Detection kit for detecting related genetic group of CML

A detection kit and the technology of the kit, which are applied in the field of molecular biology, can solve the problems such as the limitation of the diagnosis method of chronic myeloid leukemia and the lack of diagnosis of chronic myeloid leukemia, and achieve the effect of high detection rate and high detection efficiency.

Inactive Publication Date: 2018-05-18
天津见康华美医学诊断技术有限公司
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AI-Extracted Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical defects of the prior art, and provides a detection kit for detecting CML-related gene groups, so as to solve the limitations of the diagnostic methods of chronic myeloid leukemia in the prior art, and the lack of diagnosis o...
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Method used

[0062] Statistics show that: (summarized according to the above table) it can be seen that the second-generation sequencing method is used f...
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Abstract

The invention discloses a sequencing kit for screening mutation of related genes of CML and a following medical explanation database. A related genetic group of CML comprises nine genes such as ASXL1and ABL1. The sequencing kit comprises multiple PCR primers which amplify all exons of the nine related genes of CML. The kit for screening mutation of the genes of CML has the advantages of being high in detection efficiency, wide in coverage of mutant genes, high in detection rate and the like; a given medical explanation report has the advantages of being accurate and authoritative.

Application Domain

Microbiological testing/measurement

Technology Topic

Multiplex pcrsGene mutation +5

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  • Detection kit for detecting related genetic group of CML
  • Detection kit for detecting related genetic group of CML
  • Detection kit for detecting related genetic group of CML

Examples

  • Experimental program(1)
  • Effect test(1)

Example Embodiment

[0031] The specific embodiments of the present invention will be described in detail below. In order to avoid too many unnecessary details, the well-known structures or functions will not be described in detail in the following embodiments. Except for definitions, the technical and scientific terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.
[0032] Materials and Method:
[0033] Among the patients diagnosed from August 2015 to April 2017 in the Hematology Hospital of the Chinese Academy of Medical Sciences and the Union Medical Diagnostic Center, we performed continuous enrollment (40 patients) according to the following criteria.
[0034] The selection criteria were: patients diagnosed with chronic myeloid leukemia (CML) by cell morphology and flow cytometry.
[0035] The diagnosis results of 40 patients are shown in Table 3:
[0036] Table 3 List of patient diagnosis results
[0037]
[0038]
[0039]
[0040] 3mL of patient bone marrow was collected to extract genomic DNA for testing. The test was approved by the Ethics Committee of the Hospital of Hematology, Chinese Academy of Medical Sciences.
[0041] Sample processing: The genome in the sample is extracted with the Tiangen DNA Extraction Kit (Cat. No.: DP318-03). For specific operation methods, please refer to the kit's instruction manual.
[0042] Use multiple PCR primers to amplify genomic DNA. For the experimental operation method, refer to the instruction manual of the Life Kit.
[0043] Library construction: Use the Ion Torrent platform library kit produced by Life to construct the sequencing library for the amplified DNA fragments. For the experimental operation method, please refer to the instruction manual of the Life kit.
[0044] High-throughput sequencing: The constructed sequencing library is subjected to water-in-oil PCR and then high-throughput sequencing is performed on the Ion Torrent platform. For the water-in-oil PCR method and high-throughput sequencing method, please refer to the high-throughput sequencer Ion Torrent and its The instruction manual of the companion device One Touch.
[0045] Bioinformatics analysis: First, use Ion Report software (v4.6, Thermo Fisher, Carlsbad, CA, USA) to filter low-quality sequencing fragments, and compare the qualified sequencing fragments to the human reference genome hg19 (http:// hgdownload.cse.ucsc.edu/downloads.html), use the Torrent Variant CCMLer (v4.6.0.7) subroutine to detect mutation sites, and the software parameters use the default settings. This software has been optimized to handle the types of errors unique to the Ion Torrent sequencing platform. Finally, use ANNOVAR (http://annovar.openbioinformatics.org/en/latest/) software to annotate the identified mutations including SNP and Indel. The annotation content includes the location of the mutation in the genome, associated genes, and gene exons Number, nucleotide level variation, corresponding protein level variation, the mutation is in dbSNP (http://www.ncbi.nlm.nih.gov/snp/), 1000Genomes database (http://www.1000genomes.org/ ) And annotations in the cancer mutation database COSMIC (http://cancer.sanger.ac.uk/cosmic), PolyPhen (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift .jcvi.org/) function prediction results, etc. Further use the following principles to screen pathogenic mutation sites: (1) Filter out the mutations that do not affect the sequence of the protein product according to the genome location and type of the mutation; (2) Use the 1000 Genomes database to annotate each mutation in the population If the ratio is less than or equal to 1%, it is not considered as a polymorphic site; (3) Search the cancer database COSMIC to query whether each mutation is recorded in COSMIC and whether it appears in hematological tumors; (4) Use protein The function prediction software PolyPhen-2 predicts whether the mutation affects the protein function; (5) If after (2) (3) (4), a mutation satisfies "non-polymorphism", "recorded in hematological tumors" and At least two of the three items "influencing protein function" will be judged as possibly related to disease. Finally, if a mutation does not meet (5) but is recorded in the COSMIC as detected in the tumor, it will be interpreted as a mutation of unknown significance. Finally, if a mutation is clearly recorded in the literature as being highly related to the disease, it is directly judged as a hot spot mutation.
[0046] Statistical analysis: Count the proportion of disease-causing mutations in each gene, and screen out the genes with the highest mutation rate. The software used is Excel.
[0047] Medical interpretation: Use the medical interpretation database to make molecular pathological diagnosis of gene mutations obtained by bioinformatics analysis. The medical interpretation database used is as follows:
[0048] (1) ABL1 kinase mutations are mainly seen in CML patients who have been using imatinib for a long time, especially in patients with CML blasts. ABL1 kinase mutation is one of the main mechanisms of TKI resistance in CML patients. When T315I mutation occurs, it is resistant to imatinib, dasatinib, nilotinib and bosutinib, T315I, F317L, and V299L are resistant to dasatinib, Y253H, E255K/V and F359V/C are resistant to nilotinib, and T315I and V299L are resistant to bosutinib; F317L, Y253H, E255K/V and F359V/C/I are sensitive to bosutinib, Y253H, E255V, T315I and F359V are sensitive to ponatinib, other mutations consider high-dose imatinib or dasatinib or nilotinib; Y253H, E255K/ V, F359V and CT315I are closely related to disease prognosis and recurrence. (NCCN Guidelines Version 2.2017; PMID: 18403620; 19589924; 19652056)
[0049] (2) ASXL1 mutations can be found in AML, CML, MDS, MPN and other myeloid tumors. Most of the mutations are located in Exon12, and they can have many forms such as missense mutations, nonsense mutations, frame shift mutations and frameshift mutations. In AML, the ASXL1 mutation has the highest proportion in AML-MRC. Secondary AML patients are more common than primary AML patients, and the patients are older than women. It has been reported in the literature that ASXL1 mutations in AML, CMML, PMF, MDS and other diseases suggest poor chemotherapy effect and poor prognosis. (PMID: 28027687)
[0050] (3) ATM mutations are mainly found in CLL and MCL, and there are also a few reports in other hematological tumors. The ATM gene is located at 11q. Mutations can lead to loss of gene function. The main forms of mutations are frameshift, nonsense, missense, and 11q deletion. Patients with ATM-mutated CLL caused by 11q deletion have rapid disease progression and short survival. Heterozygous ATM gene germline mutations also play an important role in the rapid progression of CLL disease. At present, the literature reports that ATM mutations have no obvious prognostic significance in MCL. (PMID: 24584352; 26314984; 21933854; 12697903; 16461462)
[0051] (4) CSF3R mutations are mainly found in CNL, but also in aCML, MM and childhood AML. The 2016 WHO classification of myeloid tumors pointed out: CSF3R mutations are closely related to CNL, and can be seen in less than 10% of aCML; in addition, the frequency of CSF3R mutations in patients with severe congenital neutropenia during the conversion of chronic granulocytopenia to AML Increased (PMID: 7542747). In CNL and aCML, this gene mainly has two forms of mutations: mutations near the membrane and truncated mutations; patients with T615A and T618I mutations near the membrane are more sensitive to ruxolitinib and truncated mutations (such as: W741X; Y752X; D711fs, S783fs, W791X) patients are more sensitive to dasatinib (PMID: 23656643). The prognosis of CMML patients with CSF3R mutation is poor (PMID:23774674).
[0052] (5) FGFR3 mutations are more common in MM. The FGFR3 gene encodes fibroblast growth factor receptor 3, which can affect mitosis and cell differentiation. (PMID: 26282654; 25269480)
[0053] (6) MYH11 gene encodes myosin. Fusion of MYH11 and CBFB gene can be seen in AML patients, and AML patients with CBFB-MYH11 mutation have a better prognosis. (PMID: 26376137)
[0054] (7) PTEN mutations are mainly found in ALL and Lymphoma. PETN is a tumor suppressor gene and plays an important role in the PI3K-AKT pathway. Inactivation of this gene can activate the PI3K-AKT pathway, causing leukemia patients to be resistant to chemotherapy, and T-ALL patients with PETN gene Exon7 mutations have an increased risk of relapse. The PTEN inhibitor is temsirolimus. Both EGFR inhibitor cetuximab and mTOR inhibitor everolimus can interfere with abnormal signaling pathways caused by PTEN mutations. (PMID:19829307)
[0055] (8) TET2 mutations are found in AML, MDS, MPN, MDS/MPN and Lymphoma, mostly in Exon4 or Exon12. The main forms are missense, nonsense and frameshift mutations. TET2 mutations in AML tend to occur in older patients, and the incidence of TET2 mutations in CN-AML (AML with normal karyotype) is about 23%, indicating a poor prognosis. If the FLT3-ITD mutation is combined, it indicates a worse prognosis. Moreover, TET2 mutations and IDH mutations are usually mutually exclusive. TET2 mutations can occur in approximately 20% of MDS and 14% of MPN. TET2 mutations suggest a good prognosis in MDS, but poor prognosis in MDS/MPN and sAML. (PMID: 21828143; 19262601; 19666869; 22430270; 21828143)
[0056] (9) TP53 mutations are mainly found in MDS, AML and CLL. MDS patients with TP53 mutations have reduced survival rates and most of them have poor prognosis. The mutation frequency of TP53 in CLL is about 14%, which is related to CLL disease progression; CLL patients with 17p- and/or TP53 mutations have a very poor prognosis and poor response to fludarabine, cyclophosphamide, and rituximab treatment. The drugs acting on TP53 and its pathways are cisplatin, fluorouracil, docetaxel, paclitaxel, and aspirin. MDS or AML patients with TP53 gene mutation may respond better to dicitabine (PMID: 21714648, 27959731, 23138133, 23415222).

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