JP2012100626A - Method of detecting translocation gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detecting a translocation gene, simply detecting all translocation genes without regard to the kind of a translocation partner even when two or more kinds of translocation partners exist.SOLUTION: This method of detecting a translocation gene is a method of detecting a translocation gene made by fusion of two kinds of different genes with a cutting point as a boundary. The method includes a process of measuring an abundance ratio of first and second regions by conducting a quantitative nucleic acid amplifying method, which amplifies the first region existing upstream from the position corresponding to the cutting point in the normal type gene and a quantitative nucleic acid amplifying method, which amplifies the second region existing downstream from the position corresponding to the cutting point in the normal type gene for the genes extracted from a tested sample or its transferred material. The abundance ratio is different from the abundance ratio in the normal type gene, thereby indicating the existence of translocation genes.

Description

  The present invention relates to a method for detecting a translocation gene. The method of the present invention is useful for detecting a translocation gene known to have a plurality of translocation partners.

  Fluorescence in situ hybridization (FISH) is mainly used as a method for detecting translocation genes, but the operation is complicated and expensive. In recent years, methods for detecting translocations comprehensively with exon arrays and next-generation sequencers have been developed, but this is an experimental method that is more complicated and expensive than FISH. Development of an inexpensive and simple detection method is expected.

  Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, activates JAK / STAT, Ras / MAPK, and PI3K pathways by interacting with ligands and contributes to cell proliferation. Cancers often report ALK gene abnormalities. In particular, gene mutation and chromosomal translocation are important because of their high carcinogenicity. ALK mutations have been reported in neuroblastoma (Non-Patent Documents 1 to 4). Some mutations cause constitutive activation of ALK and are involved in cancer cell growth. As for ALK translocation, NPM-ALK was first discovered in 1994 in non-Hodgkin lymphoma (Non-patent Document 5). Since then, various translocation partners of ALK have been reported. In 2007, the translocation gene EML4-ALK with EML4 was reported for the first time in cancers other than lymphoma and lung cancer (Non-patent Document 6). These ALK translocation genes are constitutively activated by forming homodimers in a ligand-independent manner. A transgenic mouse into which EML4-ALK has been introduced forms a tumor two weeks after birth, and is considered to be extremely carcinogenic (Non-patent Document 7).

  In recent years, molecular targeting drugs for cancer targeting tyrosine kinases have attracted attention, and various clinical trials have been conducted. Research and development of inhibitors against ALK is ongoing, and some clinical trials are ongoing. When an ALK inhibitor was administered to the above-described transgenic mice, the tumor disappeared rapidly, and therefore it is considered that the ALK inhibitor is effective in treating lung cancer in ALK translocation cases. From these facts, detection of the presence or absence of ALK translocation is very important in determining the administration of an ALK inhibitor.

  Conventionally, a method using a nucleic acid amplification method such as PCR is known as a method for detecting a translocation gene. In this method, a forward primer is set on the upstream side of the translocation gene breakpoint (boundary site where two kinds of genes are bound), a reverse primer is set on the downstream side, and these primers are used. This is a method of amplifying a region straddling the cut point.

  However, in this method, when there are a plurality of types of translocation partners (for example, 11 types of translocation partners are known for translocation of the ALK gene), it is necessary to set a primer for each translocation partner. Yes, it is inconvenient.

George RE et al. Nature. 455: 975-978, 2008. Chen Y et al. Nature. 455: 971-974, 2008. Janoueix-Lerosey I et al. Nature. 455: 967-970, 2008. Mosse YP et al. Nature. 455: 930-935, 2008. Morris SW et al. Science. 263: 1281-1284, 1994 Soda M et al. Nature. 448: 561-566, 2007. Soda M et al. Proc Natl Acad Sci USA. 105: 19893-19897, 2008.

  An object of the present invention is to provide a method for detecting a translocation gene that can easily detect all translocation genes regardless of the type of translocation partner even when there are a plurality of translocation partners. Is to provide.

  As a result of diligent research, the inventors of the present application have amplified each of the upstream region and the downstream region from the breakpoint in a normal gene by a quantitative nucleic acid amplification method, and the abundance ratio of each region measured. When the gene extracted from the test sample does not have a translocation (in the normal type), the region upstream and downstream from the breakpoint is amplified, but has a translocation. In some cases, one region is not amplified regardless of the type of translocation partner, so the abundance ratio of each region is different from the normal type. As a result, the present invention has been achieved.

  That is, the present invention is a method for detecting a translocation gene in which two different genes are fused with a breakpoint as a boundary, wherein the first region existing upstream from the position corresponding to the breakpoint in a normal gene is detected. A gene or transcript thereof obtained by extracting a quantitative nucleic acid amplification method for amplification and a quantitative nucleic acid amplification method for amplifying a second region existing downstream from the position corresponding to the cleavage point in a normal gene or a transcript thereof And measuring the abundance ratio of the first and second regions, wherein the abundance ratio is different from the abundance ratio in the normal gene, indicating the presence of a translocation gene. A detection method is provided.

  The present invention provides, for the first time, a method for detecting a translocation gene that can easily detect all translocation genes regardless of the type of translocation partner even when there are multiple types of translocation partners. It was done.

It is a figure for demonstrating the principle of the method of this invention taking ALK gene as an example. In the following Example, it is a figure which shows the measurement result which performed the method of this invention with respect to the plasmid. In the following Examples, it is a figure which shows the measurement result which performed the method of this invention with respect to the cell line. In the following Example, it is a figure which shows the measurement result which performed the method of this invention with respect to the clinical specimen.

  As described above, the present invention provides a quantitative nucleic acid amplification method for amplifying a region located upstream and a region located downstream of a breakpoint of a gene known to be capable of translocation, This is performed on a gene extracted from a test sample or a transcript thereof. When the gene used as the template for the nucleic acid amplification method is a normal type, amplification occurs both upstream and downstream of the cleavage point. On the other hand, when the gene serving as the template for the nucleic acid amplification method is a translocation gene, amplification of either the upstream region or the downstream region does not occur regardless of the translocation partner. Therefore, when the ratio of the abundance of each region measured by the quantitative nucleic acid amplification method is calculated, when the translocation gene is included in the test sample, only the normal gene is included. It becomes a different value. Therefore, a translocation gene can be detected based on this ratio. For example, when a translocation gene is associated with cancer, when a normal cell having a normal gene and a cancer cell having a translocation gene are mixed in the test sample, Since the ratio varies depending on the abundance of translocation genes, the ratio of translocation genes in the test sample (ie, the number of translocation genes / (number of normal genes + translocation) The number of genes)) can also be determined. Even when the translocation gene in the test sample is quantified as described above, the translocation gene is inevitably detected by the quantification, so that it is also included in the scope of the present invention since it is also a detection method.

  The principle of the present invention will be described with reference to FIG. 1, taking the ALK gene as an example. FIG. 1 schematically shows a gene map of a normal ALK gene. As shown in FIG. 1, ALK is a transmembrane protein, and the ALK gene has an extracellular domain, a transmembrane domain, and a cytoplasmic domain from the upstream side. A “cutting point” exists in the transmembrane region. “Cleavage point” means a boundary site where a part of a normal gene and another gene (translocation partner) are linked when translocation occurs. Since there is no translocation in the normal type gene, “cutting” does not occur. However, for the normal type gene, when the translocation occurs, the site that is the boundary between the two genes is referred to as the “breaking point”. I am calling. In the ALK gene shown in FIG. 1, when a breakpoint exists in the transmembrane region and translocation occurs, the downstream is normal from the breakpoint, but other than the ALK gene upstream from the breakpoint. Other genes (translocation partners) are linked. In the ALK gene, more than 11 types of translocation partners are known. The base sequence of the normal ALK gene is described in GenBank Accession No. NM_004304.3, which is shown in SEQ ID NO: 1. In SEQ ID NO: 1, the main cleavage point exists at a position between 4079 nt (4079th base counted from the 5 ′ end, the same shall apply hereinafter) and 4080 nt.

  In the method of the present invention, as shown in FIG. 1, in a normal ALK gene, a region located upstream of the breakpoint (5 ′ amplification region (5R) indicated by a double-headed arrow, 1) ”)) and the region located downstream of the breakpoint (the 3 ′ amplification region (3R) indicated by the double-headed arrow,“ second region ”in claim 1))) Amplify by nucleic acid amplification method. Then, when the gene used as a template for the nucleic acid amplification method is a normal type, both the 5 ′ amplification region and the 3 ′ amplification region are amplified. On the other hand, when the gene serving as a template is a translocation gene, amplification of the 5 ′ amplification region does not occur regardless of the type of translocation partner. For this reason, if the ratio of the abundance of each region quantified by the quantitative nucleic acid amplification method is taken, when the translocation gene is contained in the test sample, compared to the case where only the normal type gene is contained The above ratio will be different. Therefore, the translocation gene can be detected based on this ratio. And, the above ratio is greatly different as the ratio of translocation genes contained in the test sample increases, compared with the case where only normal genes are included. It is also possible to quantify.

  Most translocation ALK genes exist between 4079 nt and 4080 nt at the breakpoint, but E14; ins11del49A20 (GenBank Accession No. AB374363.1, breakpoint between 4128 nt and 4129 nt)) and E14 There are also a few known cases in which cutting points exist at positions other than 4079 nt and 4080 nt, such as; del12A20 (Gen Bank Accession No. AB462412.1, cutting point is between 4091 nt and 4092 nt)). Thus, even in the case where it is known that the transection partner has different cutting points, the first region is set upstream from the cutting point located most upstream, and the second region is The method of the present invention described above can be carried out by setting the cutting point downstream of the most downstream cutting point. Therefore, the method of the present invention can also be applied to a case where a plurality of types of translocation genes combined with a plurality of types of translocation partners and having different cleavage points are known. It is included in the scope of the present invention.

  The translocation gene to which the method of the present invention can be applied is not particularly limited as long as it is a translocation gene in which a breakpoint is present, and in particular, the existence of a plurality of types of translocation genes bound to a plurality of types of translocation partners is known. It is very effective when applied to a gene. Examples of such genes that are known to have a plurality of types of translocation genes combined with a plurality of types of translocation partners include leukemia genes (ABR-ABL, PML-) in addition to the above-mentioned translocation ALK gene. RARA, E2A-PBX1, MLL-AF4, MLL-AF9, AML1-ETO, TEL-AML1, CBFB-MYH11, SIL-TAL, etc.), sarcoma genes (EWS-Fli1, EWS-ERG, EWS-ETV1, EWS-E1AF) , EWS-FEV, EWS-WT1, EWS-CHN, TAF2N-CHIN, TLS-CHOP, etc.), lung cancer genes (EML4-ALK, etc.) and prostate cancer (TMPRSS2-ERG, etc.). It is not something.

  The test sample used in the method of the present invention is a tissue suspected of having a translocation gene, a body fluid such as blood, or its components (white blood cells, lymphocytes, etc.).

  Various methods such as TaqMan probe (trade name) method, quenching probe method (QP method (trade name)), and MGB method (trade name) are widely known as quantitative nucleic acid amplification methods. Any of the known quantitative nucleic acid amplification methods can be employed. Among the known quantitative nucleic acid amplification methods, the above-described TaqMan probe (trade name) method, QP method (trade name), MGB method (trade name) and the like are commercially available. It can be easily carried out using a kit.

  In the following examples, among these, the QP method (trade name) is used. The QP method (trade name) uses a quenching probe (Q probe) in which a fluorescent dye called BODIPY is bound to the 3 ′ end of a probe whose 3 ′ end is cytosine. When this quenching probe hybridizes with the template DNA, the fluorescently labeled cytosine at the 3 ′ end is paired with guanine. Fluorescently labeled cytosine emits fluorescence when the probe is free, but when the fluorescently labeled cytosine is paired with guanine by hybridizing with the template DNA, the fluorescence is quenched. When the complementary strand of the template is extended, the Q probe hybridized with the template DNA is detached from the template DNA and becomes free to emit fluorescence again. For this reason, when PCR is performed in the presence of a sufficient amount of Q probe, if amplification occurs, the Q probe is quenched in the annealing step. Since the degree of quenching increases as the amount of DNA serving as a template increases, amplification can be monitored by measuring the degree of quenching in the annealing step. The degree of this quenching rapidly increases when the template DNA is amplified to a certain level or more, so based on how many cycles of PCR the quenching will be detected, The amount of template nucleic acid in the sample can be quantified. In addition, since the kit for performing QP method (brand name) is marketed from J-Bio21, Inc., it can carry out easily using this.

  The quantitative nucleic acid amplification method can be performed on a genomic gene contained in a test sample, or can be performed on a transcript (mRNA) thereof. In the case of performing on a transcript, it is usual to prepare cDNA from mRNA and subject this cDNA to PCR.

  In the method of the present invention, it is preferable that the amplification efficiencies of the 5 ′ side amplification region (first region) and the 3 ′ side amplification region (second region) are made substantially equal to increase the measurement accuracy. That is, when the amplification efficiencies are equal, the abundance ratio is 1, but this ratio is preferably as close to 1 as possible, that is, preferably in the range of 0.9 to 1.1, more preferably in the range of 0.95 to 1.05. .

  As a result of earnest research, the present inventor has found that the above ratio is approximately 1 (0.95 to 0.99 as specifically described in the following examples) when the QP method is applied to the ALK gene. A combination was found. That is, the forward primer used for amplification of the 5 ′ amplification region is an oligonucleotide having a size of 18 bases or more that hybridizes to the region of 2696nt to 2721nt of the nucleotide sequence shown in SEQ ID NO: 1, and 5 ′ amplification The reverse primer used for region amplification is an oligonucleotide having a size of 18 bases or more that hybridizes to the region 2847nt to 2822nt of the nucleotide sequence shown in SEQ ID NO: 1, and is used for measurement of the 5 'amplification region The Q probe is an oligonucleotide having a size of 18 bases or more that hybridizes to the region of 2786 nt to 2815 nt of the base sequence shown in SEQ ID NO: 1, and the forward primer used for amplification of the 3 ′ amplification region is SEQ ID NO: Oligonucleotide having a size of 18 bases or more that hybridizes to the region of 4127nt to 4152nt of the base sequence shown in FIG. Yes, the reverse primer used for amplification of the 3 ′ amplification region is an oligonucleotide having a size of 18 bases or more that hybridizes to the 4282 nt to 4258 nt region of the nucleotide sequence shown in SEQ ID NO: 1, and the 3 ′ amplification The Q probe used for the region measurement is an oligonucleotide having a size of 18 bases or more that hybridizes to the region of 4192 nt to 4221 nt of the base sequence shown in SEQ ID NO: 1.

  More preferably, as specifically described in the Examples below, the forward primer used for amplification of the 5 ′ amplification region is an oligonucleotide having the base sequence represented by SEQ ID NO: 2, The reverse primer used for amplification of the side amplification region is an oligonucleotide having a base sequence represented by SEQ ID NO: 3, and the Q probe used for measurement of the 5 ′ amplification region is represented by SEQ ID NO: 4. An oligonucleotide consisting of a base sequence, and a forward primer used for amplification of the 3 ′ side amplification region is an oligonucleotide consisting of a base sequence represented by SEQ ID NO: 5, and used for amplification of the 3 ′ side amplification region The reverse side primer is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6, and the Q probe used for measurement of the 3 ′ amplification region is Is an oligonucleotide comprising the base sequence represented by sequence number 7.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. How to operate
1-1. RNA extraction Extract RNA from the sample. As an extraction kit, RNeasy Mini kit + QIAShreddar (Qiagen) was used. The procedure was according to the Qiagen manual, but when RNA was extracted from tissue sections, the tissue sections were disrupted with zirconia beads using TissueLyser instead of QIAShreddar, and then RNA was extracted.

1-2. CDNA synthesis
For cDNA synthesis, M-MLV (Life Technologies) was used. The composition of each reagent is as follows.

1. In a microtube, add Random primers and dNTP mix to 1 μg of RNA, and adjust to 15 μL with Distilled Water.
2. Set the microtube containing the reaction solution in the thermal cycler and incubate at 65 ° C for 5 minutes.
3. After ice-cooling for 5 minutes, add M-MLV 5x Reaction Buffer, DTT, RNase OUT, and M-MLV RT, and adjust to 25 µL with Distilled Water.
4. Place the microtube containing the reaction mixture in the thermal cycler and incubate at 25 ° C for 10 minutes, 37 ° C for 50 minutes, and 70 ° C for 15 minutes. Hold at 4 ° C after the reaction.
5. The cDNA synthesis product is diluted 2-fold with Distilled Water and used in the next step. Store at -80 ° C.

1-3. QP method
1. As 3 'PCR, mix the reagents in the table below and template cDNA per 25 µL of reaction tube.

2. また、5’側のPCRとして、1反応チューブ25μLあたり下記表の試薬、および鋳型cDNAを混合する。

2. For 5 'PCR, mix the reagents in the table below and template cDNA per 25 µL of reaction tube.

  The primer sequences are shown in Table 4.

3. Set the microtube containing the reaction solution in the real-time PCR device and perform the reaction under the following conditions.

Analysis of the results calculates the rate of change in fluorescence intensity for each cycle (Kurata S et al. Nucleic Acids Research. 29: e34, 2001.). The fluorescence intensity change rate is calculated using the following formula.

Fluorescence intensity change rate = [1-(F _{64, n} / F _{95, n} ) / (F _{64, base} / F _{95, base} )] × 100

For example, F _{64, n} indicates the fluorescence value at 64 ° C. (annealing & extension) of the nth cycle. F _{95, base} indicates the number of cycles immediately before exponential amplification, that is, the fluorescence value at 95 ° C. in the baseline cycle.
The above analysis is performed using analysis software provided by J-bio21.

5. The ratio or difference of the finally obtained Ct values is evaluated as the difference in expression between the 3 'side and the 5' side of the ALK gene.

2. Results
2-1. Example 1
The measurement results of the control plasmid are shown. A control plasmid was prepared from the SK-N-DZ strain expressing the full length of ALK using the cloning vector pCR2.1-TOPO. Plasmids diluted to 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies were measured at n = 5.

The results are shown in FIG. The slope and intercept of the 3 'and 5' calibration curves were almost the same. The variation coefficient of the Ct value was found to be around 10% for 10 copies, but about 1% for 100 copies or more, and a stable result was obtained. The ratio of Ct values (Ct ^{5 ′} / Ct ^{3 ′} ) was 0.950-0.993, which was almost 1 (Table 6).

以上の結果から、ALK遺伝子における転座の切断点より3’側と5’側でほぼ同等の増幅効率を示す定量系が構築できたと考えられた。

From the above results, it was considered that a quantitative system showing almost the same amplification efficiency on the 3 ′ side and 5 ′ side from the translocation breakpoint in the ALK gene could be constructed.

2-2. Example 2
The measurement result of the cell line was shown. H2228 (EML4-ALK translocation positive strain) and SK-N-DZ (ALK high expression strain) were used as positive controls, and HCC827 and HL60 were used as negative controls. The measurement was performed at n = 2.

The results are shown in FIG. Amplification was not observed for HCC827 and HL60 that did not express ALK. On the other hand, in the H2228 having the EML4-ALK translocation, amplification was observed only on the 3 'side, but amplification was not observed on the 5' side. In addition, SK-N-DZ expressing full-length ALK gave similar amplification curves for both 3 'and 5'. From these facts, it was shown that the present invention can distinguish between expression of ALK full length and expression of ALK translocation.

2-3. Example 3
100 lung cancer tissue specimens were measured by the present invention. Clinical specimens distributed from Kanagawa Cancer Clinical Research and Information Organization were measured at n = 2. Since variation was considered in 10 copies of the plasmid from Example 1, the Ct value of 100 copies of the plasmid was set as the cut-off value in 3 ′ PCR, and samples higher than this were set as negative.

As a result of the measurement, 19 cases had a Ct value of 3 'less than 38 cycles. All Ct values and Ct ^{5 ′} / Ct ^{3 ′} ratios are shown in FIG. The # 94 specimen had the highest Ct ^{5 '} / Ct ^3' ratio, 1.221, and the 3 'PCR was faster by 7.2 cycles than 5'. This specimen was confirmed to express EML4-ALK. The # 56 specimen also had a slightly higher Ct ^{5 '} / Ct ^3' ratio of 1.173, suggesting translocation. From these facts, it became clear that the present invention can detect the presence or absence of ALK translocation in clinical specimens.

Claims (8)

  A method for detecting a translocation gene in which two different genes are linked with a breakpoint as a boundary, wherein the first region existing upstream from the breakpoint in a normal gene is amplified, A quantitative nucleic acid amplification method for amplifying a second region present downstream of the breakpoint in a normal gene, and performing the gene extracted from the test sample or a transcript thereof, and the first and second regions A method for detecting a translocation gene, comprising a step of measuring the abundance ratio of the translocation gene, wherein the abundance ratio is different from the abundance ratio in a normal gene.   The method according to claim 1, wherein the translocation gene is a gene known to have a plurality of types of translocation genes bound to a plurality of types of translocation partners.   A plurality of types of translocation genes combined with a plurality of types of translocation partners are known, which have different cleavage points, and the first region exists upstream from the most upstream cleavage point. The method according to claim 2, wherein the second region is present downstream of a cut point located most downstream.   The method according to any one of claims 1 to 3, wherein when the normal gene alone is used as a template, the abundance ratio of the first and second regions is in the range of 0.9 to 1.1.   The method according to any one of claims 2 to 4, wherein the translocation gene is a translocation gene of an anaplastic lymphoma kinase gene.   The method according to any one of claims 1 to 5, wherein the quantitative nucleic acid amplification method is a quenching probe method.   The quantitative nucleic acid amplification method is a quenching probe method, and the forward primer used for amplification of the first region hybridizes to a region of 2696nt to 2721nt of the base sequence shown in SEQ ID NO: 1. The reverse primer used for amplification of the first region is an oligonucleotide having a size of 18 bases or more that hybridizes to the region of 2847nt to 2822nt of the base sequence shown in SEQ ID NO: 1. And the Q probe used for the measurement of the first region is an oligonucleotide having a size of 18 bases or more that hybridizes to the region of 2786 nt to 2815 nt of the base sequence shown in SEQ ID NO: 1, and the second region The forward primer used for the amplification of the nucleotide hybridizes to the 4127nt to 4152nt region of the base sequence shown in SEQ ID NO: 1. It is an oligonucleotide having a size of 18 bases or more that is soybean, and the reverse primer used for amplification of the second region has a base sequence of 18 bases or more that hybridizes to the 4282 nt to 4258 nt region of the base sequence shown in SEQ ID NO: 1 A Q probe used for measuring the second region is an oligonucleotide having a size of 18 bases or more that hybridizes to a region of 4192nt to 4221nt of the base sequence shown in SEQ ID NO: 1. Item 6. The method according to Item 5.   The forward primer used for amplification of the first region is an oligonucleotide having a base sequence represented by SEQ ID NO: 2, and the reverse primer used for amplification of the first region is SEQ ID NO: 3 The Q probe used for the measurement of the first region is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4, and is used for amplification of the second region. The forward primer used is an oligonucleotide composed of a base sequence represented by SEQ ID NO: 5, and the reverse primer used for amplification of the second region is an oligo composed of a base sequence represented by SEQ ID NO: 6 The Q probe, which is a nucleotide and used for the measurement of the second region, comprises an nucleotide sequence represented by SEQ ID NO: 7. A Gore-nucleotides The method of claim 7 wherein.

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