c. 3214C>T.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The gene combination for the second-generation sequencing of hereditary thrombocytopenia provided by the present invention includes 51 major pathogenic genes and 21 potential pathogenic genes, and has newly added multiple mechanisms such as platelet skeleton abnormalities, transcription factor abnormalities, signal transduction disorders, sialylation abnormalities, mitochondrial and metabolic abnormalities, ion pathways, platelet destruction and aggregation abnormalities, etc. It is used in the detection kit, which improves the detection rate of rare gene mutations and can accurately discover the patient's genetic information.

[0018] The detection kit for second-generation sequencing of hereditary thrombocytopenia provided by the present invention has significantly improved mutation detection rate, diagnostic accuracy and clinical application value. The detection kit increases the gene coverage, including 51 major pathogenic genes and 21 potential pathogenic genes, and adds multiple mechanisms such as platelet skeleton abnormalities, transcription factor abnormalities, signal transduction disorders, sialylation abnormalities, mitochondrial and metabolic abnormalities, ion pathways, platelet destruction and aggregation abnormalities, etc., improves the detection rate of rare gene mutations, and can accurately discover the genetic information of patients.

[0019] Furthermore, in addition to the exonic regions, the test kit also detects the untranslated regions (UTRs), which helps to discover non-hotspot mutations located in these regions, including pathogenic sites in the 5'-UTR region.

[0020] The application provided by the present invention is that hereditary thrombocytopenia is caused by gene mutation, and the clinical manifestations vary greatly. Through the detection kit, specific genes related to hereditary thrombocytopenia, such as GATA1, MYH9, RUNX1, etc., can be detected, so as to accurately diagnose the disease and avoid misdiagnosis of other diseases such as immune thrombocytopenia; the present invention successfully identified patients who were previously misdiagnosed, showing a higher diagnostic accuracy rate, and can detect gene mutations before symptoms appear or when symptoms are mild, which is helpful for early diagnosis and wins precious treatment time for patients; different IT cases may involve different gene mutations, and the combined gene detection kit can identify these differences and provide a basis for personalized diagnosis; according to the gene test results, doctors can understand the patient's specific genotype and thus choose the most appropriate treatment plan. By monitoring the changes in related genes, the progression and severity of the disease can be evaluated, which not only provides new clues for the genetic diagnosis of hereditary thrombocytopenia, but also guides doctors' diagnosis and treatment options, and has significant clinical application value and technical advantages.

[0021] The method for detecting genetic mutations of hereditary thrombocytopenia using a detection kit provided by the present invention detects all exons and untranslated regions of 72 related genes, improving the detection rate of mutations and providing strong support for clinical diagnosis and treatment; adopting next-generation sequencing technology significantly improves the detection efficiency, and the detection panel includes 72 related genes, with a significantly increased number of genes compared to traditional detection methods; it not only detects the exon region but also the untranslated region (UTR), enabling the detection of previously unrecognized genetic variations, including pathogenic sites located in the 5'-UTR region (such as ANKRD26: c. -128G>T), which are difficult to detect by traditional methods, significantly improving the detection rate of mutations and helping to fill the blank areas of related gene variations; relying on large-scale clinical research and bioinformatics analysis, it has successfully identified patients who were previously misdiagnosed, achieving a higher diagnostic accuracy rate, providing new clues for the gene diagnosis of hereditary thrombocytopenia, and guiding doctors to make more accurate diagnoses for patients; it can detect the carrier status of pathogenic genes for recessive genetic diseases, helping carriers understand their genetic risks and reducing the risk of genetic diseases occurring. Brief Description of the Drawings

[0022] Figure 1 It is a mutation waterfall plot obtained by detecting using the method of the present invention. Detailed Embodiments

[0023] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0024] The patient samples involved in the present invention have obtained approval from the Medical Ethics Committee of the Second Affiliated Hospital of Army Medical University of the Chinese People's Liberation Army (ID: 2020-Research No. 074-01), and all patients have signed informed consent forms.

[0025] The following further describes the present invention in detail with reference to the accompanying drawings: Example 1 1. Gene panel design and primer design: 1. Determine the genes related to hereditary thrombocytopenia: By searching databases such as PubMed, OMIM, ClinVar, and ClinGen, consulting the latest literature and unpublished data of the research team, genes related to hereditary thrombocytopenia are screened out, including 51 major pathogenic genes and 21 potential pathogenic genes, specifically as follows: The 51 major pathogenic genes are: ABCG5, ABCG8, ACTB, ACTN1, ANKRD26, ARPC1B, CDC42, CYCS, DIAPH1, ETV6, FLI1, FLNA, FYB, GATA1, GFI1B, GNE, GP1BA, GP1BB, GP9, HOXA11, IKZF5, ITGA2B, ITGB3, MECOM, MPIG6B, MPL, MYH9, NBEAL2, ORAI1, PRKACG, PTPN11, RBM8A, RUNX1, SLFN14, SRC, STIM1, THPO, TPM4, TUBB1, WAS, WIPF1, GALE, KDSR, MASTL, PTPRJ, RNU4ATAC, SLC35A1, TRPM7, ADAMTS13, TUBA8 and VWF.

[0026] The 21 potential pathogenic genes are: THBD, GBA, LYST, SOCS1, TPP2, SMAD4, PROS1, ERCC6L2, RAP1B, AP3B1, BLOC1S6, CD36, WDR1, CFH, CFHR1, CFHR3, MCP, CFI, CFB, C3 and DGKE.

[0027] As shown in Table 1, first obtain the specific location range of the coding sequence (CDS) of the gene on the human genome. Then, based on the principle of probe centering, design a probe with a length of 120bp and cover the target area in a tiling manner for primer synthesis and design of gene hybridization probes. Combined with the second-generation sequencing technology, it can detect all exon regions and untranslated regions of the combined gene.

[0028] Table 1: Gene combinations

[0029] 2. Extraction of leukocyte DNA from peripheral blood samples of patients. The specific steps are as follows: 1 mL of peripheral blood sample was taken from the patient and passed through a 100 μm mesh, centrifuged at 3000 rpm for 3 minutes, and the supernatant was removed; Add 3 times the volume of red blood cell lysis buffer, invert and mix, place at room temperature for 5 minutes, centrifuge at 10,000 rpm for 1 minute, remove the supernatant, leave the white blood cell pellet, add 200 μL buffer GA, oscillate and mix to obtain cells suspended in buffer; Add 20 μL of proteinase K solution to each mL of cells suspended in the above buffer and mix well; add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. The solution should become clear and centrifuge instantly; add 200 μL of anhydrous ethanol, mix well, and centrifuge instantly to obtain the processed sample; Transfer the treated sample to the adsorption column, centrifuge at 12000 rmp for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube; add 500 μL buffer GD, centrifuge at 12000 rmp for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube; add 600 μL rinse solution PW, centrifuge at 12000 rmp for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube; repeat this operation once and rinse twice; centrifuge at 12000 rmp for 2 minutes, discard the waste liquid, dry at room temperature for 10 minutes, put the adsorption column into a new EP tube, and add 50 μL elution buffer TE to the middle part of the adsorption membrane in mid-air, incubate at room temperature for 5 minutes or more, and centrifuge at 12000 rpm for 2 minutes.

[0030] Take 1 μL DNA to measure the concentration. The DNA concentration is required to be ≥10 ng / μL.

[0031] Take 2 μL DNA to measure purity, and the A260 / A280 ratio is qualified when it is 1.8-2.0.

[0032] 3. DNA library construction, using the Homgen Universal Plus DNA Library Prep Kit, the specific steps are as follows: End repair: Take out ECT Buffer, ECT Enzyme Mix, and NTZ Buffer from the -20℃ refrigerator, thaw on ice, mix and centrifuge for later use. Prepare the reaction system shown in Table 2 in a sterile PCR tube. Vortex mix for 10 seconds and centrifuge. Run the PCR program as follows: 75℃ hot cover On→37℃×20min→65℃×30min→4℃ hold.

[0033] Table 2: End repair reaction system

[0034] Connector connection: Take out Rapid Ligation buffer, Rapid DNA ligase and Working Adapter from -20℃ refrigerator, thaw on ice box, mix and centrifuge for use. Add 20 μL RapidLigation buffer, 5 μL Rapid DNA ligase, 2.5 μL Working Adapter and 17.5 μL Nuclease-Free Water to each sample after end repair, vortex mix for 10 seconds and centrifuge. Run PCR program: 105℃ hot cover off → 20℃×15 minutes → 4℃ hold.

[0035] Purification of the adapter ligation product: Incubate the Homgen DNA Clean Beads magnetic beads at room temperature for 30 minutes and vortex to mix; add 60 μL of magnetic beads to each sample, vortex to mix, and incubate at room temperature for 5 minutes; briefly centrifuge the PCR tube and place it on the magnetic rack for 5 minutes. After the solution is clear, carefully remove the supernatant; remove the PCR tube from the magnetic rack and add 21 μL of ddH 2 O, vortex and mix, let stand at room temperature for 5 minutes, centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clear, transfer 21 μL of the supernatant to a clean PCR tube. Take out 2X HiFi Enzyme and UDI-ILMN-TS-XXX+XXX (Index) from the -20℃ refrigerator, thaw on an ice box, mix and centrifuge for use. Add 25 μL 2X HiFi Enzyme and 2 μLUDI-ILMN-TS-XXX+XXX (Index) to each sample after the connector connection is completed, vortex and mix for 10 s, then centrifuge. Run the PCR program as follows: 105℃ hot cover On→98℃×2min→98℃×20sec+60℃×15sec+72℃×30sec for a total of 5 cycles→72℃×3min→4℃ hold.

[0036] Purification of amplified products: Incubate Homgen DNA Clean Beads magnetic beads at room temperature for 30 minutes and oscillate to mix; add 45 μL of magnetic beads to each sample, vortex and oscillate to mix, and incubate at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it on a magnetic rack for 5 minutes. After the solution is clarified, carefully remove the supernatant; keep the PCR tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, rotate the reaction tube 180 degrees on the magnetic rack twice, let it stand for 30 seconds, and carefully remove the supernatant after the solution is clarified, rinse twice; centrifuge the reaction tube instantly, put it back on the magnetic rack, and use a 10 μL pipette to absorb the remaining liquid. Keep the PCR tube on the magnetic rack, open the lid and let it dry for about 2-5 minutes. Visually check that the surface of the magnetic beads is dry without reflection; remove the PCR tube from the magnetic rack and add 50 μL ddH 2 O, vortex to mix, let stand at room temperature for 5 minutes, centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clarified, transfer the supernatant to a clean EP tube for storage.

[0037] Library quality detection: Qubit 4 Fluorometer was used to detect the concentration, and 5 μL of the library was subjected to 2% agarose gel electrophoresis at 150V for 50 minutes. The library bands were concentrated at 300-400 bp.

[0038] Quality control standard: library concentration>50 ng / μL qualified, library bands concentrated around 300-400 bp, and there is only one main band, then the library is considered to be successfully constructed.

[0039] 4. Probe hybridization capture, using the Homgen ProbeCap® probe hybridization capture kit, the specific steps are as follows: Library mixing, blocking and concentration: Human Cot-1 DNA and MGI-Dual-Blocker were taken out from the -20℃ refrigerator, thawed on an ice box, mixed and centrifuged for use. The library was mixed according to the input amount of 250 ng per sample. In this experiment, 16 samples were mixed into one tube; 10 μL Human Cot-1 DNA and 2 μL MGI-Dual-Blocker were added to each tube, vortexed for 10 seconds and centrifuged; the PCR tube was placed in a vacuum rotary evaporator and the reaction solution was evaporated at 55~60℃.

[0040] Library hybridization: Take out HYB-Buffer, Enhancer and Panel Probe from the -20℃ refrigerator, thaw on an ice box, mix and centrifuge for use. Add 10 μL HYB-Buffer, 2 μL Enhancer and 4 μL Panel Probe to the vacuum-evaporated PCR tube, vortex and mix thoroughly, place at room temperature for 5 to 10 minutes, mix again and centrifuge. Place in a pre-set PCR instrument and incubate at 95℃ for 5 minutes, with the PCR hot cover temperature at 105℃; then take out the PCR tube from the previous step, vortex and mix, centrifuge, and immediately place in a pre-set PCR instrument and incubate at 65℃ for 2 to 4 hours, with the PCR hot cover temperature at 75℃.

[0041] Prepare elution buffer: Take out 2×BWB Buffer, 10×WI Buffer, 10×WII Buffer, 10×WIII Buffer and 10×SW Buffer from -20℃ refrigerator, thaw on ice box, mix and centrifuge for use. Prepare the above buffer into 1× system according to the corresponding ratio, and keep stable at 4℃ to room temperature for 30 days. Take 2 times the number of PCR tubes for hybridization reaction, add 150 μL 1×SW Buffer to each tube; take the same number of PCR tubes for hybridization reaction, add 120 μL 1×WI Buffer to each tube, and preheat on 65℃ PCR instrument for at least 15 minutes before use.

[0042] Prepare Streptavidin Magnetic Beads: ProbeCap ® SA Beads (streptavidin magnetic beads), incubate at room temperature for 10 minutes, and vortex to mix; use 50 μL of magnetic beads to add to the PCR tube for each hybridization reaction (1 rxns), centrifuge the PCR tube briefly and place it on a magnetic rack for 5 minutes, and carefully remove the supernatant after the solution is clarified to retain the SA Beads; add 100 μL BWB Buffer to the tube with magnetic beads for rinse, vortex to mix, centrifuge the PCR tube briefly and place it on a magnetic rack, and carefully remove the supernatant after the solution is clarified, and rinse twice; remove the PCR tube from the magnetic rack, add 50 μL BWB Buffer, pipette, mix thoroughly and set aside.

[0043] Binding of library to streptavidin magnetic beads: Place the PCR tube containing the SA Beads mixture prepared above on a magnetic rack to separate the SA magnetic beads from the solution, remove the supernatant, and retain the SA magnetic beads; Transfer all 16 μL of the product that has completed library hybridization to the PCR tube containing SA magnetic beads in the previous step, use a pipette to pipette 10 times to mix, and immediately place it in a 65°C PCR instrument to continue the reaction for 30 minutes. Take out the PCR tube every 10 minutes, shake it for 5 seconds, and immediately put it back into the PCR instrument until the total reaction time of 30 minutes is met.

[0044] Elution: Add 120 μL of 1× WI Buffer preheated at 65℃ to the completed PCR tube, use a pipette to repeatedly pipette and incubate at 65℃ for 10 to 20 seconds, centrifuge briefly and place on a magnetic stand for 5 minutes, and carefully remove the supernatant after the solution is clarified; add 150 μL of 1X SW Buffer preheated at 65℃ to the above PCR tube, use a pipette to slowly pipette 10 to 15 times to evenly mix, incubate at 65℃ for 5 minutes, centrifuge briefly and place on a magnetic stand for 5 minutes, and carefully remove the supernatant after the solution is clarified, wash twice with the shortest interval between the two washes; take the prepared 150 μL 1X WI Buffer at room temperature, add it to the PCR tube containing SA magnetic beads in the previous step, vortex and oscillate for 2 minutes (vortex for 30 seconds each, pause for 3 seconds) to fully suspend the magnetic beads, centrifuge briefly and place on a magnetic stand for 5 minutes, and carefully remove the supernatant after the solution is clarified; then take the prepared 150 μL 1×WII Buffer, add the PCR tube containing SA magnetic beads in the previous step, vortex and shake for 1 minute (vortex for 30 seconds each time, pause for 3 seconds) to fully suspend the magnetic beads, centrifuge it briefly and place it on a magnetic rack for 5 minutes, and carefully remove the supernatant after the solution is clear; then take 150 μL 1×WIII Buffer at room temperature, add it to the PCR tube containing SA magnetic beads in the previous step, use a pipette to mix, centrifuge it briefly, and place it on a magnetic rack for 5 minutes, and carefully remove the supernatant after the solution is clear; take the PCR tube out of the magnetic rack, add 23 μL Nuclease-Free H 2 O, then vortex to mix and set aside.

[0045] Post-PCR amplification: Take out 2×HIFI Enzyme and MGI-DU-FR from the -20℃ refrigerator, thaw them in an ice box, mix and centrifuge for use. Add 25 μL 2X HiFi Enzyme and 2 μL to the PCR tube containing SA magnetic beads that has been eluted and vortexed for 10 seconds and then centrifuge. Run the PCR program as follows: 105℃ hot cover On→98℃×45 seconds→98℃×15 seconds+50℃×30 seconds+72℃×30 seconds for a total of 12 cycles→72℃×1 minute→4℃ hold. After the above steps of PCR amplification, take 1μL of the product and use ABI Qubit4 to detect the concentration.

[0046] Post-PCR amplification and purification: Incubate Homgen DNA Clean Beads magnetic beads at room temperature for 30 minutes and oscillate to mix; add 50 μL of magnetic beads to the Post-PCR amplification product, vortex and oscillate to mix, and incubate at room temperature for 5 minutes; briefly centrifuge the PCR tube and place it on a magnetic rack for 5 minutes, and carefully remove the supernatant after the solution is clarified; keep the PCR tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, rotate the reaction tube 180 degrees on the magnetic rack twice, let it stand for 30 seconds, and carefully remove the supernatant after the solution is clarified; centrifuge the reaction tube instantly, put it back on the magnetic rack, and use a 10 μL pipette to aspirate the remaining liquid, keep the PCR tube on the magnetic rack, open the lid and let it dry for about 2 to 5 minutes, and visually check that the surface of the magnetic beads is dry without reflection; remove the PCR tube from the magnetic rack and add 25 μL 0.1×TE Buffer, vortex to mix, let stand at room temperature for 5 minutes, centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clarified, transfer the supernatant to a clean EP tube for storage.

[0047] Hybridization capture library quality detection: ABI Qubit4 was used for concentration detection, and 5 μL of library was subjected to 2% agarose gel electrophoresis at 140V for 40 minutes. The library bands were concentrated at 300-400 bp.

[0048] Quality control standard: the concentration is >10 ng / μL, the library bands are concentrated around 300-400 bp, and there is only one main band, then the library is considered to be successfully constructed.

[0049] The sequencing test was performed using a MGI sequencer (model MGISEQ-2000). The specific steps were as follows: the reagent cartridge was thawed at room temperature for 3 to 4 hours, 35 μL of DNB (DNA Nanoballs) library was added, the instrument was set up according to the instructions, the reagent cartridge was loaded, the run was started, and the test data was exported after the test was completed.

[0050] Bioinformatics analysis of sequencing test results, the specific steps are as follows: Data preprocessing: The SplitBarcode software in the analysis system was used to convert the files generated by MGISeq2000 sequencing into FASTQ files corresponding to the samples, and fastp was used to filter the adapters and remove low-quality sequences, and FASTQC was used for quality control.

[0051] Data alignment: The sequence alignment module of the analysis system (based on bwa 0.7.17) was used to align the base sequences in the fastq files to the hg19 (GRCh37) human reference genome to generate bam files, and the bam files were sorted according to the genome coordinates.

[0052] Primer soft excision: Use the primer excision module (translate bam) in the analysis system to soft excise the primer parts at both ends of the sequence.

[0053] Data quality control: Use the data quality control module in the analysis system to calculate the Q30 base ratio of each sample, the ratio of sequence alignment to the reference genome, the average depth of the target region, uniformity and other parameters.

[0054] Sequence realignment and mutation analysis: The VarScan software (v2.4.6) of the analysis system was used to perform local realignment, base quality correction and mutation analysis on the bam files.

[0055] Mutation Annotation: The identified point mutations and indels were annotated in HGVS format and COSMIC data frame (v86) using the annotation module (based on ANNOVAR v20180416).

[0056] Medical interpretation: Based on the genetic variation classification standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG), a molecular pathological diagnosis is made for the gene mutations obtained by bioinformatics analysis using the medical interpretation database. The medical interpretation database used is as follows: ABCG5 and ABCG8: Sitosterolemia (STSL) is an autosomal recessive genetic disease caused by homozygous or compound heterozygous mutations in the ABCG5 and ABCG8 genes located on chromosome 2p21. These two genes encode Sterolin-1 and Sterolin-2, respectively. These two ATP-binding cassette transporters are mainly expressed in the small intestine and liver and are responsible for the excretion of phytosterols through the bile and intestinal barrier. Gene mutations lead to abnormal accumulation of phytosterols, affecting erythrocyte membranes and platelet production, thereby causing hemolytic anemia and giant thrombocytopenia. Patients usually show elevated plasma phytosterol levels, xanthomas, and premature coronary atherosclerosis. Some patients may have significant hematological abnormalities in combination or alone, usually manifested as macrothrombocytopenia, anemia, and increased erythrocyte osmotic fragility.

[0057] GNE: Thrombocytopenia 12 With Or Without Myopathy (THC12) is a rare autosomal recessive genetic disease caused by mutations in the GNE gene located on chromosome 9p13. This gene encodes UDP-N-acetylglucosamine-2-epimerase / N-acetylformamide kinase (GNE / MNK), a bifunctional enzyme that plays a key role in the sialic acid biosynthesis pathway. GNE mutations are usually associated with myopathy, but recent studies have revealed that its mutations also lead to reduced platelet sialylation levels. This lack of sialylation increases the clearance rate of platelets from the circulation and shortens their lifespan, ultimately leading to thrombocytopenia. The clinical features of THC12 are usually temporal: thrombocytopenia symptoms appear in infancy or early childhood, while myopathy symptoms may be delayed until adulthood in some patients.

[0058] GP1BA and GP1BB: Bernard-Soulier syndrome (BSS) is an autosomal recessive disorder caused by homozygous or compound heterozygous mutations in the GP1BA gene located on chromosome 17p13 and the GP1BB gene located on chromosome 22q11. The mutation results in the absence of the GP Ib-IX-V complex in platelets (classical BSS) or the expression of a non-functional receptor (variant BSS), which affects the adhesion of platelets to sites of vascular injury, especially the binding to von Willebrand factor (vWF) on the vascular endothelium. It is characterized by moderate or severe thrombocytopenia, enlarged platelets, and a bleeding tendency. Mutations in the GP1BA gene can also cause PT-VWD (platelet-type von Willebrand disease), an autosomal dominant disorder in which patients have mild to severe thrombocytopenia, platelet aggregation can be seen in blood smears, and platelet aggregation response to low-dose ristocetin is enhanced.

[0059] MYH9: MYH9-related diseases are a group of autosomal dominant genetic diseases caused by mutations in the MYH9 gene located on chromosome 22q12. This gene encodes the non-muscle myosin heavy chain IIA chain (NMM-IIA protein) involved in the contractility of the platelet cytoskeleton. This protein plays a key role in the contraction of the platelet cytoskeleton and is involved in the maturation of megakaryocytes, the formation of platelet precursors, and the maintenance of platelet morphology. Mutations in this gene lead to abnormalities in the platelet cytoskeleton, impaired platelet production, and the formation of abnormally large platelets. In the peripheral blood smears of 42% to 84% of patients, Döhle-like inclusions in the cytoplasm of leukocytes can be observed. The formation of these inclusions is related to the aggregation of mutant proteins and is one of the diagnostic markers of MYH9-RD. Patients usually present with giant thrombocytopenia, and some patients may develop renal impairment, sensorineural hearing loss, and early-onset cataracts in the later stages.

[0060] RUNX1: Familial platelet abnormalities with acute myeloid leukemia susceptibility (FPD / AML) is an autosomal dominant genetic disease caused by mutations in the RUNX1 gene located on chromosome 21q22. This gene encodes the transcription factor RUNX1 (core binding factor α subunit 1), which plays a key regulatory role in the differentiation of hematopoietic stem cells into platelets, white blood cells, and red blood cells. RUNX1 gene mutations are associated with a variety of hematological diseases, especially with an increased risk of familial thrombocytopenia and acute myeloid leukemia (AML). Patients usually show mild to moderate decreases in platelet counts and decreased secretion of dense granules, and some patients are prone to developing myelodysplasia or leukemia.

[0061] SLFN14: Bleeding Disorder, Platelet-type, 20 (BDPLT20) is an autosomal dominant genetic disease caused by mutations in the SLFN14 gene located on chromosome 17q12. The SLFN14 protein has endoribonuclease activity and is essential for translational regulation during platelet maturation. This gene mutation affects ribosome homeostasis and protein translation, leading to impaired platelet production, reduced ATP secretion, and reduced dense granules, ultimately causing thrombocytopenia and bleeding tendency. Patients present with giant thrombocytopenia and moderate to severe bleeding tendency. Depending on the specific mutation type of the SLFN14 gene, the severity of the disease may vary.

[0062] TUBB1: Macrothrombocytopenia and chronic hemolytic anemia, type 1 (MACTHC1) is a rare autosomal dominant genetic disease caused by mutations in the TUBB1 gene located on chromosome 20q13. The TUBB1 gene encodes β1-tubulin, an important component of the cytoskeleton of platelets and megakaryocytes, and plays a key role in the formation and maintenance of platelets. Mutations in the TUBB1 gene can lead to abnormal microtubule function, affecting the division of megakaryocytes and the release of platelets, leading to insufficient platelet production and thrombocytopenia. Patients usually show a decrease in the number of platelets and abnormally enlarged platelets with irregular morphology. Affected individuals are usually not accompanied by bleeding tendencies and have normal platelet function.

[0063] CD36: Platelet Glycoprotein IV Deficiency is an autosomal recessive genetic disease caused by homozygous or compound heterozygous mutations in the CD36 gene located on chromosome 7q21. This gene mutation leads to a defect in platelet membrane glycoprotein IV, which affects platelet adhesion and aggregation. The disease is divided into three types: Type I deficiency: CD36 is completely absent on both platelets and monocytes. Type II deficiency: CD36 is absent on platelets but present on monocytes. Type III deficiency: Some cells lack CD36 expression, and the symptoms are usually mild. Most patients have thrombocytopenia but no obvious clinical abnormalities. Some patients may experience mild bleeding tendencies, thrombotic disorders, and rare transfusion reactions caused by anti-CD36 antibodies.

[0064] ERCC6L2: Bone Marrow Failure Syndrome 2 (BMFS2) is an autosomal recessive genetic disease caused by homozygous or compound heterozygous mutations in the ERCC6L2 gene located on chromosome 9q22. The protein encoded by the ERCC6L2 gene is involved in DNA damage repair, especially by regulating chromosome segregation and DNA damage repair pathways to maintain the genomic stability of hematopoietic stem cells. Mutations in this gene are associated with hereditary bone marrow failure, and patients present with thrombocytopenia.

[0065] GBA: Gaucher disease (GD) is an autosomal recessive lysosomal storage disease caused by insufficient activity of β-glucocerebrosidase due to homozygous or compound heterozygous mutations in the GBA gene. This enzyme is normally responsible for degrading the cell membrane component glucocerebroside. When its function is defective, it causes glucocerebroside to accumulate in macrophages to form characteristic Gaucher cells. Gaucher cells accumulate mainly in the spleen and bone marrow, which on the one hand causes splenomegaly and increases platelet destruction, and on the other hand inhibits bone marrow hematopoiesis and reduces platelet production, ultimately causing patients to exhibit clinical symptoms such as thrombocytopenia, anemia, and hepatosplenomegaly.

[0066] TPP2: Immunodeficiency 78 With Autoimmunity And Developmental Delay (IMD78) is an autosomal recessive genetic disease caused by homozygous or compound heterozygous mutations in the TPP2 gene located on chromosome 13q33. The TPP2 gene encodes tripeptidyl peptidase 2, a lysosomal protease that cleaves tripeptidyl peptides. The main functions of this gene involve the immune system and protein metabolism. Patients show features of immunodeficiency, recurrent sinopulmonary or skin infections, as well as cytopenia, hemolytic anemia, and thrombocytopenia.

[0067] A method for detecting a genetic mutation associated with hereditary thrombocytopenia using the kit of the invention.

[0068] The results of the detection of hereditary thrombocytopenia pathogenicity-related gene mutations using the kit of the present invention are shown in Table 3 below: Table 3: List of patient outcomes

[0069] Note: LP = Likely Patheogenic; P = Patheogenic.

[0070] Specifically, the method of the present invention was used to detect 23 samples (541 samples were tested, 6 normal controls, and 23 were detected). The mutation waterfall diagram was obtained by using 6 normal members of the hereditary thrombocytopenia family confirmed by the previous study of this laboratory (doi.10.3389 / fgene.2020.00340) as the control group. A routine peripheral blood examination was performed when thrombocytopenia was awaiting diagnosis at the initial diagnosis, when immune thrombocytopenia recurred, or when thrombocytopenia was caused by unknown reasons. The sequencing was performed using the MGI instrument, and the gene mutation was determined using the bioinformatics analysis system to generate the gene mutation. Figure 1 . Figure 1 The left vertical axis shows the 13 genes detected, the first 9 of which are major pathogenic genes, and the last 4 are potential pathogenic genes. The right vertical axis shows the detection frequency of the gene. The blue represents De novo variant, which means that the mutation site of the gene has not been reported before (n=8), and Reported variant, which means that the mutation site of the gene has been reported before (n=15). The main pathogenic genes are detected at a high frequency in different samples, and potential pathogenic genes also have a certain frequency of mutations. These data provide an important basis for the formulation of clinical diagnosis and treatment plans.

[0071] The use of patient samples involved in the present invention has been approved by the Medical Ethics Committee of the Second Affiliated Hospital of Army Medical University of the Chinese People's Liberation Army (ID: 2020-Research No. 074-01), and the patients have all signed informed consent forms.

[0072] Actual clinical examples of the application value of the present invention: A patient initially diagnosed with sick sinus syndrome and finally diagnosed with depleted pacemaker battery, female, 82 years old, was enrolled due to unexplained thrombocytopenia. The patient's platelet volume was normal. According to the test results of the present invention, a frameshift mutation of GP1BA:c.1856_1857del was detected in this patient, in heterozygous state. According to the ACMG guidelines, the pathogenicity level of this mutation site is recommended to be rated as Likely Pathogenic. The GP1BA gene mutation causes PT-VWD (platelet-type von Willebrand disease) in an autosomal dominant inheritance pattern. The degree of thrombocytopenia in the patient ranges from mild to severe, and the platelet volume is normal. Carriers of a single pathogenic mutation may develop into patients. Currently, no relevant clinical evidence of this mutation has been found in ClinVar, nor has it been reported in relevant literature; A patient initially diagnosed and finally diagnosed with thrombocytopenia and leukopenia, female, 36 years old. According to the test results of the present invention, a frameshift mutation of GP1BB:c.53dup was detected in this patient, in heterozygous state. According to the ACMG guidelines, the pathogenicity level of this mutation site is recommended to be rated as Likely Pathogenic. The Bernard-Soulier syndrome caused by the GP1BB gene mutation is in an autosomal recessive inheritance pattern. Carriers of homozygous or compound heterozygous pathogenic mutations may develop into patients, and this patient is a carrier of this pathogenic gene. Currently, no relevant clinical evidence of this mutation has been found in ClinVar, nor has it been reported in relevant literature.

[0073] A patient initially diagnosed and finally diagnosed with acute myeloid leukemia, male, 69 years old. According to the test results of the present invention, a deletion mutation of RUNX1:c.508+2del was detected in this patient, in heterozygous state. According to the ACMG guidelines, the pathogenicity level of this mutation site is recommended to be rated as Likely Pathogenic. The Familial Platelet Disorder With Associated Myeloid Malignancies (FPD-PMM) caused by the RUNX1 gene mutation is mainly in an autosomal dominant inheritance pattern. Carriers of a single pathogenic mutation may develop into patients, and the pathogenic germline variation of this gene also has a risk of developing into hematological malignancies. Currently, no relevant clinical evidence of this mutation has been found in ClinVar, nor has it been reported in relevant literature. The patient was diagnosed with pancytopenia at the first visit and aplastic anemia at the last visit, male, 35 years old; the patient was diagnosed with immune thrombocytopenic purpura at the first visit and primary thrombocytopenia at the last visit, female, 52 years old; the patient was diagnosed with thrombocytopenia and osteoporosis at the first and last visit, female, 50 years old. According to the test results of the present invention, the first two patients were detected with SLFN14: c.277C>T missense mutation, heterozygous state; the third patient was detected with SLFN14: c.1120C>T missense mutation, heterozygous state. According to the ACMG guidelines, the pathogenicity of these two mutation sites is recommended to be rated as Likely Pathogenic. SLFN14 gene mutations cause platelet-type hemorrhagic disease type 20, which is mainly autosomal dominant inheritance mode, and carriers of a single pathogenic mutation may develop into patients. No clinical evidence related to these two mutations in ClinVar has been found so far, and there is no relevant literature report.

[0074] The patient was a 60-year-old female with posterior circulation ischemic syndrome and thrombocytopenia at the initial and final diagnosis. According to the test results of the present invention, the patient was detected with a TUBB1:c.22C>T missense mutation in a heterozygous state. According to the ACMG guidelines, the pathogenicity of the mutation site was analyzed and it was recommended to be rated as Likely Pathogenic. TUBB1 gene mutations lead to macrothrombocytopenia and chronic hemolytic anemia, and type 1 (Macrothrombocytopenia And Chronic Hemolytic Anemia, Type 1, MACTHC1) is mainly inherited in an autosomal dominant manner, and carriers of a single pathogenic mutation may develop into patients. No clinical evidence related to this mutation has been found in ClinVar, and no relevant literature has been reported.

[0075] The patient was a 30-year-old male with pancytopenia at the initial and final diagnosis. According to the test results of the present invention, the patient was detected with ERCC6L2: c.3438dup nonsense mutation, heterozygous state. According to the ACMG guidelines, the pathogenicity level of the mutation site was analyzed and it was recommended to be rated as Likely Pathogenic. The protein encoded by the ERCC6L2 gene plays an important role in DNA repair and mitochondrial function. The gene mutation is associated with hereditary bone marrow failure, and the patient has thrombocytopenia, but the direct relationship between ERCC6L2 mutation and thrombocytopenia is currently unclear (PMID: 29633571, 29987015). The gene mainly presents an autosomal recessive inheritance pattern, and carriers of homozygous or compound heterozygous pathogenic mutations may develop into patients. This patient is a carrier of the pathogenic gene. No clinical evidence related to the mutation in ClinVar has been found so far, and there is no relevant literature report.

[0076] The patient was diagnosed with primary immune thrombocytopenia at the first diagnosis and refractory chronic ITP at the last diagnosis. The patient was a 37-year-old female. According to the test results of the present invention, the patient was detected with TPP2: c.3214C>T nonsense mutation, heterozygous state. According to the ACMG guidelines, the pathogenicity of the mutation site was analyzed and it was recommended to be rated as Likely Pathogenic. The TPP2 gene encodes tripeptidyl peptidase 2, a lysosomal protease that cleaves tripeptidyl peptides. The main functions of this gene involve the immune system and protein metabolism. Diseases associated with TPP2 include immunodeficiency type 78 with autoimmune diseases and developmental delay, and patients show chronic thrombocytopenia (PMID: 33586135), but no clear link was found between TPP2 mutations and thrombocytopenia. The gene mainly presents an autosomal recessive inheritance pattern, and carriers of homozygous or compound heterozygous pathogenic mutations may develop into patients. This patient is a carrier of the pathogenic gene. No clinical evidence related to this mutation in ClinVar has been found so far, and there is no relevant literature report.

[0077] Compared with the patent "A detection kit for detecting hereditary thrombocytopenia-related gene groups" (CN107916290 A), the invention uses multiple PCR primers to detect all exon regions of 18 genes related to hereditary thrombocytopenia, including 198 hotspot mutation sites. The present invention uses the second-generation sequencing method to detect all exons (Exon) and untranslated regions (UTR) of 72 genes related to hereditary thrombocytopenia, and improves the mutation detection rate through the following three points: (1) 55 pathogenic genes are added, which increases the detection of rare gene mutations. The 55 genes added specifically include 9 genes related to platelet skeleton abnormalities, 7 genes related to transcription factor abnormalities, 5 genes related to signal transduction disorders, 3 genes related to sialylation abnormalities, 2 genes related to mitochondrial and metabolic abnormalities, 3 genes related to ion pathways, 2 genes related to platelet destruction and aggregation abnormalities, 3 genes related to other mechanism abnormalities, and 21 potential pathogenic genes; RASGRP2 gene mutations have been reported to cause congenital platelet dysfunction, but they mainly affect platelet function rather than platelet count, so they were not included; (2) Detection of exons and non-coding regions can detect non-hotspot mutations of genes. For example, the previously confirmed pathogenic site ANKRD26:c.-128G>T is located in the 5'-UTR region (doi.10.3389 / fgene.2020.00340); (3) Through the new detection method, the present invention successfully identified patients who were previously misdiagnosed and achieved a higher diagnostic accuracy. This method has a significantly improved detection rate and can detect mutation types that are difficult to identify with traditional methods, thereby improving the diagnosis rate of genetic diseases.

[0078] In summary, the present invention is based on the relevant gene database, and can formulate a targeted gene detection combination suitable for patients with hereditary thrombocytopenia IT. Relying on large-scale clinical research and bioinformatics analysis, previously unidentified genetic variations are detected, which helps to fill the blank area of ​​related gene variations and provide new clues for the genetic diagnosis of the disease, thereby guiding doctors to diagnose IT patients, and is also an important step for IT patients to enter precision treatment; the carrier status of the pathogenic gene of the recessive genetic disease is detected, which helps the carrier understand their own genetic risk, and can provide preventive guidance for the health of the next generation, reduce the risk of genetic diseases, and provide a scientific basis for genetic counseling before marriage or pregnancy. The main and potential pathogenic genes of patients with IT gene mutation detection combination provided by the present invention are detected, and the guiding value of mutant genes for differential diagnosis, treatment selection and genetic evaluation is explored, so as to further optimize the current system. Therefore, the second-generation sequencing gene mutation detection combination scheme for hereditary thrombocytopenia disclosed in the present invention includes 72 genes including the main pathogenic genes and potential pathogenic genes, and is verified by 23 results, indicating that the gene mutation detection combination is feasible and accurate, and has clinical application value.

[0079] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A combination gene for second-generation sequencing of hereditary thrombocytopenia, characterized in that: The combined gene test includes 72 related genes, which are composed of 51 major pathogenic genes and 21 potential pathogenic genes; The 51 major pathogenic genes include: ABCG5, ABCG8, ACTB, ACTN1, ANKRD26, ARPC1B, CDC42, CYCS, DIAPH1, ETV6, FLI1, FLNA, FYB, GATA1, GFI1B, GNE, GP1BA, GP1BB, GP9, HOXA11, IKZF5, ITGA2B, ITGB3, MECOM, MPIG6B, MPL, MYH9, NBEAL2, ORAI1, PRKACG, PTPN11, RBM8A, RUNX1, SLFN14, SRC, STIM1, THPO, TPM4, TUBB1, WAS, WIPF1, GALE, KDSR, MASTL, PTPRJ, RNU4ATAC, SLC35A1, TRPM7, ADAMTS13, TUBA8 and VWF; The 21 potential pathogenic genes include: THBD, GBA, LYST, SOCS1, TPP2, SMAD4, PROS1, ERCC6L2, RAP1B, AP3B1, BLOC1S6, CD36, WDR1, CFH, CFHR1, CFHR3, MCP, CFI, CFB, C3 and DGKE.

2. Use of the combination gene for second-generation sequencing of hereditary thrombocytopenia as described in claim 1 in the preparation of products related to the diagnosis, treatment prediction or prognosis evaluation of hereditary thrombocytopenia.

3. A detection kit for second-generation sequencing of hereditary thrombocytopenia, characterized in that: The detection kit comprises the combined gene for second-generation sequencing of hereditary thrombocytopenia according to claim 1.

4. The detection kit for second generation sequencing of hereditary thrombocytopenia according to claim 3, characterized in that: The test kit detects all exons and untranslated regions of the 72 related genes.

5. The detection kit for second generation sequencing of hereditary thrombocytopenia according to claim 3, characterized in that: The detection kit includes reagents for amplifying all exons and untranslated regions of the 72 related genes and for second-generation sequencing.

6. The detection kit for second generation sequencing of hereditary thrombocytopenia according to claim 3, characterized in that: The detection kit includes reagents for preparing genomic DNA extracted from the detection object into a library for sequencing.

7. The detection kit for second generation sequencing of hereditary thrombocytopenia according to claim 3, characterized in that: The detection kit also includes bioinformatics software or system for processing and analyzing sequencing data, and the software or system can determine gene mutations or identify new genetic variations and generate corresponding reports.

8. A method for detecting hereditary thrombocytopenia gene mutation using the detection kit according to any one of claims 3 to 5, characterized in that: include: The steps of extracting genomic DNA from the test object, preparing the sequencing library, performing second-generation sequencing, and applying bioinformatics software or system to analyze the sequencing data to determine the gene mutation status and identify new gene mutations.

9. The method for detecting hereditary thrombocytopenia gene mutation using the detection kit according to claim 8, characterized in that: The detection of hereditary thrombocytopenia gene mutations includes at least one of hereditary thrombocytopenia pathogenic gene mutations, treatment prediction gene mutations and prognosis-related gene mutations.

10. The method for detecting hereditary thrombocytopenia gene mutation using the detection kit according to claim 8, characterized in that: The identified new gene mutations include: GP1BA: c. 1856-1857del, GP1BB: c. 53dup, RUNX1: c. 508+2del, SLFN14: c. 277C>T, SLFN14: c. 1120C>T, TUBB1: c. 22C>T, ERCC6L2: c. 3438dup and TPP2: c. 3214C>T.

Images