Method for detecting tumorigenic clones of human T-cell leukemia virus type I-infected cells

Abstract

To provide methods for detecting tumorigenic clones of HTLV-1-infected cells with a small sample volume simply and in a short time.SOLUTION: Provided is a method for detecting tumorigenic clones of human T-cell leukemia virus type I infected cells, the method comprising the steps of: (1) extracting genomic DNA from a biological sample of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) adding an adapter to the obtained DNA fragment; (4) performing PCR using a forward primer that hybridizes to a human T-cell leukemia virus type I provirus and a reverse primer that hybridizes to an adapter sequence; and (5) detecting amplification products derived from the tumorigenic clones.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting oncogenic clones of cells infected with human T-cell leukemia virus type I. [Background technology]

[0002] Human T-cell leukemia virus type I (HTLV-1) is a retrovirus first discovered in humans, and infects human CD4-positive T lymphocytes. The HTLV-1 genomic RNA that invades the cell is reverse transcribed into genomic DNA, which is then integrated into the human genome at random locations to become a provirus. Therefore, the HTLV-1 integration site characterizes each individual HTLV-1-infected cell. People infected with HTLV-1 are called HTLV-1 carriers. Approximately 95% of HTLV-1 carriers end their lives as asymptomatic carriers, but approximately 5% develop adult T-cell leukemia (ATL) as a result of tumorigenesis of HTLV-1-infected cells. To evaluate tumorigenesis of HTLV-1-infected cells, it is necessary to demonstrate monoclonal proliferation of HTLV-1-infected cells.

[0003] Southern blotting is currently the most widely used method for diagnosing ATL. However, the Southern blotting method has problems such as complicated procedures that require skill, time-consuming testing (4-5 days), and the large amount of sample required. For these reasons, there is a need for a method that can easily and quickly detect tumorigenic clones of HTLV-1-infected cells using a small amount of sample.

[0004] Patent Document 1 discloses a method for identifying the insertion site of HTLV-1 into the host genome by PCR, but this method is not intended to detect tumorigenic clones of HTLV-1-infected cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2020-5549 Summary of the Invention [Problem to be solved by the invention]

[0006] The objective of the present invention is to provide a method for detecting tumorigenic clones of HTLV-1-infected cells simply and quickly using a small amount of sample, and to provide a method for assisting in the diagnosis of ATL. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention includes the following inventions. [1] A method for detecting a tumorigenic clone of a cell infected with human T-cell leukemia virus type I, comprising the steps of: (1) extracting genomic DNA from a biological sample of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) adding an adapter to the obtained DNA fragment; (4) performing PCR using a forward primer that hybridizes to the provirus of human T-cell leukemia virus type I and a reverse primer that hybridizes to the adapter sequence; and (5) detecting an amplification product derived from the tumorigenic clone. [2] The method according to [1] above, wherein the recognition sequence of the restriction enzyme is 4 or 5 bases. [3] The method according to [1] or [2] above, wherein the restriction enzyme does not have a recognition sequence within 50 bases upstream from the 3' end of the proviral sequence. [4] The method according to any one of [1] to [3] above, wherein the PCR is nested PCR or semi-nested PCR. [5] A method for assisting in the diagnosis of adult T-cell leukemia, comprising: (1) extracting genomic DNA from peripheral blood mononuclear cells of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) adding an adapter to the obtained DNA fragment; (4) performing PCR using a forward primer that hybridizes to the human T-cell leukemia virus type I provirus and a reverse primer that hybridizes to the adapter sequence; and (5) detecting an amplification product derived from the tumorigenic clone. [6] The method according to [5] above, wherein the recognition sequence of the restriction enzyme is 4 or 5 bases. [7] The method according to [5] or [6] above, wherein the restriction enzyme does not have a recognition sequence within 50 bases upstream from the 3' end of the proviral sequence. [8] The method according to any one of [5] to [7] above, wherein the PCR is nested PCR or semi-nested PCR. Effect of the Invention

[0008] The present invention provides a method for detecting tumorigenic clones of HTLV-1-infected cells simply and quickly using a small amount of sample. The detection method of the present invention can be applied to aid in the diagnosis of ATL, and can contribute to the rapid and accurate diagnosis of ATL. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows that when genomic DNA extracted from three types of cells infected with HTLV-1 was treated with restriction enzymes, the size of the resulting DNA fragment containing the provirus differed depending on the cell. [Diagram 2] This figure shows that in samples where no tumorigenic clones are present (HTLV-1 carriers), the lanes after electrophoresis are stained as a smear, whereas in samples where a tumorigenic clone is present (ATL patients), amplification products of the same size derived from the tumorigenic clone are stained as bands. [Diagram 3] FIG. 1 shows the results of detecting tumorigenic clones in peripheral blood mononuclear cells or lymph node tissues of HTLV-1 carriers and ATL patients using the detection method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention provides a method for detecting a tumorigenic clone of an HTLV-1-infected cell (hereinafter, referred to as the "detection method of the present invention"). The detection method of the present invention may include the following steps (1) to (5): (1) extracting genomic DNA from a biological sample of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) Adding adapters to the resulting DNA fragments (4) performing PCR using a forward primer that hybridizes to the HTLV-1 provirus and a reverse primer that hybridizes to the adapter sequence; and (5) detecting the amplification products derived from the tumorigenic clones.

[0011] In step (1), genomic DNA is extracted from a biological sample of a subject. The subject is not particularly limited, but since the detection method of the present invention is not a method for distinguishing between HTLV-1 infected and non-infected individuals, it is preferable to use HTLV-1 carriers and / or ATL patients as subjects. The biological sample is not particularly limited, but examples thereof include peripheral blood mononuclear cells, lymph nodes, skin, bone marrow fluid, pleural effusion, ascites, and cerebrospinal fluid.

[0012] Peripheral blood mononuclear cells are separated from blood collected from a subject by a known method such as density gradient centrifugation, and the obtained peripheral blood mononuclear cells are subjected to genomic DNA extraction. When lymph nodes are used, the lymph nodes are cut into small pieces, and white blood cells are separated using a cell strainer or the like, and the obtained white blood cells are subjected to genomic DNA extraction. When bone marrow fluid is used, it is treated with a hemolytic agent, centrifuged to separate cells, and the obtained cells are subjected to genomic DNA extraction. When pleural effusion, ascites, or cerebrospinal fluid is used, it is centrifuged to separate cells, and the obtained white blood cells are subjected to genomic DNA extraction. When skin is used, it is directly subjected to genomic DNA extraction.

[0013] The method for extracting genomic DNA from each biological sample is not particularly limited, and may be appropriately selected from known methods. A commercially available DNA extraction kit can be suitably used. The purity of the extracted DNA is preferably such that the absorbance at 260 nm / absorbance at 280 nm is 1.8 to 2.0.

[0014] In step (2), the obtained genomic DNA is digested with restriction enzymes. HTLV-1 that has infected human CD4-positive T lymphocytes is integrated into the human genome at random locations to become a provirus, and the size (fragment length) of the fragments containing both the provirus sequence and the human genome sequence after restriction enzyme digestion varies depending on the infected cell clone (see Figure 1).

[0015] The restriction enzyme used in step (2) is not particularly limited, but it is preferable to use a restriction enzyme whose recognition sequence is 5 or 4 bases. Examples of the restriction enzyme whose recognition sequence is 5 bases include Bcn I, BscG I, Fin I, Fnu4H, Mae III, Mva I, ScrF I, Tau I, Tfi I, Tse I, Tsp4C I, and Tsp45 I. Examples of the restriction enzyme whose recognition sequence is 4 bases include Acc II, Alu I, CviR I, Hha I, HpyCH4III, HpyCH4V, Mae II, Mbo I, Mse I, Nla III, Taq I, and Xsp I. The restriction enzyme used in step (2) may be HpyCH4V, HpyCH4III, or Mse I.

[0016] The restriction enzyme used is preferably one that produces fragments whose maximum fragment length is amplifiable by PCR when cleaving human genomic DNA. For example, it is preferable to use a restriction enzyme that produces fragments whose maximum fragment length is about 1000 bp or less, about 900 bp or less, about 800 bp or less, about 700 bp or less, about 600 bp or less, or about 500 bp or less. The fragment length of the DNA fragment treated with the restriction enzyme can be confirmed using a microchip electrophoresis device (e.g., Agilent 2100 Bioanalyzer, etc.) or a capillary electrophoresis device.

[0017] The restriction enzyme to be used is selected from one that has a length from the 3'-most cleavage site of the HTLV-1 provirus to the 3'-end of the provirus sufficient to design a primer. It is preferable that the restriction enzyme does not have a recognition sequence within at least 50 bases upstream from the 3'-end of the HTLV-1 provirus integrated into human genomic DNA. More preferably, a restriction enzyme is used that does not have a recognition sequence within 60 bases, 70 bases, 80 bases, 90 bases, or 100 bases upstream from the 3'-end of the provirus.

[0018] It is preferable to use a restriction enzyme that has been confirmed through preliminary studies to have the desired cleavage site and fragment length.

[0019] In step (3), adapters are added to both ends of the obtained DNA fragment. The type of adapter is not particularly limited, and the base sequence and base length (size) thereof are not particularly limited. The method for adding the adapter is not particularly limited. It can be carried out by appropriately using a known genetic engineering technique. For example, the adapter may be added using a library preparation kit for next-generation sequencing or the like.

[0020] In step (4), PCR is performed using a forward primer that hybridizes to the HTLV-1 provirus and a reverse primer that hybridizes to the adapter sequence. The nucleotide sequence of the forward primer can be designed based on the nucleotide sequence of the HTLV-1 provirus, and the nucleotide sequence of the reverse primer can be designed based on the nucleotide sequence of the adapter added downstream of the DNA fragment. The nucleotide sequence of the HTLV-1 provirus can be obtained from a known database. For example, the forward primer may be designed based on the nucleotide sequence assigned the accession number of "GenBank: J02029.1".

[0021] PCR can be performed by a known method using the above primer set. If a band derived from the amplified product cannot be detected in one PCR, nested PCR or semi-nested PCR may be performed. When nested PCR is performed, the forward primer and reverse primer used in the second PCR are designed inside the position where the primer in the first PCR hybridizes. That is, the forward primer is designed closer to the 3' end of the HTLV-1 provirus, and the reverse primer is designed closer to the 3' end of the adapter base sequence. When semi-nested PCR is performed, the forward primer is designed closer to the inside of the position where the primer in the first PCR hybridizes, and the second PCR is performed. A part of the reaction solution of the first PCR is used as a template for the second PCR.

[0022] In step (5), the amplification product derived from the tumorigenic clone is detected. The method for confirming the PCR amplification product is not particularly limited, and can be appropriately selected from known methods. For example, a method of performing agarose gel electrophoresis, staining with a DNA staining solution such as ethidium bromide, and confirming the band of the amplification product derived from the tumorigenic clone with a transilluminator can be suitably used.

[0023] In samples without tumorigenic clones (HTLV-1 carriers), PCR amplified products of various sizes are obtained for each clone, and the lane after electrophoresis is stained as a smear. On the other hand, in samples with tumorigenic clones (ATL patients), amplified products of the same size derived from the tumorigenic clone are obtained, and these amplified products are stained as bands (see Figure 2).

[0024] Therefore, in step (5), if the amplified product is stained as a band when the PCR product is subjected to agarose gel electrophoresis, it can be determined that the subject from which the sample was taken contains tumorigenic HTLV-1-infected cells.

[0025] When the detection method of the present invention was used to detect tumorigenic clones of HTLV-1-infected cells in HTLV-1 carriers (8 cases) and ATL patients (11 cases), tumorigenic clones were not detected in the HTLV-1 carriers, but tumorigenic clones were detected in all ATL patients. In other words, when a tumorigenic clone of an HTLV-1-infected cell is detected by the detection method of the present invention, it is considered that the subject can be determined to have developed ATL. Therefore, the present invention provides a method for assisting in the diagnosis of ATL.

[0026] The method for assisting in the diagnosis of ATL of the present invention may comprise the following steps (1) to (5). (1) extracting genomic DNA from peripheral blood mononuclear cells of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) Adding adapters to the resulting DNA fragments (4) performing PCR using a forward primer that hybridizes to the HTLV-1 provirus and a reverse primer that hybridizes to the adapter sequence; and (5) detecting the amplification products derived from the tumorigenic clones. Each step can be carried out in the same manner as each step in the above-mentioned detection method of the present invention. EXAMPLES

[0027] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0028] Example 1: Comparison between ATL patients and HTLV-1 carriers using the method of the present invention 1-1 Experimental materials and methods (1) Subjects The subjects were HTLV-1 carriers (8 cases) and ATL patients (11 cases) who had been requested to undergo genetic testing at the Nagasaki University Hospital Laboratory. One HTLV-1 non-infected individual served as a negative control.

[0029] (2) Extraction of DNA from samples and restriction enzyme treatment The subjects' blood or lymph nodes were used as samples. Mononuclear cells were isolated from the subjects' blood using lymphocyte separation solution Lymphoprep (Abbott Diagnostics). The subjects' lymph nodes were also cut into small pieces, and white blood cells were isolated using a cell strainer (20 μm). Genomic DNA was extracted from the obtained peripheral blood mononuclear cells or white blood cells using a QIAamp DNA Mini Kit (QIAGEN). The obtained genomic DNA was fragmented with the restriction enzyme HpyCH4V (New England Biolabs).

[0030] (3) Addition of adapter sequences to DNA fragments Adaptor sequences were added to the DNA fragmented with restriction enzymes using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs). Specifically, 1. NEBNext End Prep, 2. Adaptor Ligation, and 3B. Cleanup of Adaptor-ligated DNA without Size Selection were performed according to the protocol of NEBNext Ultra DNA Library Prep Kit for Illumina (https: / / international.neb.com / protocols / 2014 / 05 / 22 / protocol-for-use-with-nebnext-ultra-dna-library-prep-kit-for-illumina-e7370).

[0031] (4) Amplification of HTLV-1 integration sites by PCR To amplify the HTLV-1 integration site, semi-nested PCR was performed using the following primers: First forward primer: 5'-CCTTTCATTCACGACTGACTGCCG-3' (SEQ ID NO: 1) Second forward primer: 5'-TGGCTCGGAGCCAGCGACAGCCCAT-3' (SEQ ID NO: 2) Reverse primer (NEBNext Index 1 Primer for Illumina): 5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-sT-3' (SEQ ID NO: 3, -s- indicates phosphorothioate bond)

[0032] The DNA polymerase used was Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Both the first and second PCR reactions were performed under the PCR conditions shown in Table 1 below. After the second PCR was completed, the reaction solution was subjected to agarose gel electrophoresis and stained with ethidium bromide to confirm the amplified products.

[0033] [Table 1]

[0034] 1-2 Results The results are shown in Figure 3. In electrophoresis of the PCR reaction solution of the HTLV-1 carrier, all lanes were stained in a smear shape, indicating the generation of a large number of amplification products of different sizes. In electrophoresis of the PCR reaction solution of the ATL patient, dark bands were stained in all lanes, indicating the presence of tumorigenic clones of HTLV-1-infected cells. These results demonstrate that the detection method of the present invention can detect tumorigenic clones of HTLV-1-infected cells in ATL patients.

[0035] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. In addition, all academic documents and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for detecting a tumorigenic clone of a human T-cell leukemia virus type I-infected cell, comprising: (1) extracting genomic DNA from a biological sample of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) adding an adaptor to the obtained DNA fragment; (4) performing PCR using a forward primer that hybridizes to the human T-cell leukemia virus type I proviral sequence and a reverse primer that hybridizes to the adapter sequence; and (5) Confirming the PCR amplification product by electrophoresis Including, The restriction enzyme used in step (2) is HpyCH4V, HpyCH4III and / or Mse I; method.

2. The method of claim 1, wherein the restriction enzyme is HpyCH4V.

3. 3. The method according to claim 1, wherein the restriction enzyme does not have a recognition sequence within 50 bases upstream from the 3' end of the proviral sequence.

4. The method according to any one of claims 1 to 3, wherein the PCR is nested PCR or semi-nested PCR.

5. 1. A method for aiding in the diagnosis of adult T-cell leukemia, comprising: (1) extracting genomic DNA from peripheral blood mononuclear cells of a subject; (2) treating the obtained genomic DNA with a restriction enzyme; (3) adding an adaptor to the obtained DNA fragment; (4) performing PCR using a forward primer that hybridizes to the human T-cell leukemia virus type I proviral sequence and a reverse primer that hybridizes to the adapter sequence; and (5) Confirming the PCR amplification product by electrophoresis Including, The restriction enzyme used in step (2) is HpyCH4V, HpyCH4III and / or Mse I; method.

6. The method according to claim 5, wherein the restriction enzyme is HpyCH4V.

7. The method according to claim 5 or 6, wherein the restriction enzyme does not have a recognition sequence within 50 bases upstream from the 3' end of the proviral sequence.

8. The method according to any one of claims 5 to 7, wherein the PCR is nested PCR or semi-nested PCR.

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