# Patent

## Title
Method and kit for detection of microsatellite instability-positive cell

## Abstract

A method for detecting an abnormal cell based on gene expression analysis, which is useful for cancer diagnosis, is provided. A gene expression analysis method, comprising: measuring the expression level of a transcript of a human MLH1 gene containing the nucleotide sequence as shown in SEQ ID NO: 1 at the 5′-terminus thereof and the expression level of a transcript of a human MLH1 gene containing the nucleotide sequence as shown in SEQ ID NO: 2 at the 5′-terminus thereof, in a biological sample; and comparing the expression levels, thereby detecting a cell positive for microsatellite instability.

## Background
CLAIM OF PRIORITY
The present application claims priority from Japanese application JP 2006-226665 filed on Aug. 23, 2006, the content of which is hereby incorporated by reference into this application.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for detecting a cell having microsatellite instability based on gene expression analysis, which is useful for cancer diagnosis.
2. Background Art
One of the unexpected findings clarified by the human genome project is a report that the number of genes contained in the human genome is much smaller than that has been predicted, and that it is only approximately 25,000. This gene number does not significantly differ from those of lower organisms, and it is insufficient for explanation of the complicated brain system of human and functions of the immune system thereof. Thus, it is considered that various expression mechanisms derived from a single gene play an important role in the complicated biological functions of higher animals.
The variability of the expression of mRNA and a protein from a single gene is mainly controlled by an alternative splicing or a usage of an alternative promoter at the stage of mRNA transcription or RNA processing (see, Non-Patent Document 1). The term “alternative splicing” is used to mean a mechanism for generating several types of mature mRNAs (splicing variants) from a single mRNA precursor as a result of a phenomenon whereby a splicing reaction that generally occurs between splice sites adjacent to each other occurs between various splice sites, when several introns exist in an mRNA precursor. On the other hand, the term “alternative promoter” is used to refer to the presence of several alternative promoters for controlling the transcription of a single gene. When such an alternative promoter exists, several transcription initiation sites are present in the gene. As a result, several types of transcripts having different 5′-terminal sequences are generated. Recently, as a result of searching for 1,780,295 types of full-length cDNA sequences that are contained in an oligo-cap cDNA clones, it has been unexpectedly reported that approximately 52% of genes assigned to the RefSeq database at NCBI, U.S.A. are under the control of alternative promoters, and that 3.1 alternative promoters exist for a single gene on average (see, Non-Patent Document 2). As a result of such a variety of expressions from a single gene, the diversity of organisms or complicated biological functions have been generated in the evolutionary process. On the other hand, it has also been known that such a variety of expressions may cause generation of abnormal proteins or deletion of such a protein itself in individuals, and as a result, it may induce malignant transformation of cells (see, Non-Patent Document 3).
The genetic understanding of hereditary non-polyposis colorectal cancer (HNPCC) has dramatically progressed as a result of discovery of various types of genes associated with mismatch repair. Such a mismatch repair mechanism is a mechanism for recognizing an abnormal base pairing (mismatch base pairs) generated during DNA replication or genetic recombination, and eliminating and repairing the mismatched base pairs. With regard to HNPCC, one of the most typical diseases among familial neoplastic diseases, it has been known that the microsatellite instability (MSI) in the DNA of tumor tissues, which reflects deficiency in a mismatch repair system, is positive in 90% or more of the cases thereof (see, Non-Patent Document 4). The term “microsatellite” is used to mean a repetitive sequence formed by repeating several to several tens of repeat units each consisting of 2 to 5 bases. The term “instability” is used herein to mean a phenomenon in which repeat numbers of microsatellites are abnormally increased or decreased. MSI can be typically detected by assaying microsatellite markers (BAT25, BAT26, D2S123, D5S346, D17S250, etc.) using PCR method. Thus, detection of MSI contributes to HNPCC diagnosis (see, Non-Patent Document 5). In addition, in an international consensus conference regarding MSI detection in 1997, tumor in which instability is detected from two or more markers as mentioned above was defined as MSI-H (a state where 30% or more MSI is detected in tumor), and it was determined that such tumor has clinicopathologic characteristics.
Genetic diagnosis of cancer is based on the results of analysis of mutation of a causative gene in many cases. However, differing from familial adenomatous polyposis (FAP) whose single causative gene is APC, five causative genes have been identified (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6) in the case of HNPCC. However, since the ratio of developing the disease (penetrance rate) when such causative genes are mutated is not 100%, the genetic diagnosis of HNPCC based on mutation of the aforementioned causative genes has not yet reached a clinical level at the present time, and it is considered that such genetic diagnosis is still at a study level in the classification of American Society of Clinical Oncology (ASCO). With regard to cancer-associated genes such as hMLH1, a method, which comprises detecting genetic mutation or methylation on their genomic sequences and detecting cancer using such genetic mutation or methylation as an indicator, has been proposed (Patent Documents 1 and 2). However, the disease cover ratio of this method is not very high, and the method has also been problematic in terms of simplicity and promptness. Hence, it has been desired that a simple and highly reliable method for testing cells, which is useful for the diagnosis of cancers such as HNPCC as a typical example, be developed. If a cell test method capable of screening of cancer at a gene expression level were developed, it means that the real-time detection system in that method would be effective. However, such a useful test method has not yet been developed.

    [Patent Document 1] JP Patent Publication (Kokai) No. 2005-304497 A
    [Patent Document 2] JP Patent Publication (Kohyo) No. 2002-533061 A
    [Non-Patent Document 1] Landry J. R. et al., Trends in Genetics, (2003) 19(11) p. 640-648
    [Non-Patent Document 2] Kimura K. et al., Genome Research, (2006) 16: p. 55-65
    [Non-Patent Document 3] Brinkman B. M., Clin. Biochem., (2004) 37(7): p. 584-594
    [Non-Patent Document 4] Liu B. et al., Nature Med., (1996) 2: p. 169-174
    [Non-Patent Document 5] Laiho P. et al., Cancer Research, (2002) 62: p. 1166-1170
    [Non-Patent Document 6] Deng G. et al., Cancer Research, (1999) 59: p. 2029-2033


SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for testing a cell based on gene expression analysis, which is useful for cancer diagnosis.
As a result of concentrated studies for achieving the aforementioned object, the present inventors have found that at least 3 types of transcriptional variants (variant type 1, variant type 2, and variant type 3) are expressed from a human MLH1 gene (hMLH1 gene), and further that in a cancer cell positive for microsatellite instability, the expression level of variant type 3 is relatively higher than that of variant type 1 that is a major transcript, thereby completing the present invention.
That is to say, the present invention includes the following embodiments.
[1] A gene expression analysis method comprising: measuring the expression level of a transcript of a human MLH1 gene containing the nucleotide sequence as shown in SEQ ID NO: 1 at the 5′ terminus thereof and the expression level of a transcript of a human MLH1 gene containing the nucleotide sequence as shown in SEQ ID NO: 2 at the 5′ terminus, in a biological sample; and comparing the expression levels, thereby detecting a cell positive for microsatellite instability.
In this method, the expression level of the transcripts can be measured using a real-time PCR method. The measurement using the real-time PCR method can be preferably carried out by TaqMan® probe method.
In the method of the present invention, the measurement is also preferably carried out using the following primer pairs (a) and (b):
(a) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion of an exon 1 sequence spanning from nucleotides 1 to 183 from the nucleotide sequence as shown in SEQ ID NO: 1, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5; and
(b) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion of exon 1 and 2 spanning from nucleotides 1 to 132 from the nucleotide sequence as shown in SEQ ID NO: 2, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from a exon 2 sequence as shown in SEQ ID NO: 5.
Preferred examples of the above primer pairs (a) and (b) include:
the primer pair (a), which is a combination of a primer comprising the nucleotide sequence as shown in SEQ ID NO: 6 with a primer comprising the nucleotide sequence as shown in SEQ ID NO: 7, and
the primer pair (b), which is a combination of a primer comprising the nucleotide sequence as shown in SEQ ID NO: 8 with a primer comprising the nucleotide sequence as shown in SEQ ID NO: 7. In the case of using the TaqMan® probe method in the measurement using such primer pairs, a probe comprising the nucleotide sequence as shown in SEQ ID NO: 9, to one end of which a fluorescent substance is added and to the other end of which a quenching substance is added, can be preferably used.
The cell positive for microsatellite instability, which is detected by the above described method of the present invention, is a cancer cell in many cases. This cancer may be colorectal cancer. Further, this colorectal cancer may be hereditary non-polyposis colorectal cancer.
[2] A primer set for use in analyzing a gene expression level to detect a cell positive for microsatellite instability, comprising the following primer pairs (a) and (b):
(a) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion of an exon 1 sequence spanning from nucleotides 1 to 183 the nucleotide sequence as shown in SEQ ID NO: 1, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5; and
(b) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion of exon 1 and 2 spanning from nucleotides 1 to 132 from the nucleotide sequence as shown in SEQ ID NO: 2, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5.
[3] A kit for analyzing a gene expression level to detect a cell positive for microsatellite instability, comprising the following primer pairs (a) and (b):
(a) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion of an exon 1 sequence spanning from positions 1 to 183 from the nucleotide sequence as shown in SEQ ID NO: 1, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5; and
(b) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion of exon 1 and 2 spanning from positions 1 to 132 from the nucleotide sequence as shown in SEQ ID NO: 2, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5.
The primer pairs (a) and (b) contained in this kit are preferably:
the primer pair (a), which is a combination of a primer comprising the nucleotide sequence as shown in SEQ ID NO: 6 with a primer comprising the nucleotide sequence as shown in SEQ ID NO: 7, and
the primer pair (b), which is a combination of a primer comprising the nucleotide sequence as shown in SEQ ID NO: 8 with a primer comprising the nucleotide sequence as shown in SEQ ID NO: 7.
This kit may further comprise a probe comprising the nucleotide sequence as shown in SEQ ID NO: 9, to one end of which a fluorescent substance is added and to the other end of which a quenching substance is added.
The above described cell that is a detection target of this kit is preferably a cancer cell. This kit is particularly useful as a kit for diagnosing cancer, is more particularly useful as a kit for diagnosing colorectal cancer, and further more particularly useful as a kit for diagnosing hereditary non-polyposis colorectal cancer.
The detection method of the present invention is capable of simply and clearly testing the expression level of an hMLH1 gene even in a case where the above method is applied to a biological sample that contains only small quantities of cancer cells. Thus, the above method is capable of rapidly and simply detecting a cell positive for microsatellite instability, in particular, a cancer cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 sets forth the exon structures of 3 types of transcripts of the hMLH1 gene; and
FIG. 2A shows the expression levels of transcriptional variant types 1 and 3 of the hMLH1 genes in 6 types of cancer cell lines. The filled bar indicates variant type 1, and the open bar indicates variant type 3.
FIG. 2B shows a result of making a comparison between the expression levels of variant type 1 and of variant type 3 in each cancer cell line as shown in the graph in FIG. 2A. In FIG. 2B, when the expression level of variant type 1 was lower than that of variant type 3, it was indicated as V1<V3, when variant type 1 was not detected and only variant type 3 was detected, it was indicated as V1(−), V3(+), and when the expression level of variant type 1 was higher than that of variant type 3, it was indicated as V1>V3. In addition, in FIG. 2B, whether microsatellite instability (MSI) is positive (+) or negative (−), and whether methylation was observed (+) or was not observed (−) in the promoter region of the hMLH1 gene, are also shown.



DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in detail below.
A human MLH1 gene (hMLH1 gene) is a human homolog (homologue) of the DNA mismatch repair gene, mutL, of Escherichia coli (E. coli). The hMLH1 gene has been known as a gene locus in which mutation occurs with high frequency in hereditary non-polyposis colorectal cancer (HNPCC). The RefSeq sequence of the MRNA of the hMLH1 gene is submitted to GenBank under Accession No. NM000249. In the present specification, the RefSeq sequence is shown in SEQ ID NO: 13 for reference. This RefSeq sequence (SEQ ID NO: 13) of the mRNA of the hMLH1 gene contains 19 exons.
In the present invention, as a variant of the transcript of the hMLH1 gene, variant type 2 and variant type 3 were discovered, in addition to variant type 1 which is a major transcript and almost corresponds to the aforementioned RefSeq sequence (FIG. 1). This variant type 1 contains the nucleotide sequence as shown in SEQ ID NO: 1 (exon 1 and 2) at the 5′ terminus thereof, followed by the sequence of exon 3 to 19 of the hMLH1 gene, which corresponds to nucleotides 268 to 2524 of SEQ ID NO: 13. Variant type 2 has an exon structure that is almost the same as that of variant type 1, but 5 nucleotides at the 5′ end of the exon 2 (SEQ ID NO: 5) have been deleted. On the other hand, variant type 3 is derived from an mRNA precursor transcribed from a transcription initiation site that is located at approximately 300 bp downstream from that of variant type 1, and thus the 5′ terminus of variant type 3 comprises the nucleotide sequence of SEQ ID NO: 2 (exon 1 and 2) that differs from that of variant type 1, and exon 3 to 19 sequences that are shared with variant types 1 and 2 follow backward. Since there exist a translation initiation site, ATG in the sequence of exon 1 of variant type 1, an amino acid sequence at N-terminus encoded by variant type 3 differs from that by variant type 1. It is predicted that such a protein encoded by variant type 3 will be produced as a defective protein, or such a protein will not be produced at all.
In the present invention, it was also shown that the expression level of variant type 3 was relatively increased when compared with the expression level of the aforementioned variant type 1, in cancer cells such as colorectal cancer, and particularly, in cells positive for microsatellite instability. It is considered that the expression of variant type 1 is generally found in the largest amount in vivo. Thus, a change in the ratio between the expression levels of the transcript of the hMLH1 gene shows abnormality in the expression state of the hMLH1 gene in cells in a biological sample. Such abnormality was considered to be correlated with microsatellite instability and malignant transformation in cells.
The method of the present invention is based on the aforementioned findings. The present method is a gene expression analysis method, comprising: measuring the expression level of variant type 1 (a transcript of the human MLH1 gene containing the nucleotide sequence as shown in SEQ ID NO: 1 at the 5′ terminus) and the expression level of variant type 3 (a transcript of the human MLH1 gene containing the nucleotide sequence as shown in SEQ ID NO: 2 at the 5′ terminus), in a biological sample; and making a comparison between the expression levels, thereby detecting cells which are positive for microsatellite instability.
In the present invention, a biological sample may be any tissue, cell, or body fluid, collected from a human subject. Examples of the biological sample may include skin, mucosal tissues, and gastrointestinal tissues. Such examples may also include blood, lymph, saliva, and seminal fluid. Preferred biological samples may particularly include tumor, cancer tissues, and tissues that are suspected of becoming malignant transformed. The biological sample may be an established cultured cell line. Particularly preferred biological samples may include tissues or cells derived from colorectal cancer, and colorectal tissues or cells that are suspected to be affected by colorectal cancer, which may be, but not limited to, for example, hereditary non-polyposis colorectal cancer (HNPCC).
The term “transcript” is used herein include an mRNA precursor transcribed from a certain gene, a modified product of such an mRNA precursor (which is modified by addition of a cap structure, polyadenylation, etc.), mature mRNA, and cDNA reversely transcribed from mature RNA. When RNA is referred to as a transcript, “T” (thymine) in the nucleotide sequence as shown in each sequence number used to specify the transcript is read as “U” (uracil). The term “the expression level of a transcript (variant)” is used herein to mean the abundance of a transcript that is expressed from a certain gene in a biological sample.
The expression levels of the transcript, variant type 1 and variant type 3, contained in a biological sample, may be measured according to a known method in the art of the present invention. Examples of the method of measuring an expression level may include, but not limited to, the real-time PCR method, the semi-quantitative RT-PCR method, the competitive PCR method using a capillary electrophoresis apparatus, a gene expression level measurement method using a DNA chip or microarray, differential display method, and the Northern blot method. Using such methods, the abundance of each transcript contained in a nucleic acid sample (e.g. RNA or cDNA) prepared from a biological sample can be measured. Such a nucleic acid sample may be prepared from a biological sample according to a routine method, and it may also be purified before analysis. In the present invention, genomic DNA or RNA may be prepared in accordance with standard experimental instructions used in the molecular biological field, such as J. Sambrook et al. (Eds.), Molecular Cloning, 3rd ed. Cold Spring Harbor Laboratory Press (2001).
Any type of method can be used to measure the expression levels of variant type 1 and variant type 3, as long as it is able to conduct an assay while distinguishing a transcript from another transcript. In the present invention, determination quantity of the exon 1 whose sequence is significantly different between variant type 1 and variant type 3 is conveniently used, for example.
As an example, the measurement of an expression level using the real-time PCR method will be described in detail. The real-time PCR method is a technique of monitoring an increase in a PCR amplified product in real time and then analyzing it. In general, the above method is carried out using a real-time PCR apparatus formed by unifying a thermal cycler and a spectrophotofluorometer. When compared with the conventional PCR method, the real-time PCR does not need electrophoresis. Thus, the real-time PCR enables rapid and easy analysis, and further it is excellently quantitative. The principle of an assay by the real-time PCR method is as follows. First, known amounts of DNA that have been subjected to serial dilution are used as standard samples. While performing PCR, the amounts of a PCR product in each of the standard samples are monitored, and the number of cycles (threshold cycle; Ct value), at which each dilution sample becomes a certain amount of amplified product in an exponential amplification phase, is detected. The number of cycles is plotted on a horizontal axis, and the amount of an initial template in the standard sample is then plotted on a vertical axis, so as to produce a calibration curve. Further, regarding a test nucleic acid sample containing an unknown concentration of a transcript, the amount of a PCR product is monitored with performing a PCR reaction under the same conditions as those for the standard sample, the number of cycles (Ct value) of the same amplification product amount as above is obtained. The Ct value obtained in the test nucleic acid sample is applied to the calibration curve as obtained above, and thus the amount of an initial template contained in the test nucleic acid sample can be estimated. This initial template amount corresponds to the expression level of a transcript.
Monitoring (detection) of a PCR product in the real-time PCR is preferably carried out using a fluorescent substance, but a monitoring method is not limited thereto. As such a fluorescence monitoring method, known techniques such as the intercalator method or the TaqMan® probe method can be used, but examples of such a method are not limited thereto. The intercalator method comprises addition of SYBR® Green I that is a substance emitting fluorescence as a result of binding to double-stranded DNA (intercalator), to a real-time PCR reaction system. In this method, an intercalator binds to double-stranded DNA synthesized by a PCR reaction and it emits florescence as a result of irradiation with excitation light. Thus, the intensity of such fluorescence is measured, so as to monitor the amount of a PCR amplified product generated. Since any given double-stranded DNA is detected by this intercalator method, the method does not require production of a detection probe for each gene. Thus, the experiment cost is inexpensive, and a reaction system is easily constructed. However, the detection specificity is not very high. On the other hand, the TaqMan® probe method is a method, which comprises addition of an oligonucleotide probe (TaqMan® probe), which is typically labeled with (bound to) a fluorescent substance (FAM, etc.) at the 5′-terminus and a quenching substance (TAMRA etc.) at the 3′-terminus, to a real-time PCR reaction system. In this method, the TaqMan® hybridizes specifically with template DNA in an annealing step. At that time, since a fluorescent substance and a quenching substance coexist on the probe, development of fluorescence is suppressed, although excitation light is emitted from the fluorescent substance. However, when the TaqMan® probes hybridizing with the templates are successively removed by the 5′→3′ exonuclease activity of Taq DNA polymerase in the subsequent elongation step, the fluorescent substances are released from the probes, and suppression by the quenching substances is removed, so as to generate fluorescence. By measuring such fluorescence intensity, the amount of amplified PCR product can be monitored. This TaqMan® probe method causes a high experiment cost, but it has high detection specificity and is excellently (or particularly) quantitative.
In the method of the present invention, a quantitative assay that is based on the aforementioned real-time PCR method can preferably be used to measure the expression level of each variant. In such a case, the TaqMan® probe method can preferably be used to monitor the PCR amplification product, but the usable method is not limited thereto. In the TaqMan® probe method, a TaqMan® probe, to one end (generally 5′-terminus) of which a fluorescent substance is added and to the other end (generally 3′-terminus) of which a quenching substance is added, is used. This probe is designed such that it can be annealed to a PCR amplification product that is generated by real-time PCR. As a fluorescent substance added to the probe, FAM or VIC can be used. As a quenching substance, TAMRA or the like can be used. Reaction conditions applied in the real-time PCR can be determined by persons skilled in the art, as appropriate. The reaction conditions as described later in the examples can also be used.
In the method of the present invention, in order to measure the expression level of each variant, the following primer pair (a) is used for specifically amplifying variant type 1 in PCR and the following primer pair (b) is used for specifically amplifying variant type 3 in PCR, so as to amplify both variants:
(a) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion (preferably 10 or more base pairs in length, for example, 10 to 30 base pairs in length) of an exon 1 sequence spanning from nucleotides 1 to 183 (This region is better for designing PCR primers than any other region. This region corresponds to the region of the almost all the sequence of exon 1 shown in SEQ ID NO: 1 and includes 3′ end of appropriate sense primer for amplifying variant type 1 shown in SEQ ID NO: 6) from the nucleotide sequence as shown in SEQ ID NO: 1, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5; and
(b) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion (for example, 10 to 30 base pairs in length) of exon 1 and 2 spanning from nucleotides 1 to 132 (This region is better for designing PCR primers than any other region. This region corresponds to the region containing the whole sequence of exon 1 and 5′end of exon 2 shown in SEQ ID NO:2 and includes 3′ end of appropriate sense primer for amplifying variant type 3 shown in SEQ ID NO:8) from the nucleotide sequence as shown in SEQ ID NO: 2, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5.
With regard to such primer pairs (a) and (b), a specific example is preferably as follows:
primer pair (a): a combination of a primer having the nucleotide sequence as shown in SEQ ID NO: 6 with a primer having the nucleotide sequence as shown in SEQ ID NO: 7, and
primer pair (b): a combination of a primer having the nucleotide sequence as shown in SEQ ID NO: 8 with a primer having the nucleotide sequence as shown in SEQ ID NO: 7.
When the expression levels of the aforementioned variant types 1 and 3 are measured by real-time PCR that is based on the TaqMan® probe method, using a combination of primers having the sequences as shown in SEQ ID NOS: 6 to 8, a probe having the nucleotide sequence as shown in SEQ ID NO: 9, to one end (generally 5′-terminus) of which a fluorescent substance is added and to the other end (generally 3′-terminus) of which a quenching substance is added, is particularly preferably used as a TaqMan® probe.
A method of measuring the expression level of a variant, using such primers and probes, comprises the following steps, for example:
a step of amplifying variant type 1 from cDNA derived from a biological sample, using a primer having the nucleotide sequence as shown in SEQ ID NO: 6 and a primer having the nucleotide sequence as shown in SEQ ID NO: 7;
a step of amplifying variant type 3 from cDNA derived from a biological sample, using a primer having the nucleotide sequence as shown in SEQ ID NO: 8 and a primer having the nucleotide sequence as shown in SEQ ID NO: 7; and
a step of monitoring the amount of the PCR amplification product in each amplification reaction, and then calculating the initial content of variant type 1 and that of variant type 3 in the biological samples.
In this monitoring step, the TaqMan® probe is added to each amplification reaction system, so that the amount of a PCR amplification product can be measured by fluorescence intensity.
In the method of the present invention, after the expression level of variant type 1 and that of variant type 3 have been measured as described above, a comparison is made between both expression levels, so as to detect a cell positive for microsatellite instability. Specifically, when the expression level of variant type 1 is compared with that of variant type 3, if the measurement value of the expression level of variant type 3 is higher than the measurement value of the expression level of variant type 1, there is a high possibility that a cell positive for microsatellite instability may be contained in the biological sample examined. Even when variant type 1 is not detected and only variant type 3 is detected, it is deemed that the expression level of variant type 3 is higher than that of variant type 1. In such a case, it can be determined that a cell positive for microsatellite instability may be contained in the biological sample examined. Thus, in the method of the present invention, the presence of a cell positive for microsatellite instability can be detected in a biological sample collected from a subject.
Microsatellite instability is a phenomenon whereby the repeat number of repeat units (microsatellites) consisting of 2 to 5 nucleotides on the genome increases or decreases abnormally. Severe microsatellite instability occurs in 12% to 16% of all types of colorectal cancers, and in 90% or more of hereditary non-polyposis colorectal cancer. Such microsatellite instability is observed in various types of cancers such as cancer of uterine body, stomach cancer, esophageal cancer, multiple primary cancer, lymphoid tumor, or squamous cell carcinoma. Thus, microsatellite instability has a strong correlation with cancers. Since it is considered that microsatellite instability occurs due to a deficiency in a DNA mismatch repair mechanism, when such microsatellite instability is exhibited, there is a concern regarding a tendency to develop multiple cancers or poor prognosis. Accordingly, when a cell positive for microsatellite instability is detected by the method of the present invention, it can be determined that there is a high possibility that the cell may be a cancer cell. Detected cell like this positive for microsatellite instability may be a cancer cell, in particular, a cell of cancer such as colorectal cancer, hereditary non-polyposis colorectal cancer, cancer of uterine body, stomach cancer, esophageal cancer, multiple primary cancer, lymphoid tumor, squamous cell carcinoma, endometrial cancer, or pancreatic cancer, and preferably, colorectal cancer, and particularly preferably hereditary non-polyposis colorectal cancer. Thus, the present invention also relates to a method of detecting a cancer cell, which comprises measuring the expression level of variant type 1 of the hMLH1 gene and the expression level of variant type 3 thereof in a biological sample, and making a comparison between such expression levels.
In addition, the present invention also relates to a primer set comprising primer pairs (a) and (b), which can preferably be used in the aforementioned method. These primers sets are ones for use in the analysis of a gene expression level for the purpose of the detection of a cell positive for microsatellite instability, which is a combination of the following primer pairs (a) and (b):
(a) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion (for example, 10 to 30 base pairs in length) of an exon 1 sequence spanning from nucleotides 1 to 183 from the nucleotide sequence as shown in SEQ ID NO: 1, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5; and
(b) a primer pair, which is a combination of a primer comprising a nucleotide sequence consisting of 15 to 50 contiguous nucleotides comprising a portion (for example, 10 to 30 base pairs in length) of exon 1 and 2 spanning from nucleotides 1 to 132 from the nucleotide sequence as shown in SEQ ID NO: 2, with a primer comprising a nucleotide sequence complementary to a nucleotide sequence consisting of 15 to 50 contiguous nucleotides from an exon 2 sequence as shown in SEQ ID NO: 5.
A specific example of such a primer set is a combination of primers, which includes:
a primer comprising the nucleotide sequence as shown in SEQ ID NO: 6;
a primer comprising the nucleotide sequence as shown in SEQ ID NO: 7; and
a primer comprising the nucleotide sequence as shown in SEQ ID NO: 8.
These primers can be easily produced by an oligonucleotide synthesis technique known in the art, or by the PCR method using, as a template, a sequence contained in the aforementioned hMLH1 gene variant types 1 and 3, such as SEQ ID NO: 1 or 2, or other methods. The “primer” of the present invention may be labeled so as to be favorably used in detection, as long as it can be used in PCR or the like. For example, the primer of the present invention may be a polynucleotide, to the 5′- or 3′-terminus of which a marker such as a fluorescence dye, enzyme, protein, radioactive isotope, chemiluminescence substance, or biotin, has been added. In the present invention, an oligonucleotide that is added to a polynucleotide so as to be used in detection (for example, the sequence “flap” irrelevant to a template, used in the invader method for mutation detection) is also included in such a “marker.”
Moreover, the present invention also relates to a kit for analyzing a gene expression level, which is used for detection of a cell positive for microsatellite instability, and which comprises the aforementioned primer pairs (a) and (b). This kit may further comprise a TaqMan®, which is a probe comprising the nucleotide sequence as shown in SEQ ID NO: 9, to one end of which a fluorescent substance is added and to the other end of which a quenching substance is added. The detection target of this kit is preferably a cancer cell, and in particular, this kit can be used in detection of a cancer cell positive for microsatellite instability. Accordingly, this kit is useful as a kit for diagnosing cancer. Further, this kit can be advantageously used as a kit for diagnosing colorectal cancer, and particularly hereditary non-polyposis colorectal cancer (HNPCC).
EXAMPLES
The present invention will be more specifically described in the following examples. However, these examples are not intended to limit the technical scope of the present invention.
Example 1
Searching for Transcriptional Variants of hMLH1 Gene
In order to examine a possibility that the hMLH1 gene would be expressed as several transcriptional variants (mutants) as a result of alternative splicing or the usage of an alternative promoter, the present inventors conducted the following experiment.
As described in Non-Patent Document 2, sequence information of approximately 1,800,000 of human full-length cDNAs including the 5′-terminus has been revealed based on the obtainment of human gene full-length cDNAs by construction of a cDNA clones using the oligo-cap method (Suzuki and Sugano (2003) Methods Mol. Biol. 221: p. 73-91). Such sequence information has been assigned to GenBank, and it is disclosed to the public. The present inventors collected such sequence information from GenBank, and they formatted it into multi-FASTA. Thereafter, homologous searching was performed against the thus obtained sequence data with a blastn program, using the RefSeq sequence (NCBI Reference Sequence) of hMLH1 mRNA (GenBank Accession No. NM—000249; SEQ ID NO: 13) as a query. Among the sequences of a high degree of similarity, sequences having an E-value of 10−10 or less were selected as hMLH1-associated cDNA sequences. Subsequently, a sequence corresponding to a region that is assigned to the hMLH1 gene in human 3rd chromosome was selected from GenBank human genomic sequence database Build 35. This genomic sequence of the hMLH1 gene and each hMLH1-associated cDNA sequence selected as described above were processed with b12seq of the BLAST program, so as to obtain an alignment for each case. Subsequently, the alignments that were correctly aligned ahead of 2nd exonic region of hMLH1 RefSeq (a sequence corresponding to nucleotides 177 to 267 of NM—000249) and that were also closely matched (the identity is 98% or more) were selected among the whole alignments. The selected alignments were analyzed. As a result, 3 patterns were observed in terms of the correlation between the hMLH1-associated cDNA sequence and the hMLH1 genomic sequence. That is to say, it was revealed that there are at least 3 types of variations (namely, variant types 1, 2, and 3) in terms of the transcripts of the hMLH1 gene. The structure on the 5′-terminal side of each of these 3 types of transcripts are shown in FIG. 1. It is to be noted that only the first 3 exons out of 19 exons are shown in FIG. 1.
As shown in FIG. 1, exon 1, exon 2, and exon 3 are included in this order from the 5′-terminal end in transcriptional variant type 1. A translation initiation site (ATG) is located in exon 1. As a result of this analysis, it was clarified that the nucleotide sequence of NM000249 that is the RefSeq sequence of the hMLH1 gene lacks only a little portion of the 5′-terminus sequence of the exon 1 of variant type 1. In contrast, variant type 2 has almost the same exonic structure as that of variant type 1, but the exon 2 of variant type 2 is 5 nucleotides shorter than that of variant type 1 on the 5′-terminal end. Thus, it was clarified that variant type 2 is an alternative splicing variant. Further, the exon 2 and 3 sequences of variant type 3 are the same as those of variant type 1, but the sequence of exon 1 of variant type 3 is completely different from those of variant type 1 and variant type 2. As a result, it was found that the hMLH1 gene is controlled by an alternative promoter, and thus the transcription of variant type 3 is found to be started approximately 300 bp downstream from a transcription initiation site of variant type 1.
It was shown from the aforementioned analysis that there are found 141 variant type 1 (75%), 14 variant type 2 (7.4%), and 32 variant type 3 (17.1%) in the selected hMLH1-associated cDNA sequences, and that variant type 1 is the most major transcript. The hMLH1-associated cDNA sequences classified into each variant are shown in Table 1. Each hMLH1-associated cDNA sequence is represented by GenBank Accession No. in Table 1.







TABLE 1







accession number









variant 1
variant 2
variant 3

















AU131219
DA031179
DA050403
DA110166
DB243737
DA385243
AU127122
DA517219


AU127758
DA716690
DA125431
DB040429
DA909925
DA868597
DA935122
DA343206


DA619769
DA472218
DA043349
DA880601
DA453337
DA815001
DB253300


DA024420
DA983131
DA695612
DA475144
DA055019
DA070867
DB204422


DA953555
DA702671
DB272759
DA762271
DA209147
DB139980
DA930241


DA023897
DA701340
DB090691
DA503831
DA914770
DB287548
DA566031


DA486475
DB200826
DA556556
DA494857
DA472331
DB157098
DA814707


DA932049
DA304561
DB274178
DB252001
DA609156
DB012065
DA166268


DA426942
DA658037
DA659112
DB120181
DA752464
DB114802
DA748219


DA555173
DA624450
DB270772
DA666173
DB014982
DB018927
DA927898


DA479521
DA489729
DB269584
DA631956
DA958303
DA866298
DA921733


DA524635
DA476333
DA559708
DA518707
DA952805
DA352218
DA963961


DA447945
AU280370
DA913772
DA698928
DA783187
DA506267
DA430514


DA952842
DA374168
DA619525
DB246315
DA884873
DB252510
DA303777


DA350100
DA201970
DA478068
DA106361
DA794400

DA664790


DA915432
DA577087
DA503277
DA712069
DA287058

DA922309


DA790495
DA926525
DA937827
DA493353
DB040297

DA476068


DA050020
DA814813
DA650335
DA911958
DA348650

DA580525


DA729732
DB011897
DA554567
DA765712
DA086654

DB012054


DA972163
DA582565
DA690019
DA656699
DA660079

DA296090


DA563477
DA287218
DA738557
DA592612
DA510241

DB259565


DA707133
DA173423
DA262636
DB266774


DA955738


AU128432
DA501808
DA819642
DA877866


DA821467


DA555707
DA881017
DA356018
DB055056


DA275297


DB092229
DB000482
DA919830
DA887994


DA172498


DA555444
DA546685
DA751926
DA997466


DA582466


DA880449
DA349545
DA207023
DA874359


DA750838


DA033654
DB008432
DA659014
DA478450


DA749519


DA305360
DA970996
DA599881
DB089707
total
total
DA073874
total


DA013759
DA308229
DA503294
DA004869
141 clones
14 clones
DA156778
32 clones









The nucleotide sequences of the exon 1 and 2 portions of variant type 1 and variant type 3, which were discovered in the present examples, are shown in Table 2.









TABLE 2







Variant type 1, exon 1
AAGAACGTGAGCACGAGGCACT



(SEQ ID NO: 3)
GAGGTGATTGGCTGAAGGCACT



TCCGTTGAGCATCTAGACGTTT



CCTTGGCTCTTCTGGCGCCAAA



ATGTCGTTCGTGGCAGGGGTTA



TTCGGCGGCTGGACGAGACAGT



GGTGAACCGCATCGCGGCGGGG



GAAGTTATCCAGCGGCCAGCTA



ATGCTATCAAAGAGATGATTGA



GAACTG


 


Variant type 3, exon 1
GCATGCCCACAACGGCGGAGGC


(SEQ ID NO: 4)
CGCCGGGTTGCCTGACGTGCCA



GTCAGGCCTTCTCCTTTTCCGC



AGACCGTGTGTTTCTTTACCGC



TCTCCCCCGAGACCTTTTAAGG



GTTGTTTGGAGT


 


Variant types 1 and 3,
TTTAGATGCAAAATCCACAAGT


exon 2
ATTCAAGTGATTGTTAAAGAGG


(SEQ ID NO: 5)
GAGGCCTGAAGTTGATTCAGAT



CCAAGACAATGGCACCGGGATC



AGG


 


Variant type 1, exons 1+ 2
AAGAACGTGAGCACGAGGCACT


(SEQ ID NO: 1)
GAGGTGATTGGCTGAAGGCACT



TCCGTTGAGCATCTAGACGTTT



CCTTGGCTCTTCTGGCGCCAAA



ATGTCGTTCGTGGCAGGGGTTA



TTCGGCGGCTGGACGAGACAGT



GGTGAACCGCATCGCGGCGGGG



GAAGTTATCCAGCGGCCAGCTA



ATGCTATCAAAGAGATGATTGA



GAACTGTTTAGATGCAAAATCC



ACAAGTATTCAAGTGATTGTTA



AAGAGGGAGGCCTGAAGTTGAT



TCAGATCCAAGACAATGGCACC



GGGATCAGG


 


Variant type 3, exons 1 + 2
GCATGCCCACAACGGCGGAGGC


(SEQ ID NO: 2)
CGCCGGGTTCCCTGACGTGCCA



GTCAGGCCTTCTCCTTTTCCGC



AGACCGTGTGTTTCTTTACCGC



TCTCCCCCGAGACCTTTTAAGG



GTTGTTTGGAGTTTTAGATGCA



AAATCCACAAGTATTCAAGTGA



TTGTTAAAGAGGGAGGCCTGAA



GTTGATTCAGATCCAAGACAAT



GGCACCGGGATCAGG









Example 2
As a result of the analysis of Example 1, it was found that 3 types of variants as the transcripts of the hMLH1 gene are present. The hMLH1 gene had previously been known as a gene associated with malignant transformation of cells. This time, the presence of transcriptional variants has been clarified for the first time, and thus, association of such variants with cancer has been anticipated.
Thus, focusing attention on variant types 1 and 3, the expression levels of such variants were measured by real-time PCR, and the expression level in cancer cell lines was compared.
As test materials, cultured cell lines LoVo, COLO320DM, COLO201, SW48, RKO, and HCT116 (all of which were cells derived from human colorectal cancer) were purchased from ATCC (American Type Culture Collection) and the Health Science Research Resources Bank, the Japan Health Sciences Foundation, and such cultured cell lines were then used. Each type of cells were cultured in an Advanced DMEM medium (Invitrogen) supplemented with 10% FBS (Invitrogen). Thereafter, total RNA was extracted from the cell lines using RNeasy Tissue Kit manufactured by QIAGEN. The extracted total RNA was used as a test sample in a reverse transcription and real-time PCR, as described later.
In addition, primers and probes used in the real-time PCR were designed. Such primers and probes were designed using Primer Express Software included with a real-time PCR apparatus, ABI PRISM 7900HT (Applied Biosystems). From the searching results of primers and probes obtained by inputting various conditions, the top 10 pairs having higher scores were selected. Using such 10 pairs, the real-time PCR was actually carried out for evaluation. As a result, good results were obtained from all of the 10 pairs. Thus, using the combination of primers and probes having the highest score, the following experiment was carried out.
SuperScriptTM III Reverse Transcriptase (Invitrogen) was used in a reverse transcription (cDNA synthesis reaction). First, 2.0 μl of oligo(dT)20primers (50 μM), 2.0 μg of total RNA, and 4.0 μl of dNTPs (5 mM) were added to a tube, and a DEPC-treated water was then added thereto, so as to prepare a mixture having a total volume of 26 μl. Subsequently, the mixture was incubated at 65° C. for 5 minutes, and it was then rapidly transferred onto ice. Thereafter, 5× First Strand Buffer (8.0 μl), 0.1 M DTT (2.0 μl), SuperScriptTM III Reverse Transcriptase (2.0 μl), and a DEPC-treated water (2.0 μl) were added thereto, so as to prepare a mixture having a total volume of 40 μl. This mixture was vortexed, and it was then centrifuged, so as to collect a solution to the bottom of the tube, followed by incubation at 50° C. for 60 minutes. Thereafter, it was further incubated at 70° C. for 15 minutes, so as to terminate the reaction.
Subsequently, using the above obtained reverse transcript as a template, the real-time PCR was carried out employing ABI PRISM 7900HT (Applied Biosystems), so as to quantify each of variant type 1 and variant type 3 contained in the reverse transcript derived from each type of cells.
In order to amplify variant type 1 by PCR, 1.0 μl of a reverse transcript as template DNA, 10 μl of 1× Mastermix, 3.5 μl of a 5.0 μM sense primer (5′-CAGCGGCCAGCTAATGCTAT-3′; SEQ ID NO: 6), 3.5 μl of a 5.0 μM antisense primer (5′-CCATTGTCTTGGATCTGAATCAACTTC-3′; SEQ ID NO: 7), 5.0 μl of 5.0 μM TaqMan® probe (5′-CAAGTATTCAAGTGATTGTTAAAGAGGGAGGCC-3′; SEQ ID NO: 9), and 1.0 μl of extra pure water were mixed, so as to prepare a reaction solution having a total volume of 20 μl. Likewise, in order to amplify variant type 3 by PCR, 1.0 μl of a reverse transcript as template DNA, 10 μl of 1× mastermix, 3.5 μl of a 5.0 μM sense primer (5′-GGGTTGTTTGGAGTTTTAGATGCA-3′; SEQ ID NO: 8), 3.5 μl of a 5.0 μM antisense primer (5′-CCATTGTCTTGGATCTGAATCAACTTC-3′; SEQ ID NO: 7), 5.0 μl of 5.0 μM TaqMan® probe (5′-CAAGTATTCAAGTGATTGTTAAAGAGGGAGGCC-3′; SEQ ID NO: 9), and 1.0 μl of extra pure water were mixed, so as to prepare a reaction solution having a total volume of 20 μl. It is to be noted that the TaqMan® probe used herein was capable of detecting both variant types 1 and 3. The above TaqMan® probe was manufactured by Applied Biosystems Japan Ltd. The 5′-terminus thereof was labeled with FAM (a fluorescent substance), and the 3′-terminus thereof was labeled with TAMRA (a quenching substance). The prepared reaction solution was subjected to a PCR using ABI PRISM 7900HT (Applied Biosystems), wherein after a reaction at 95° C. for 10 minutes, a reaction consisting of 95° C., 15 seconds, and 60° C., 1 minute, was repeated for 40 cycles. Thereafter, the fluorescence intensity was measured.
Moreover, in order to produce a calibration curve that is used to calculate the amount of DNA contained in the sample based on the fluorescence intensity measured in the real-time PCR, before carrying out the real-time PCR, a PCR product had previously been prepared from the aforementioned reverse transcript derived from the COLO320DM in the following process. First, in order to amplify variant type 1 by PCR, 2.0 μl of a reverse transcript as template DNA, 5.0 μl of a 10×PCR buffer, 4.0 μl of dNTP Mix (2.5 mM each), 3.5 μl of a 5.0 μM sense primer (5′-CTGGACGAGACAGTGGTGAAC-3′; SEQ ID NO: 10), 3.5 μl of a 5.0 μM antisense primer (5′-ATACTGGCTAAATCCTCAAAGGACTG-3′; SEQ ID NO: 11), 0.25 μl of Ex Taq polymerase, and 37 μl of extra pure water were mixed, so as to prepare a reaction solution having a total volume of 50 μl. In order to amplify variant type 3 by PCR, a reaction solution was prepared with the same composition as that used in PCR amplification of the aforementioned variant type 1, with the exception that the sense primer was changed to that as shown in SEQ ID NO: 12 (5′-TTTCTTTACCGCTCTCCCCCG-3′). Each of the prepared reaction solutions was reacted at 95° C. for 10 minutes, and thereafter, a reaction consisting of 95° C., 30 seconds, 63° C., 1 minute, and 72° C., 1 minute, was repeated for 35 cycles. Finally, a reaction at 72° C. for 10 minutes was carried out. With regard to the thus obtained PCR product derived from the COLO320DM, the amount of DNA thereof was measured, and it was then subjected to serial dilution. Using the resultant as template DNA, the same amplification as for other reverse transcripts was carried out in the aforementioned real-time PCR. Thereafter, the fluorescence intensity was measured as described above, and a calibration curve was then produced based on the obtained value according to a routine method.
From the Ct (Threshold Cycle) value obtained from the fluorescence intensity measured as described above and the above produced calibration curve, the amounts of variant types 1 and 3 contained in an initial transcript contained in total RNA extracted from each cell were calculated. The measurement values of the amounts of variant types 1 and 3 correspond to their expression levels, respectively.
The results obtained by quantifying the expression levels of variant types 1 and 3 in 6 types of colorectal cancer cell lines, as described above, are shown in FIG. 2. As shown in FIG. 2, the expression of variant type 1 was detected in LoVo, HCT116, COLO320DM, and COLO201. However, the expression thereof was not detected in RKO and SW48 (less than detection limit). These results correspond to the previous report (Non-Patent Document 5). On the other hand, the expression pattern of variant type 3, the existence of which had been discovered by the present inventors, clearly differed from the expression pattern of variant type 1. The expression of variant type 3 was detected in RKO or SW48, in which the expression of variant type 1 that was hMLH1 had not been detected in the previous report. Further, it was also shown that the expression level of variant type 3 was significantly higher than that of variant type 1 in LoVo and HCT116. Such results were contrary to the prediction in the analysis of Example 1 that variant type 1 was a major transcript of the hMLH1 gene and that the expression level of variant type 3 was low. Thus, LoVo and HCT116 in which the expression level of variant type 3 was higher than that of variant type 1, and RKO and SW48 in which variant type 3 was detected but variant type 1 was not detected, were microsatellite instability (MSI) positive whose information was obtained from Non-Patent Document 5 (FIG. 2; Non-Patent Document 5). Furthermore, the degree of methylation of the promoter region of the hMLH1 gene, which has an influence upon the expression level of the above gene, was examined based on the descriptions of Non-Patent Document 5. As a result, it was found that methylation of the promoter region of the hMLH1 gene was detected only in RKO and SW48, in which variant type 3 had been detected but variant type 1 had not been detected. That is, it was considered that such methylation of the promoter region of the hMLH1 gene suppresses the expression of variant type 1, but that it does not suppress the expression of variant type 3.
Taking into consideration the facts that the hMLH1 gene has been known to be associated with hereditary non-polyposis colorectal cancer (HNPCC), and that it has been known that microsatellite instability becomes positive with high frequency in various types of cancers including HNPCC, it is considered that a relative increase in the expression level of variant type 3 compared to the transcriptional variant type 1 of the hMLH1 gene is in correlation with MSI positive in cancer and methylation of a promoter region.
The conditions for expression of variant type 3 and the specific actions of the expression itself on the occurrence of cancer still remain unknown. There may be a possibility that the transcriptional variant type 3 itself could not affect MSI, and thus the occurrence of cancer, but that a decrease in the expression level of variant type 1 or the loss thereof could become a major factor for inducing MSI. However, in spite of estimation of such mechanisms, when compared with the transcriptional variant type 1, if the expression level of variant type 3 was relatively increased, it was clear that there was the presence of cancer cells associated with MSI.
INDUSTRIAL APPLICABILITY
The detection method of the present invention enables simple and clear detection of the presence of a cancer cell such as colorectal cancer in a cancer sample even at an extremely early stage. The detection results obtained by the present method are useful in diagnosing cancer with microsatellite instability, such as HNPCC, at an early stage.
SEQUENCE LISTING FREE TEXT
SEQ ID NOS: 6 to 8 and 10 to 12 represent primers.
SEQ ID NO: 9 represents a probe.

## Claims

1. A method for detecting the possible presence of microsatellite instability in a biological sample comprising:
(i) measuring, in the biological sample, the mRNA expression level of a first transcript of a human MLH1 gene containing the nucleotide sequence of SEQ ID NO: 1 at the 5′-terminus thereof and the mRNA expression level of a second transcript of a human MLH1 gene containing the nucleotide sequence of SEQ ID NO: 2 at the 5′-terminus thereof using a primer pair (a) and a primer pair (b), wherein the primer pair (a) is a combination of a primer comprising the nucleotide sequence as shown in SEQ ID NO: 6 with a primer comprising the nucleotide sequence of SEQ ID NO: 7, and wherein the primer pair (b) is a combination of a primer comprising the nucleotide sequence as shown in SEQ ID NO: 8 with a primer comprising the nucleotide sequence of SEQ ID NO: 7, and wherein the expression levels are measured using a real-time PCR method; and
(ii) comparing the expression levels measured in (i), wherein a mRNA expression level of the second transcript that is higher than the mRNA expression level of the first transcript indicates the possible presence of microsatellite instability.

2. The method according to claim 1, wherein said real-time PCR method comprises the use of an oligonucleotide probe labeled with a fluorescent substance and a quenching substance.

3. The method according to claim 1, wherein a probe comprising the nucleotide sequence as shown in SEQ ID NO: 9, to one end of which a fluorescent substance is added and to the other end of which a quenching substance is added, is used in the real-time PCR method.