JP6108571B2 - Method and kit for determining risk of developing esophageal cancer - Google Patents
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Description
本発明は、食道癌の発症リスクを判定する方法及びそのキットに関する。 The present invention relates to a method for determining the risk of developing esophageal cancer and a kit thereof.
遺伝子診断は、癌、糖尿病等の疾患のリスクを判定するのに有用であり、疾患の予防、早期発見の観点から非常に重要である。 Genetic diagnosis is useful for determining the risk of diseases such as cancer and diabetes, and is very important from the viewpoint of disease prevention and early detection.
BAP1（BRCA1 associated protein-1）は細胞周期に関与する蛋白質であり、癌抑制遺伝子として知られている。特許文献１には、ＢＡＰ１遺伝子の変異によって、癌になりやすい旨が記載されている。乳癌患者、腎臓明細胞癌患者、卵巣癌患者において、ＢＡＰ１遺伝子のイントロン部分に生殖細胞系列変異が見つかった旨が開示されている。また、イントロン６の変異によって、スプライシング工程でエクソン７が転写されなくなる（スキップされる）ことが開示されている。しかし、BAP1と悪性黒色腫、乳癌、腎臓明細胞癌、卵巣癌以外の癌との関連性については知られていない。
BAP1 (BRCA1 associated protein-1) is a protein involved in the cell cycle and is known as a tumor suppressor gene.
一方、食道癌のリスクファクターとしてはアルコール等が知られており、アルコール分解酵素の活性が低いと食道癌のリスクが高くなることが知られている。 On the other hand, alcohol and the like are known as risk factors for esophageal cancer, and it is known that the risk of esophageal cancer increases when the activity of the alcohol-degrading enzyme is low.
しかし、食道癌は自覚症状があまりなく早期発見の困難な癌種であり、また現在知られている遺伝子診断法でも食道癌の十分なリスク評価はできず、例えば、複数の診断法を組合わせることにより精度の高いリスク評価が可能となるため、食道癌のリスクを評価するための新たな指標の開発が所望されている。 However, esophageal cancer is a type of cancer that has few subjective symptoms and is difficult to detect at an early stage, and the currently known genetic diagnostic methods cannot sufficiently evaluate the risk of esophageal cancer. For example, multiple diagnostic methods are combined. Therefore, it is possible to evaluate the risk with high accuracy. Therefore, it is desired to develop a new index for evaluating the risk of esophageal cancer.
本発明が解決すべき課題は、遺伝子診断により被験者における食道癌の発症リスクを判定するための新規の指標を提供することである。 The problem to be solved by the present invention is to provide a novel index for determining the risk of developing esophageal cancer in a subject by genetic diagnosis.
本発明者は、上記のような状況の下、鋭意研究を重ねた結果、多種多様な遺伝子のなかから、ＢＡＰ１遺伝子上の変異が食道癌のリスク判定の指標となり得ることを見出した。本発明は当該新規の知見に基づくものである。 As a result of intensive studies under the circumstances as described above, the present inventor has found that mutations in the BAP1 gene can serve as an index for esophageal cancer risk determination from a wide variety of genes. The present invention is based on the novel findings.
本発明によれば、これまで知られていた食道癌のリスク判定で用いられてきたものとは全く異なるBAP1遺伝子上の変異を指標として用いた、食道癌のリスク判定法を提供することができる。食道がんは初期症状に乏しく、早期発見が難しいため、本発明の方法は、非常に有用である。 According to the present invention, it is possible to provide a risk determination method for esophageal cancer using, as an index, a mutation on the BAP1 gene that is completely different from that used in the conventional risk determination for esophageal cancer. . Since the esophageal cancer has few initial symptoms and is difficult to detect early, the method of the present invention is very useful.
食道癌の発症リスクを判定する方法
本発明は、被験者のＢＡＰ１遺伝子における変異を検出し、該変異が検出された被験者を食道癌の発症リスクが存在すると判定する、食道癌の発症リスクを判定する方法を提供する。 Method for determining risk of developing esophageal cancer The present invention detects a mutation in the BAP1 gene of a subject and determines that the subject in which the mutation is detected is at risk of developing esophageal cancer. Provide a method.
本発明において、「（食道癌の）発症リスクの判定」とは、将来食道癌を発症する可能性の有無の判定することを意味する。「判定」は、「検査」と換言することもできる。 In the present invention, “determining the risk of developing (esophageal cancer)” means determining whether or not there is a possibility of developing esophageal cancer in the future. “Determining” can be rephrased as “inspection”.
被験者（被験対象）は、特に限定されるものではないが、ヒトを含む哺乳類が例示される。非ヒト哺乳類としては、マウス、ラット、イヌ、ネコ、ウシ、ヒツジ、ウマ等が挙げられる。本発明の判定方法の好ましい被験対象は、ヒトである。被験対象の性別、年齢、人種は特に限定されない。 The subject (test subject) is not particularly limited, and examples include mammals including humans. Examples of non-human mammals include mice, rats, dogs, cats, cows, sheep and horses. A preferred test subject of the determination method of the present invention is a human. The sex, age, and race of the test subject are not particularly limited.
本発明において「遺伝子」とは、特に言及しない限り、２本鎖DNA、１本鎖DNA(センス鎖又はアンチセンス鎖)、及びそれらの断片が含まれる。また、本発明において「遺伝子」とは、特に言及しない限り、調節領域、コード領域、エクソン、及びイントロンを区別することなく示すものとする。 In the present invention, “gene” includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof unless otherwise specified. In the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.
また、本明細書中において、「ヌクレオチド」、「オリゴヌクレオチド」及び「ポリヌクレオチド」は、核酸と同義であって、ＤＮＡおよびＲＮＡの両方を含むものとする。また、これらは２本鎖であっても１本鎖であってもよく、ある配列を有する「ヌクレオチド」（または「オリゴヌクレオチド」、「ポリヌクレオチド」）といった場合、特に言及しない限り、これに相補的な配列を有する「ヌクレオチド」（または「オリゴヌクレオチド」及び「ポリヌクレオチド」）も包括的に意味するものとする。さらに、「ヌクレオチド」（または「オリゴヌクレオチド」及び「ポリヌクレオチド」）がＲＮＡである場合、配列表に示される塩基記号「Ｔ」は「Ｕ」と読み替えられるものとする。 Further, in this specification, “nucleotide”, “oligonucleotide” and “polynucleotide” are synonymous with nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.
本発明において、ＢＡＰ１遺伝子としては、配列番号１で表される塩基配列が挙げられる（図６〜９）。本発明において、食道癌の発症リスクの指標となるＢＡＰ１遺伝子の変異とは、配列番号１で表される塩基配列に対し少なくとも１個（好ましくは１〜３個）の塩基が欠失、置換、付加及び／又は挿入されていること、好ましくは少なくとも１個（好ましくは１〜３個）の塩基が欠失していることを意味する。食道癌の発症リスクの指標となるＢＡＰ１遺伝子の変異が２個以上の塩基の場合、それらの塩基は連続していることが好ましい。ＢＡＰ１遺伝子の変異としては、将来の悪性腫瘍発生のリスク診断の観点からは生殖細胞系列の遺伝子変異を指標とすることが好ましい。 In the present invention, the BAP1 gene includes the base sequence represented by SEQ ID NO: 1 (FIGS. 6 to 9). In the present invention, the BAP1 gene mutation that serves as an index of the risk of developing esophageal cancer is a deletion or substitution of at least one (preferably 1 to 3) bases from the base sequence represented by SEQ ID NO: 1. It means that it has been added and / or inserted, and preferably at least one (preferably 1 to 3) base has been deleted. When the mutation of the BAP1 gene serving as an index of the risk of developing esophageal cancer is 2 or more bases, it is preferable that these bases are continuous. As a BAP1 gene mutation, germline gene mutation is preferably used as an index from the viewpoint of risk diagnosis of future malignant tumor development.
ＢＡＰ１遺伝子は、１７個のエクソン及び１６個のイントロンを有し、本発明の好ましい実施形態において、当該ＢＡＰ１遺伝子の変異としては、９番目のイントロンであるイントロン９における変異が挙げられる。本発明の好ましい実施形態において、当該ＢＡＰ１遺伝子の変異としては、当該ＢＡＰ１遺伝子のイントロン９内の塩基配列ctctから塩基配列ctまたはtcが欠失したものが挙げられる。また当該変異は、Human Genome variation societyが規定する表記（http://www.hgvs.org/mutnomen/examplesDNA.html#del）のうち、nucleotide numbering coding DNA Reference Sequenceに従えば、c.784-26〜c.784-23の塩基配列ctctから塩基配列ctまたはtcが欠失したものと換言することができる。当該欠失は、塩基配列ctctの５’の塩基配列ctが欠失したものでも、塩基配列ctctの３’の塩基配列ctが欠失したものでも、塩基配列ctctの中央の塩基配列tcが欠失したものでもよい。これらの欠失により、いずれの場合もＢＡＰ１遺伝子のイントロン９内の塩基配列ctctは、塩基配列ctとなる。 The BAP1 gene has 17 exons and 16 introns. In a preferred embodiment of the present invention, the BAP1 gene includes a mutation in intron 9, which is the ninth intron. In a preferred embodiment of the present invention, the BAP1 gene mutation includes a deletion of the base sequence ct or tc from the base sequence ctct in intron 9 of the BAP1 gene. In addition, the mutation is c.784-26 according to the nucleotide numbering coding DNA Reference Sequence in the notation (http://www.hgvs.org/mutnomen/examplesDNA.html#del) defined by the Human Genome variation society. In other words, the nucleotide sequence ct or tc is deleted from the nucleotide sequence ctct of ˜c.784-23. The deletion may be the deletion of the 5 ′ base sequence ct of the base sequence ctct or the deletion of the 3 ′ base sequence ct of the base sequence ctct, or the lack of the central base sequence tc of the base sequence ctct. It may be lost. Due to these deletions, in any case, the base sequence ctct in the intron 9 of the BAP1 gene becomes the base sequence ct.
本発明において、特に明記しない限り、塩基配列及び遺伝子変異の記載法についてはHuman Genome Variation Societyが2013年1月26日に改訂したDescription of sequence changes: examples DNA-levelのnucleotide numbering coding DNA Reference Sequenceに従った。ここで、nucleotide numbering coding DNA Reference Sequenceの表記法に従うと、エクソンに含まれる塩基配列Ｘは、５’ＵＴＲ及びイントロンを除いたコーディング領域の塩基のうちＸ番目であることを意味する。ここでＢＡＰ１遺伝子のコーディング領域は、配列番号１でいう116〜152番目、251〜280番目、386〜440番目、1388〜1520番目、1917〜2036番目、2534〜2595番目、2678〜2820番目、3087〜3165番目、3618〜3741番目、4082〜4229番目、4700〜4884番目、5408〜5541番目、6100〜6578番目、6696〜6856番目、7123〜7215番目、7320〜7392番目、及び7573〜7706番目の塩基配列で表される。従って、例えば、エクソン３の最初の塩基（配列番号１でいう３８６番目の塩基ｇ）は、nucleotide numbering coding DNA Reference Sequenceの表記法に従うと、c.６８と表記される。イントロンの場合、直前のエクソンの最後の塩基よりいくつ後にあるか、または直後のエクソンの最初の塩基よりいくつ前にあるかで示される。例えば、イントロン２のうち最後から１０個目の塩基（配列番号１でいう３７６番目の塩基ｃ）は、c.６８−１０と表記される。 In the present invention, unless otherwise stated, the description of sequence changes: examples DNA-level nucleotide numbering coding DNA Reference Sequence revised by the Human Genome Variation Society on January 26, 2013 for the description of nucleotide sequences and gene mutations. I followed. Here, according to the notation method of nucleotide numbering coding DNA Reference Sequence, it means that the base sequence X contained in the exon is the Xth of the bases of the coding region excluding the 5 'UTR and the intron. Here, the coding region of the BAP1 gene is 116 to 152, 251 to 280, 386 to 440, 1388 to 1520, 1917 to 2036, 2534 to 2595, 2678 to 2820, 3087 in SEQ ID NO: 1. ~ 3165th, 3618 ~ 3741st, 4082 ~ 4229th, 4700 ~ 4884th, 5408 ~ 5541st, 6100 ~ 6578th, 6696 ~ 6856th, 7123 ~ 7215th, 7320 ~ 7392th, and 7573 ~ 7706th It is represented by the base sequence. Therefore, for example, the first base of exon 3 (the 386th base g in SEQ ID NO: 1) is expressed as c.68 according to the notation of nucleotide numbering coding DNA Reference Sequence. In the case of an intron, it is shown how many times after the last base of the immediately preceding exon, or how long before the first base of the immediately following exon. For example, the 10th base from the end of intron 2 (the 376th base c in SEQ ID NO: 1) is expressed as c.68-10.
また、本発明において、ＢＡＰ１遺伝子中の塩基は、特に明記しない限り、nucleotide numbering coding DNA Reference Sequenceを参照配列として特定した塩基を示す。従って、被験者の検体におけるＢＡＰ１遺伝子に、本発明で食道癌の発症リスク判定の指標とする変異以外にも１個又は複数の塩基の付加、欠失、置換等の変異が存在する場合も、配列番号１を参照配列として特定した遺伝子座にある塩基に対応する塩基を示す。例えば、検体のＢＡＰ１遺伝子において配列番号１でいう４０５９〜４０８１番目の塩基配列の領域に１個又は複数の塩基の付加、欠失等があっても、「検体のＢＡＰ１におけるnucleotide numbering coding DNA Reference Sequenceの表記法でc.784-26〜c.784-23の塩基配列」とは、配列番号１でいう４０５６〜４０５９番目のctctに対応する塩基配列を意味する（図６）。
Further, in the present invention, the base in the BAP1 gene indicates a base specified using the nucleotide numbering coding DNA reference sequence as a reference sequence unless otherwise specified. Therefore, the sequence of the BAP1 gene in the subject sample also includes a mutation such as addition, deletion or substitution of one or more bases in addition to the mutation used as an index for determining the risk of developing esophageal cancer in the present invention. The base corresponding to the base at the gene locus identified with the
ＢＡＰ１遺伝子のイントロン９内の塩基配列ctctとは、ＢＡＰ１遺伝子のイントロン９内の塩基配列tttctcctctgagcc（配列番号２）における７〜１０番目の塩基配列ctctと換言できる。 The base sequence ctct in the intron 9 of the BAP1 gene can be referred to as the 7th to 10th base sequence ctct in the base sequence tttctcctctgagcc (SEQ ID NO: 2) in the intron 9 of the BAP1 gene.
ＢＡＰ１における変異を検出する方法としては、特に限定されず、例えば、サザンブロット法、ノーザンブロット法、ＰＣＲを使用する方法等が挙げられる。ＰＣＲを使用する方法としては、変異を含むオリゴヌクレオチドをプライマーとして用いる方法、ＰＣＲとシークエンスとを組合わせる方法、ＰＣＲ−ＳＳＣＰ法、ＰＣＲ後に制限酵素を利用する方法、対立遺伝子特異的オリゴヌクレオチドプローブ（ＡＳＯ）を利用する方法等が挙げられ、real time ＰＣＲ法、ＰＣＲとシークエンスとを組合わせる方法等が好ましい。 A method for detecting a mutation in BAP1 is not particularly limited, and examples thereof include a Southern blot method, a Northern blot method, a method using PCR, and the like. As a method of using PCR, a method using an oligonucleotide containing a mutation as a primer, a method combining PCR and a sequence, a PCR-SSCP method, a method using a restriction enzyme after PCR, an allele-specific oligonucleotide probe ( ASO) is used, and a real time PCR method, a method combining PCR and a sequence, and the like are preferable.
変異を含むオリゴヌクレオチドをプライマーとして用いるＰＣＲ法を用いる場合、ＢＡＰ１遺伝子中における所定の変異部位を含むオリゴヌクレオチドをプライマーの一方に用いる。そして、当該プライマーを用い、ＢＡＰ１遺伝子が所定の変異部位を有する場合に、ＢＡＰ１遺伝子中の当該変異部位を含む部分が増幅され、かつ、ＢＡＰ１遺伝子が所定の変異部位を有さない場合に当該部分が有意に増幅されないような条件をあらかじめ設定しておく。当該設定された条件で検体をＰＣＲにかけ、ＢＡＰ１遺伝子中の当該変異部位を含む部分が増幅されるか否かで変異の有無を決定する。当該方法においてreal time ＰＣＲ法を用いると、短時間で評価ができ、簡便なため、特に初期のスクリーニング等として有効である。real time ＰＣＲ法を用いる場合には、例えば、ＲＦＵ（相対蛍光単位）等により示される目的のヌクレオチドの濃度が閾値を超えるまでのサイクル数を指標とし、当該サイクル数があらかじめ設定したcut off値よりも小さい場合に変異を有するとし、当該サイクル数があらかじめ設定したcut off値以上の場合もしくは目的のヌクレオチドの濃度が閾値を超えなかった場合に変異を無とする。 When the PCR method using an oligonucleotide containing a mutation as a primer is used, an oligonucleotide containing a predetermined mutation site in the BAP1 gene is used as one of the primers. When the primer is used and the BAP1 gene has a predetermined mutation site, the portion containing the mutation site in the BAP1 gene is amplified, and the BAP1 gene does not have the predetermined mutation site. A condition is set in advance so that is not significantly amplified. The specimen is subjected to PCR under the set conditions, and the presence or absence of mutation is determined by whether or not the portion containing the mutation site in the BAP1 gene is amplified. When the real time PCR method is used in this method, it can be evaluated in a short time and is simple, so it is particularly effective as an initial screening. When using the real time PCR method, for example, the number of cycles until the concentration of the target nucleotide indicated by RFU (relative fluorescence unit) exceeds a threshold is used as an index, and the number of cycles is determined from a preset cut off value. If the number of cycles is greater than or equal to the preset cut-off value, or if the concentration of the target nucleotide does not exceed the threshold value, no mutation is determined.
ＰＣＲとシークエンスとを組合わせる方法としては、所定の変異をはさむ位置にプライマーセットを設計し、当該プライマーを用いて検体をＰＣＲにかけ、増幅したヌクレオチドの塩基配列をシークエンスにより決定する方法が挙げられる。 As a method for combining PCR and sequencing, a primer set is designed at a position sandwiching a predetermined mutation, a sample is subjected to PCR using the primer, and the base sequence of the amplified nucleotide is determined by sequencing.
本発明において、変異の検出に用いるプローブ又はプライマーとして用いられるオリゴヌクレオチドの塩基数は、15〜50塩基、好ましくは、15〜40塩基、より好ましくは18〜23塩基、より好ましくは19〜22塩基である。 In the present invention, the number of bases of an oligonucleotide used as a probe or primer used for detecting a mutation is 15 to 50 bases, preferably 15 to 40 bases, more preferably 18 to 23 bases, more preferably 19 to 22 bases. It is.
各検出方法に用いる検体は、本発明の判定方法の被験対象に由来する。検体は被験対象に由来する血液検体を好適に使用することができ、血液検体には、例えば血液（全血）及び血液に由来する血清、血漿、白血球などが含まれる。本発明の好ましい態様の一つにおいて、検体は全血または白血球である。 The specimen used for each detection method is derived from the test subject of the determination method of the present invention. As the sample, a blood sample derived from a test subject can be preferably used. Examples of the blood sample include blood (whole blood) and blood-derived serum, plasma, leukocytes, and the like. In one preferred embodiment of the present invention, the specimen is whole blood or white blood cells.
検体は、当業者に公知の方法で採取することができる。例えば、血液は、注射器などを用いた採血によって採取することができる。なお、採血は、医師、看護師などの医療従事者が行うことが望ましい。血清は、血液から血球及び特定の血液凝固因子を除去した部分であり、例えば、血液を凝固させた後の上澄みとして得ることができる。血漿は、血液から血球を除去した部分であり、例えば、血液を凝固させない条件下で遠心分離に供した際の上澄みとして得ることができる。白血球は、血液から血清および血餅を除去した部分であり、例えば、全血を遠心分離し、中間層としてのbuffy coatとして得ることができる。検体はこれらのものに限られず、公知の方法により全血から抽出されたgenomic DNAも対象となり得る。検体は、被験対象の動脈由来、静脈由来、末梢血管由来のいずれであってもよい。
上記方法により、食道癌の発症リスクが存在すると判定された被験者には、上記ＢＡＰ遺伝子の変異を検出する方法以外の方法で食道癌の診断を行うことが有効である。例えば、上部消化管内視鏡検査、 上部消化管透視（バリウム）検査等の診断方法を行うことが有効であり、これらの診断方法を定期的に行うことにより食道癌の早期発見が期待できる。
従って、本発明は、被験者のＢＡＰ１遺伝子における変異を検出し、該変異が検出された被験者を食道癌の発症リスクが存在すると判定する、食道癌の発症リスクを判定する工程、及び
前記工程により、食道癌の発症リスクが存在すると判定された被験者に、食道癌を発見するための診断を行う工程
を含む、食道癌の診断方法も提供する。The specimen can be collected by a method known to those skilled in the art. For example, blood can be collected by blood collection using a syringe or the like. Note that blood collection is preferably performed by medical personnel such as doctors and nurses. Serum is a part obtained by removing blood cells and a specific blood coagulation factor from blood, and can be obtained, for example, as a supernatant after coagulating the blood. Plasma is a portion obtained by removing blood cells from blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not coagulate blood. The leukocyte is a portion obtained by removing serum and blood clot from blood. For example, whole blood can be centrifuged to obtain a buffy coat as an intermediate layer. The specimen is not limited to these, and genomic DNA extracted from whole blood by a known method can also be a target. The specimen may be derived from the subject's arteries, veins, or peripheral blood vessels.
It is effective to diagnose esophageal cancer by a method other than the method for detecting a mutation in the BAP gene for a subject who has been determined by the above method to be at risk of developing esophageal cancer. For example, it is effective to perform diagnostic methods such as upper gastrointestinal endoscopy and upper gastrointestinal fluoroscopy (barium) examination, and early detection of esophageal cancer can be expected by regularly performing these diagnostic methods.
Therefore, the present invention detects a mutation in the BAP1 gene of a subject, determines that the subject in which the mutation is detected is at risk of developing esophageal cancer, and determines the risk of developing esophageal cancer, Also provided is a method for diagnosing esophageal cancer, comprising the step of conducting a diagnosis for discovering esophageal cancer in a subject determined to be at risk of developing esophageal cancer.
食道癌の発症リスクを判定するためのキット
本発明は、ＢＡＰ１遺伝子における変異を検出するための手段を含む、食道癌の発症リスクを判定するためのキットを提供する。 Kit for determining risk of developing esophageal cancer The present invention provides a kit for determining the risk of developing esophageal cancer, comprising means for detecting a mutation in the BAP1 gene.
ＢＡＰ１遺伝子における変異を検出するための手段としては、特に限定されないが、例えば、前述したサザンブロット法、ノーザンブロット法、ＰＣＲを使用する方法等に用いる手段を挙げることができる。好ましくは、当該ＢＡＰ１遺伝子における変異を検出するための手段には、プライマー又はプローブとして、変異を有するＢＡＰ１遺伝子における当該変異部位を含むオリゴヌクレオチドが含まれ得る。 The means for detecting a mutation in the BAP1 gene is not particularly limited, and examples thereof include means used in the aforementioned Southern blot method, Northern blot method, PCR method and the like. Preferably, the means for detecting a mutation in the BAP1 gene may include an oligonucleotide containing the mutation site in the BAP1 gene having a mutation as a primer or a probe.
また、本発明のキットには、必要に応じて他の成分を含めることができる。他の成分は、例えば検体を採取するための道具（例えば、注射器等）、ポジティブコントロール試料及びネガティブコントロール試料などが挙げられるが、これに限定されない。上記判定方法を行うための手順を書き記した書面等を含むこともできる。 The kit of the present invention can contain other components as necessary. Examples of other components include, but are not limited to, a tool for collecting a specimen (for example, a syringe), a positive control sample, a negative control sample, and the like. It can also include a document in which a procedure for performing the above determination method is written.
実施例１
本発明者は、倫理委員会承認の元に（承認番号；2005-124、2008-31、2011-80、2011-432、2012-442）食道扁平上皮癌患者69名の切除標本（腫瘍部および正常粘膜部）からgenomic DNAを抽出し研究を行ってきた。具体的には、直接シークエンス法により、上記末梢血白血球由来または切除標本中のBAP1遺伝子中の変異を調べた。Example 1
The present inventor has obtained an excised specimen of 69 patients with squamous cell carcinoma of the esophagus (oncology and tumor) with approval by the Ethics Committee (approval numbers; 2005-124, 2008-31, 2011-80, 2011-432, 2012-442). We have been researching genomic DNA extracted from normal mucosa. Specifically, mutations in the BAP1 gene in the peripheral blood leukocytes or in the excised specimen were examined by direct sequencing.
この研究の結果、これまでに報告の無い、BAP1遺伝子のイントロン領域にある２塩基欠失を発見した（図１）。これはイントロン９に存在し、イントロン９とエクソン１０のexon-intron junctionの上流２３塩基のCTの欠失である。尚、２５塩基上流のCTまたは２４塩基上流のTCの欠失でも結果として同じ塩基配列になるため、これら３種類のいずれかの欠失が生じていることが分かる。 As a result of this study, we discovered a 2-base deletion in the intron region of the BAP1 gene that has not been reported so far (FIG. 1). This is a deletion of the CT of 23 bases upstream of the exon-intron junction between intron 9 and exon 10. In addition, since deletion of CT of 25 bases upstream or TC of 24 bases upstream also results in the same base sequence, it can be seen that any one of these three types of deletions has occurred.
実施例２ real time PCR法によるスクリーニング法の確立
real time PCR法（Biorad社）を応用した迅速で簡便なスクリーニング法を確立した。具体的には、実施例１で示した２塩基欠失に特異的に反応するPCRプライマー（5’-GGGTCTACCCTTTCTCCTGA-3’（配列番号３）及び5’-GTCCACAAGAGGTCCCAAAC-3’（配列番号４））を作成し、この変異を有するＤＮＡ（ポジティブコントロール、Ｎｏ．１２２の検体）又はこの変異を有さない正常配列のDNA（Ｎｏ．１７０の検体）をtemplateとして、アニール温度を６０℃から７５℃まで変動させて、realtimePCR解析をおこなった。その結果、ＲＦＵが閾値を超えるまでのサイクル数を３０回でcut offとするとアニール温度が69℃で変異ありのDNAで陽性、変異無しの正常塩基配列のDNAで陰性の結果を得た。なお、このスクリーニング方法が有用であることは直接シークエンス法によって確認することが出来た。すなわち、下記実施例３において2例のPCR陽性例ではいずれも当該2塩基欠失を確認し、PCR陰性のうち，ランダムに抽出した29例はいずれも正常の塩基配列であった。Example 2 Establishment of screening method by real time PCR
A rapid and simple screening method applying the real time PCR method (Biorad) was established. Specifically, PCR primers (5′-GGGTCTACCCTTTCTCCTGA-3 ′ (SEQ ID NO: 3) and 5′-GTCCACAAGAGGTCCCAAAC-3 ′ (SEQ ID NO: 4)) that react specifically with the 2-base deletion shown in Example 1 And using the DNA having this mutation (positive control, No. 122 specimen) or the normal sequence DNA not having this mutation (No. 170 specimen) as a template, annealing temperature from 60 ° C. to 75 ° C. Realtime PCR analysis was carried out with variation. As a result, when the number of cycles until the RFU exceeded the threshold value was cut off at 30 times, the annealing temperature was 69 ° C., positive for the DNA with mutation, and negative for the normal base sequence DNA without mutation. The usefulness of this screening method could be confirmed by direct sequencing. That is, in Example 3 below, the two PCR-positive cases were confirmed to have the 2-base deletion, and among the PCR-negative cases, 29 cases extracted at random were normal base sequences.
なお，このRealtimePCRの詳細は以下の通りである：
反応液：１０μｌ
反応液組成：テンプレートＤＮＡ20ｎｇ（ナノグラム）、プライマーＤＮＡ（２０microM）各々0.25μL、SsoAdvanced SYBR Green Supermix 5 microL（Bio-rad 社製）
温度設定：
1: 95.0℃ for 3:00
2: 95.0℃ for 0:01
3: 69.0℃ for 0:30
Plate Read
4: GOTO 2, 39 more times
実施例３
上記条件に従い、実施例１及び２で検査したのとは別の食道扁平上皮癌患者２１７例（各検体をＳＴ１〜ＳＴ２１７とする）の末梢血白血球由来のgenomic DNAをスクリーニングした。Real time PCRの結果を図２に示す。その結果、上記ポジティブコントロールとしたＮｏ．１２２の検体と、ほぼ同じサイクル数でＳＴ２８とＳＴ１６６で陽性の結果を得た。尚、この２例についてはdirect sequence法により、２塩基欠失を持つことを確認した。食道扁平上皮癌患者２１７例中、ＳＴ２８及びＳＴ１６６以外の２１５例については、Real time PCRの結果、陰性であった。陰性例はランダムに選択した29例で同様にシークエンスを行い、２塩基欠失を持たないことを確認した。The details of this RealtimePCR are as follows:
Reaction solution: 10 μl
Composition of reaction solution:
Temperature setting:
1: 95.0 ℃ for 3:00
2: 95.0 ℃ for 0:01
3: 69.0 ℃ for 0:30
Plate Read
4:
Example 3
According to the above conditions, genomic DNA derived from peripheral blood leukocytes of 217 patients with squamous cell carcinoma of the esophagus (each sample is designated as ST1 to ST217) different from those examined in Examples 1 and 2 was screened. The result of Real time PCR is shown in FIG. As a result, no. Positive results were obtained in ST28 and ST166 at approximately the same number of cycles as 122 samples. These two cases were confirmed to have a 2-base deletion by the direct sequence method. Of 217 patients with squamous cell carcinoma of the esophagus, 215 cases other than ST28 and ST166 were negative as a result of Real time PCR. Negative cases were selected from 29 randomly selected sequences, and it was confirmed that there were no 2-base deletions.
実施例４
前述の実施例１〜３に示すように、食道扁平上皮癌患者２８６名中、４名において、イントロン９とエクソン１０のexon-intron junctionの上流２３塩基のCTの欠失が見られた。Example 4
As shown in Examples 1-3 above, in 286 patients with squamous cell carcinoma of the esophagus, CT deletion of 23 bases upstream of the exon-intron junction of intron 9 and exon 10 was observed.
一方、米国政府機関（National Center for Biotechnology Information, U.S. National Library of Medicine. 8600 Rockville Pike, Bethesda MD, 20894 USA、以下NCBI）の発行する遺伝子多型の検索サイト（http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=SNP&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome）で検索したが、この変異の報告は正常人1089名ではない。この情報から統計学的解析を行うとChi二乗検定ではp<0.001、Fisherの正確法検定ではp=0.002で有意に食道扁平上皮癌患者と相関があるとの結果であった。 On the other hand, a genetic polymorphism search site (http: //blast.ncbi.nlm) issued by a US government agency (National Center for Biotechnology Information, US National Library of Medicine. 8600 Rockville Pike, Bethesda MD, 20894 USA, NCBI). .nih.gov / Blast.cgi? PROGRAM = blastn & BLAST_SPEC = SNP & BLAST_PROGRAMS = megaBlast & PAGE_TYPE = BlastSearch & SHOW_DEFAULTS = on & LINK_LOC = blasthome). Statistical analysis of this information showed that the Chi-square test was significantly correlated with patients with squamous cell carcinoma of the esophagus at p <0.001 and Fisher's exact test at p = 0.002.
以上の結果から、この遺伝子変異（あるいは多型）を上記に示した迅速かつ簡便な方法で末梢血白血球由来のgenomic DNAで検出することにより、将来の食道扁平上皮癌発生のリスクを明らかにすることが出来ることが分かる。高リスクと診断されれば、内視鏡検査などにより早期発見が可能となり、その結果として、リスクが高く、また術後の愁訴も極めて多い食道切除術の回避、あるいは副作用のリスクも高く、そのための支持療法や抗がん薬や分子標的薬に代表される高額医薬品による医療費の高騰に繋がる放射線化学療法の抑制も可能となる。個人および社会にとって，極めて意義の大きい発見であると考えられる。 Based on the above results, the risk of developing future esophageal squamous cell carcinoma is clarified by detecting this genetic mutation (or polymorphism) with genomic DNA derived from peripheral blood leukocytes using the rapid and simple method described above. I understand that I can do it. If it is diagnosed as high risk, it can be detected early by endoscopy, etc., and as a result, there is a high risk and avoidance of esophagectomy with many postoperative complaints, or a high risk of side effects. It is also possible to suppress radiation chemotherapy that leads to a rise in medical costs due to supportive treatment of drugs and high-priced drugs represented by anticancer drugs and molecular targeted drugs. This is a very significant discovery for individuals and society.
実施例５
前述のイントロン９における変異は、Genomeにおける位置からはsplicingに影響を与える可能性が予想される。これを直接、評価するために、functional splicing assay法を使用し（図３ および参考文献；Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay Hiromu Naruse et al. Familial Cancer DOI 10.1007/s10689-009-9280-6 ）、このイントロン内の２塩基欠失の生物学的意義を評価した。上記文献に沿って、functional splicing assayを行い、電気泳動で評価したところ、２塩基欠失を導入した場合、正常の塩基配列をもつプラスミドに比べて、エクソン１０の転写効率が著しく低下することが示された（図４及び５）。
以上から病的な意義、すなわち、２塩基欠失ではエクソン１０の転写効率が低下しているために食道扁平上皮癌発癌の可能性が高まっていると考えられる。Example 5
The aforementioned mutation in intron 9 is expected to affect splicing from the position in Genome. To directly evaluate this, we used a functional splicing assay (Figure 3 and references; Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay Hiromu Naruse et al. Familial Cancer DOI 10.1007 / s10689-009-9280-6), the biological significance of a two base deletion in this intron was evaluated. When functional splicing assay is performed according to the above-mentioned literature and evaluated by electrophoresis, the transcription efficiency of exon 10 may be remarkably reduced when a 2-base deletion is introduced compared to a plasmid having a normal base sequence. (Figures 4 and 5).
From the above, it is considered that the possibility of carcinogenesis of esophageal squamous cell carcinoma is increased because the transcriptional efficiency of exon 10 is reduced due to pathological significance, that is, deletion of 2 bases.
食道癌を含め癌の早期発見に対する要望は非常に大きいところ、本発明は、新規な食道癌リスクの判定方法を提供するものであるため、産業利用上、非常に有用である。 Since there is a great demand for early detection of cancer including esophageal cancer, the present invention provides a novel method for determining the risk of esophageal cancer, and thus is very useful for industrial use.
配列番号３はプライマーである。 SEQ ID NO: 3 is a primer.
配列番号４はプライマーである。 SEQ ID NO: 4 is a primer.
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