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BCR-ABL1 kinase structural domain compound mutant and kit

A technology of BCR-ABL1 and kinase domain, applied in the field of BCR-ABL1 kinase domain mutation and detection kits for the mutation, can solve the problem of inability to analyze the relationship of subcloning evolution of multiple mutation points, low sensitivity and difficulty in accuracy Mutation frequency and other issues to achieve the effect of improving the level of molecular detection and shortening the diagnosis time

Inactive Publication Date: 2018-03-30
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of SS is low (mutation abundance cannot be less than 20%), it is difficult to accurately determine the mutation frequency, and it is impossible to analyze the relationship between subclonal evolution between multiple mutation points

Method used

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  • BCR-ABL1 kinase structural domain compound mutant and kit

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Effect test

Embodiment 1

[0024] 1. NGS detection of mutations in the kinase domain of BCR-ABL1

[0025] Firstly, RNA was extracted from the collected 40 drug-resistant samples, reverse-transcribed into cDNA, and the BCR-ABL1 gene was amplified by PCR. The upstream and downstream primers for amplification were shown in Table 1. The amplified products were sequenced by next-generation sequencing (NGS) (depth greater than 10000×). NGS sequencing improves the sensitivity of mutation detection by increasing the throughput and repeating multiple scans. In CML patients who have failed multiple TKI treatments, NGS can identify low-level mutations below the detection limit of SS (abundance <10%-15% ), and in many cases reveal complex clonal textures, thereby identifying compound mutations. NGS test results showed that two mutations, F359C and E450A, were present in the same specimen of patient CP-05, which belonged to multiple mutations (Table 2).

[0026] Table 1: Primer sequences

[0027]

[0028] In T...

Embodiment 2

[0038] 1. Verify the TKIs sensitivity of the new compound mutants discovered

[0039] According to the results of the detected mutation sites, the overexpressed plasmids and viruses of each mutant were constructed to obtain the BaF3 cell line stably expressing wild-type and mutant BCR-ABL1 fusion proteins, and the effect of TKIs drugs on cytotoxicity was determined by CCK method.

[0040] The results showed that, compared with the wild type, the mutants had different degrees of drug resistance to imatinib, dasatinib and nilotinib. Mutants F359C and E450A showed moderate resistance to three TKI drugs, while T315I showed obvious high resistance. The compound mutant F359C / E450A showed more significant resistance to TKIs than single mutants, and had similar biological responses to T315I. This BCR-ABL1 compound mutant shows a high degree of TKI drug resistance, as shown in Table 3 and Figure 2 to Figure 4 shown.

[0041] Table 3. IC 50 of Mutations on TKIs

[0042]

[0043] A...

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Abstract

The invention discloses a BCR-ABL1 kinase structural domain compound mutant and a kit. Mutation loci are located in an ABL1 gene, and are F359C and E450A, the base sequence of the mutant is shown in SEQ ID NO:9, and the kit for detecting the compound mutation comprises multiple pairs of amplimers. The detection method of the BCR-ABL1 kinase structural domain compound mutation improves the molecular detection level, is particularly suitable for detection of BCR-ABL mutation by a next-generation sequencing platform, improves the molecular detection level, and effectively shortens the diagnosis time of a patient.

Description

technical field [0001] The invention relates to a BCR-ABL1 kinase domain mutation and a kit for detecting the mutation. Background technique [0002] Chronic myelogenous leukemia (CML) is a malignant clonal proliferative disease of the blood system that occurs in hematopoietic stem cells. Chromosome Ph1 appears in CML patients, manifested as the recombination of the proto-oncogene abl on chromosome 9 and the breakpoint concentration region bcr gene on chromosome 22 into the fusion gene BCR-ABL1. This fusion gene produces a new mRNA, and the encoded protein P210 has enhanced tyrosine kinase activity, which mainly changes the tyrosine phosphorylation level of the cell, makes the cell lose its responsiveness to the surrounding environment, and inhibits the process of apoptosis. occur. [0003] Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 protein are standard therapy for CML patients, and imatinib, nilotinib, and dasatinib are approved for the treatment of newly di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/11C12Q1/6869
CPCC12N9/1205C12Q1/6869C12Q2531/113C12Q2535/122
Inventor 张巧霞杜新王恒肖鸿莉楼瑾
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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