阅读说明：本技术 一种bcr-abl1激酶结构域突变筛查方法 (BCR-ABL1 kinase domain mutation screening method ) 是由 刘红星 滕文 谭印成 陈佳琦 于 2020-08-06 设计创作，主要内容包括：本发明公开了一种BCR-ABL1激酶结构域突变筛查方法,包括如下步骤：从样本中分离单个核细胞并提取RNA；将提取的RNA进行逆转录为cDNA,并进行PCR反应、纯化,获得包括BCR-ABL1激酶结构域的扩增产物；对扩增产物进行测序文库构建；将测序文库在PGM测序平台上进行测序,根据测序结果确定突变位点及其变异等位基因频率,再根据突变位点的变异等位基因频率判断是否出现对TKI耐药的突变位点以及突变模式；本发明筛查方法能够准确获得样本序列中突变位点及其变异等位基因频率,确定低水平突变(＜20％)以及复合突变和多克隆突变；此外,该筛查方法结合巢式PCR的特点,采用特异性引物搭配使用,使得本发明筛查方法具有较高灵敏度和特异性具有较高的筛查灵敏度。(The invention discloses a BCR-ABL1 kinase domain mutation screening method, which comprises the following steps: isolating mononuclear cells from the sample and extracting RNA; carrying out reverse transcription on the extracted RNA to obtain cDNA, carrying out PCR reaction and purification to obtain an amplification product comprising a BCR-ABL1 kinase structure domain; constructing a sequencing library for the amplification product; sequencing the sequencing library on a PGM sequencing platform, determining mutation sites and mutation allele frequencies of the mutation sites according to a sequencing result, and judging whether mutation sites resistant to TKI and a mutation mode occur according to the mutation allele frequencies of the mutation sites; the screening method can accurately obtain mutation sites and the frequency of the variant alleles thereof in the sample sequence, and determine low-level mutation (less than 20 percent), compound mutation and polyclonal mutation; in addition, the screening method combines the characteristics of nested PCR and adopts specific primers to match, so that the screening method has higher sensitivity and specificity and higher screening sensitivity.)

1. A BCR-ABL1 kinase domain mutation screening method is characterized by comprising the following steps:

(a) isolating mononuclear cells from the sample and extracting RNA;

(b) carrying out reverse transcription on the extracted RNA to obtain cDNA, carrying out PCR reaction and purification to obtain an amplification product comprising a BCR-ABL1 kinase structure domain;

(c) constructing a sequencing library for the amplification product;

(d) sequencing the sequencing library on a PGM sequencing platform, determining mutation sites and variant allele frequencies thereof according to sequencing results, and judging whether mutation sites resistant to TKI and mutation modes occur according to the mutation sites and the variant allele frequencies thereof.

2. The screening method of claim 1, wherein determining whether a mutation occurs based on the sequencing result comprises:

and comparing the sequencing result with the data of hg19 genome, and determining the mutation sites and the variant allele frequency thereof.

3. The screening method of claim 1, wherein the number of mononuclear cells is (5-8) x 10⁶And (4) respectively.

4. The screening method of claim 1, wherein the PCR reaction comprises a nested first-round PCR reaction and a nested second-round PCR reaction, and the nested second-round PCR reaction uses a product of the nested first-round PCR reaction as a reaction raw material.

5. The screening method of claim 4, wherein the nested first round PCR reaction employs primer sequences that are:

BCR190F：5’-TCCTTCGACAGCAGCAGTCC-3’；

BCR210F：5’-GAGCAGCAGAAGAAGTGTTTCAGA-3’；

ABL1R：5’-CAGGCACGTCAGTGGTGTCTC-3’。

6. the screening method of claim 4 or 5, wherein the first round PCR reaction control conditions are: pre-deforming for 20s at 98 ℃ once; deformation at 98 ℃ for 1s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, and 15 PCR cycles.

7. The screening method of claim 4, wherein the nested second round PCR reaction comprises:

respectively amplifying by adopting two primer pools, and mixing products amplified by the two primer pools to obtain an amplification product, wherein:

the primer sequence in one primer pool is as follows:

ABL1-1F：5’-ACAAGTGGGAGATGGAACGCA-3’；

ABL1-1R：5’-AGGTAGTCCAGGAGGTTCCC-3’；

ABL1-3F：5’-GGAGAACCACTTGGTGAAGGT-3’；

ABL1-3R：5’-CTTCTCTGGGCAGCCTTCTG-3’；

the sequence of the primer in the other primer pool is as follows:

ABL1-2F：5’-AGCTCCTTGGGGTCTGCAC-3’；

ABL1-2R：5’-CCCTGTCATCAACCTGCTCAG-3’；

ABL1-4F：5’-CCCTTACCCGGGAATTGACC-3’；

ABL1-4R：5’-TTCCCCAGCTCCTTTTCCAC-3’。

8. the screening method of claim 7,

the reaction conditions of each primer pool are as follows: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, performing 23 PCR cycles, cooling at 72 deg.C for 2min, and cooling to 10 deg.C.

9. The screening method of claim 1, wherein the sequencing library is constructed as follows:

selecting an amplification product to perform adaptor ligation amplification reaction, purification and quantification in sequence;

the primers used in the ligation amplification reaction included the X-terminus, and P1-terminus, required for sequencing of the Ion Torrent PGM system.

10. The screening method of claim 9, wherein the ligation amplification reaction conditions are: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, performing 5 PCR cycles, cooling at 72 deg.C for 2min, and cooling to 10 deg.C.

Technical Field

The invention relates to the technical field of biology, in particular to a BCR-ABL1 kinase domain mutation screening method.

Background

The appearance of BCR-ABL1 Kinase Domain (KD) mutations in BCR-ABL1 fusion gene positive leukemia patients is a common mechanism of resistance to Tyrosine Kinase Inhibitors (TKIs) therapy. In almost all studies, mutations in BCR-ABL1 KD were detectable in 50% -80% of patients when the disease recurred. These mutations suggested higher IC50 values in biochemical and cellular analyses. The most common drug-resistant mutation site of imatinib among BCR-ABL1 positive leukemia patients is T315I, and except ponatinib or monoclonal antibody, the rest of the second generation TKIs (2G-TKIs) are ineffective on T315I. Patients positive for mutations show a very high genetic instability, which may facilitate obtaining more mutations in a subpopulation of cells positive for the same ("compound" mutations) or different ("polyclonal" mutations) BCR-ABL1 fusion genes, resulting in a complex mutation pattern.

There is increasing evidence that rational use of Minimal Residual Disease (MRD) monitoring and BCR-ABL1 KD mutation screening plays an important role in the optimization of BCR-ABL1 positive leukemia therapy. However, Sanger Sequencing (SS) assay, although a gold standard for BCR-ABL1 KD mutation screening, has the property that it is not suitable for identifying low-level variations (< 20% VAF), nor is it generally possible to unambiguously distinguish multiple mutations occurring in the same clone (multiple mutations) or in individual clones (multiple clonal mutations). This is because the sequences generated by the SS are mixed and it is not possible to analyze the underlying subcloned infrastructure and relationships.

Complex or polyclonal analysis is particularly important for severe patients who have received multiple treatments with consecutive TKIs, and for patients who may have complex underlying clonal structures, and subclones of these patients often exhibit multiple patterns of mutation. BCR-ABL1 complex mutations can lead to high drug resistance, even in the context of new generation, highly active TKIs, such as ponatinib. In vitro studies showed that E255V/T315I, occurring as a compound mutation, had higher ponatinib IC50 values than either mutation occurring alone. Clinical studies have supported the adverse effects of the emergence of the E255V/T315I complex mutation in ponatinib therapy. The importance of determining the status of complex and polyclonal mutations is becoming increasingly severe as patients are sequentially exposed to more and more TKIs and develop complex patterns of mutations that may be more difficult to treat.

Disclosure of Invention

The invention aims to provide a BCR-ABL1 kinase domain mutation screening method, which can accurately obtain mutation sites and the mutation allele frequency thereof in a sample sequence, determine low-level mutation (less than 20 percent), compound mutation and polyclonal mutation, and has higher screening sensitivity.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a BCR-ABL1 kinase domain mutation screening method in a first aspect, which comprises the following steps:

(a) isolating mononuclear cells from the sample and extracting RNA;

(b) carrying out reverse transcription on the extracted RNA to obtain cDNA, carrying out PCR reaction and purification to obtain an amplification product comprising a BCR-ABL1 kinase structure domain;

(c) constructing a sequencing library for the amplification product;

(d) sequencing the sequencing library on a PGM sequencing platform, determining the variant allele frequency of the mutation site according to the sequencing result, and judging whether the mutation site resistant to TKI and the mutation mode appear according to the mutation site and the variant allele frequency.

The screening method can accurately obtain mutation sites and the mutation allele frequency thereof in the sample sequence, determine low-level mutation (less than 20 percent), compound mutation and polyclonal mutation, and has higher screening sensitivity.

Preferably, the determining whether a mutation occurs according to the sequencing result includes:

and comparing the sequencing result with the data of hg19 genome, and determining the mutation sites and the variant allele frequency thereof.

Preferably, the number of the mononuclear cells is (5-8) multiplied by 10⁶And (4) respectively. By limiting the number of mononuclear cells, RNA which is enough to be used for experiments can be extracted for subsequent successful amplificationAnd the database building provides powerful preconditions.

Preferably, the PCR reaction comprises a nested first-round PCR reaction and a nested second-round PCR reaction, and the nested second-round PCR reaction takes the products of the nested first-round PCR reaction as reaction raw materials.

Preferably, the primer sequences adopted by the nested first round PCR reaction are as follows:

BCR190F：5’-TCCTTCGACAGCAGCAGTCC-3’(SEQ ID NO:1)；

BCR210F：5’-GAGCAGCAGAAGAAGTGTTTCAGA-3’(SEQ ID NO:2)；

ABL1R：5’-CAGGCACGTCAGTGGTGTCTC-3’(SEQ ID NO:3)。

preferably, the first round of PCR reaction control conditions are: pre-deforming for 20s at 98 ℃ once; deformation at 98 ℃ for 1s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, and 15 PCR cycles.

Preferably, the amplification system of the nested first round PCR reaction is 20 μ l, which specifically includes:

reagent	Volume of
		Q5 Mix	10μl
One-round primer	2μl
		Water (W)	4μl
cDNA	4μl
		Total	20μl

。

Preferably, the nested second round PCR reaction comprises:

respectively amplifying by adopting two primer pools, and mixing products amplified by the two primer pools to obtain an amplification product, wherein:

the primer sequence in one primer pool is as follows:

ABL1-1F：5’-ACAAGTGGGAGATGGAACGCA-3’(SEQ ID NO:4)；

ABL1-1R：5’-AGGTAGTCCAGGAGGTTCCC-3’(SEQ ID NO:5)；

ABL1-3F：5’-GGAGAACCACTTGGTGAAGGT-3’(SEQ ID NO:6)；

ABL1-3R：5’-CTTCTCTGGGCAGCCTTCTG-3’(SEQ ID NO:7)；

the sequence of the primer in the other primer pool is as follows:

ABL1-2F：5’-AGCTCCTTGGGGTCTGCAC-3’(SEQ ID NO:8)；

ABL1-2R：5’-CCCTGTCATCAACCTGCTCAG-3’(SEQ ID NO:9)；

ABL1-4F：5’-CCCTTACCCGGGAATTGACC-3’(SEQ ID NO:10)；

ABL1-4R：5’-TTCCCCAGCTCCTTTTCCAC-3’(SEQ ID NO:11)；

preferably, the reaction conditions of each primer pool are: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, performing 23 PCR cycles, cooling at 72 deg.C for 2min, and cooling to 10 deg.C.

Preferably, the amplification system of each primer pool in the nested second round PCR reaction is 20 μ l, and specifically comprises:

reagent	Volume of
		Q5 Mix	10μl
Two-round primer (1+3)/(2+4)	2μl
		Water (W)	6μl
One round of amplification products	2μl
		Total	20μl

. The invention ensures that PCR amplification has higher sensitivity and specificity by selecting the specific primer and limiting the reaction condition.

Preferably, the sequencing library is constructed as follows:

selecting an amplification product to perform adaptor ligation amplification reaction, purification and quantification in sequence;

the primers used in the ligation amplification reaction included an X-terminus and a P1-terminus required for sequencing of the Ion Torrent PGM system,

preferably, the ligation amplification reaction conditions are: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, performing 5 PCR cycles, cooling at 72 deg.C for 2min, and cooling to 10 deg.C.

Preferably, the amplification system of the ligation amplification reaction is 20 μ l, and specifically comprises:

reagent	Volume of
		Q5 Mix	10μl
Primer (X + P1)	2μl
		Water (W)	0μl
Two rounds of purification of the product	8μl
		Total	20μl

。

The Q5 Mix in the present invention is Q5 High-Fidelity 2X Master Mix (New England BioLabs, USA).

Preferably, the purification is that AMPure XP Beads are added into the amplified product for purification, and then Low TE is adopted for elution to obtain a purified amplification product.

Preferably, the quantification is performed by diluting the purified ligation amplification product and then quantifying on ABI-7500 by using Ion LibraryTaqMan quantification Kit.

Compared with the prior art, the invention has the beneficial effects that at least:

the screening method can accurately obtain mutation sites and VAFs thereof in a sample sequence, and can accurately determine low-level mutation (less than 20%), compound mutation and polyclonal mutation; in addition, the screening method combines the characteristics of the nested PCR and adopts specific primers to match, so that the screening method has higher sensitivity and specificity and higher screening sensitivity.

The screening method can select chips with different fluxes according to the number of the samples, can obtain data with higher flux by one-time detection, and has low cost.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a schematic diagram of a BCR-ABL1 kinase domain mutation screening method provided by the embodiment of the invention;

FIG. 2 is a graph showing the results of sequencing two samples of the same patient in example 1 of the present invention;

FIG. 3 is a graph showing the results of sequencing two samples from a patient in example 2 of the present invention;

FIG. 4 is a graph showing the results of sequencing two samples from another patient in example 2 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

BCR-ABL1 kinase domain mutations are the most common mechanism of resistance to TKIs in Chronic Myeloid Leukemia (CML) and Ph + ALL patients treated with Tyrosine Kinase Inhibitors (TKIs); classical Sanger gene sequencing is the most common method for screening BCR-ABL1 Kinase Domain (KD) mutations at present, but has low detection sensitivity and cannot reveal mutations with Variant Allele Frequencies (VAF) lower than 20%; and it is difficult to distinguish complex mutations or polyclonal mutations.

Accordingly, the embodiment of the present invention provides a BCR-ABL1 kinase domain mutation screening method, as shown in fig. 1, the screening method comprises the following steps:

(a) separating (5-8) × 10 from sample⁶Single nuclear cell and extracting RNA;

(b) carrying out reverse transcription on the extracted RNA to obtain cDNA, carrying out PCR reaction, adding AMPureXP Beads into the amplification product for purification, and eluting by adopting Low TE to obtain the amplification product comprising a BCR-ABL1 kinase structural domain;

the PCR reaction comprises a nested first round PCR reaction and a nested second round PCR reaction, and the nested second round PCR reaction takes the products of the nested first round PCR reaction as reaction raw materials;

the primer sequences adopted by the nested first round PCR reaction are as follows:

BCR190F：5’-TCCTTCGACAGCAGCAGTCC-3’(SEQ ID NO:1)；

BCR210F：5’-GAGCAGCAGAAGAAGTGTTTCAGA-3’(SEQ ID NO:2)；

ABL1R：5’-CAGGCACGTCAGTGGTGTCTC-3’(SEQ ID NO:3)；

the control conditions are as follows: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, and performing 15 PCR cycles;

the amplification system is 20 μ l, and specifically comprises:

reagent	Volume of
		Q5 Mix	10μl
One-round primer	2μl
		Water (W)	4μl
cDNA	4μl
		Total	20μl

The nested second round PCR reaction comprises:

respectively amplifying by adopting two primer pools, and mixing products amplified by the two primer pools to obtain an amplification product, wherein:

the primer sequence in one primer pool is as follows:

ABL1-1F：5’-ACAAGTGGGAGATGGAACGCA-3’(SEQ ID NO:4)；

ABL1-1R：5’-AGGTAGTCCAGGAGGTTCCC-3’(SEQ ID NO:5)；

ABL1-3F：5’-GGAGAACCACTTGGTGAAGGT-3’(SEQ ID NO:6)；

ABL1-3R：5’-CTTCTCTGGGCAGCCTTCTG-3’(SEQ ID NO:7)；

the sequence of the primer in the other primer pool is as follows:

ABL1-2F：5’-AGCTCCTTGGGGTCTGCAC-3’(SEQ ID NO:8)；

ABL1-2R：5’-CCCTGTCATCAACCTGCTCAG-3’(SEQ ID NO:9)；

ABL1-4F：5’-CCCTTACCCGGGAATTGACC-3’(SEQ ID NO:10)；

ABL1-4R：5’-TTCCCCAGCTCCTTTTCCAC-3’(SEQ ID NO:11)；

the reaction conditions of each primer pool are as follows: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, performing 23 PCR cycles, cooling at 72 deg.C for 2min, and cooling to 10 deg.C;

the amplification system of each primer pool is 20 mu l, and specifically comprises:

reagent	Volume of
		Q5 Mix	10μl
Two-round primer (1+3)/(2+4)	2μl
		Water (W)	6μl
One round of amplification products	2μl
		Total	20μl

(c) And (3) carrying out sequencing library construction on the amplification products:

selecting amplification products to carry out adaptor connection amplification reaction in sequence, then adding AMPure XP Beads into the products after connection amplification for purification, and then eluting by adopting Low TE to obtain purified connection amplification products; then, diluting the purified ligation amplification product, and quantifying on ABI-7500 by adopting an Ion Library TaqMan quantification Kit to obtain a sequencing Library;

the primers used in the ligation amplification reaction included an X-terminus and a P1-terminus required for sequencing of the Ion Torrent PGM system,

n in the X-terminal sequence of the invention represents any one of A, T, C, G.

The ligation amplification reaction conditions were: pre-deforming for 20s at 98 ℃ once; deforming at 98 deg.C for 1s, annealing at 60 deg.C for 20s, extending at 72 deg.C for 30s, performing 5 PCR cycles, cooling at 72 deg.C for 2min, and cooling to 10 deg.C;

the amplification system of the ligation amplification reaction is 20 μ l, and specifically comprises:

reagent	Volume of
		Q5 Mix	10μl
Primer (X + P1)	2μl
		Water (W)	0μl
Two rounds of purification of the product	8μl
		Total	20μl

(d) Sequencing the sequencing library on a PGM sequencing platform, comparing and analyzing the sequencing result with hg19 genome data to determine the variant allele frequency of the mutation site, and judging whether the mutation site resistant to TKI and the mutation mode occur according to the mutation site and the variant allele frequency.

The screening method can accurately obtain mutation sites and the mutation allele frequency thereof in the sample sequence, determine low-level mutation (less than 20 percent), compound mutation and polyclonal mutation, and has higher screening sensitivity.

The technical solution of the present invention is further described in detail by the following specific examples.