JP6243342B2 - 上皮増殖因子受容体キナーゼ・ドメイン内の新規な複合突然変異 - Google Patents
上皮増殖因子受容体キナーゼ・ドメイン内の新規な複合突然変異 Download PDFInfo
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Description
本発明は、がんの診断と、がんを治療するためのコンパニオン診断に関する。特に本発明は、診断と予後予測に役立つとともに、がん治療の有効性を予測するのに役立つ突然変異の検出に関する。
上皮増殖因子受容体(EGFR)はHER1またはErbB1としても知られており、増殖因子受容体の1型チロシンキナーゼ・ファミリーのメンバーである。これらの膜結合タンパク質は、さまざまなシグナル伝達経路と相互作用する細胞内チロシンキナーゼ・ドメインを有する。このファミリーの受容体はリガンドが結合すると二量体化し、それに続いてチロシンキナーゼ・ドメインの自己リン酸化が起こる。この自己リン酸化によって細胞内シグナル伝達経路(例えばRas/MAPK経路、PI3K経路、AKT経路)でイベントのカスケードが開始される。HERファミリーのタンパク質は、これらの経路を通じて細胞の増殖、分化、生存を調節している。
一実施態様では、本発明は、配列番号1の中の2236_2248>ACCC、2237_2244>CGCCC、2252_2277>AC、2240_2264>CGAAAGA、2239_2240TT>CC、2264C>Aからなるリストから選択した突然変異を含む核酸に特異的にハイブリダイズするオリゴヌクレオチドである。
定義
開示内容を理解しやすくするため、この明細書で使用する表記、用語に関して以下のように定義する。
選択的プライマー=突然変異した核酸だけを増幅し、野生型核酸は増幅しないプライマー
E=N4-t-ブチル-ベンジル-dC
F=N6-t-ブチル-ベンジル-dA
M=FAM
Q=BHQ2
P=リン酸
肺がん患者サンプル中の突然変異の同定
組織サンプルを肺がん(NSCLC)患者から取得した。サンプルは、ホルマリン固定パラフィン包埋組織(FFPET)として保管した。核酸をサンプルから単離し、ゲノム・シークエンサFLX装置(454 Life Sciences社、ブランフォード、コネティカット州)で直接シークエンシングした。
対立遺伝子特異的オリゴヌクレオチドを用いた突然変異の検出
この実施例では、突然変異標的と野生型標的のそれぞれを、それぞれ突然変異インサートと野生型インサートを含むプラスミドによって代表させた。標的は、突然変異特異的プライマーを用いて増幅するか、非選択的“共通”プライマー(対照反応の場合)を用いて増幅した。
Claims (7)
- ヒトに由来するサンプル中の上皮増殖因子受容体(EGFR)遺伝子内の1以上の突然変異を検出するための突然変異特異的オリゴヌクレオチドの使用であって、該オリゴヌクレオチドが、配列番号1の中の2236_2248>ACCC、2237_2244>CGCCC、2252_2277>AC、及び2264C>Aからなるリストから選択した突然変異を含む核酸に特異的にハイブリダイズし、ここで、2236_2248>ACCCの突然変異を含む標的核酸に特異的にハイブリダイズするオリゴヌクレオチドは、配列番号23〜25で表される核酸配列から選択され、2237_2244>CGCCCの突然変異を含む標的核酸に特異的にハイブリダイズするオリゴヌクレオチドは、配列番号28〜30で表される核酸配列から選択され、2252_2277>ACの突然変異を含む核酸に特異的にハイブリダイズするオリゴヌクレオチドは、配列番号17〜20で表される核酸配列から選択され、及び、2264C>Aの突然変異を含む標的核酸に特異的にハイブリダイズするオリゴヌクレオチドは、配列番号33〜35で表される核酸配列から選択され、当該突然変異は肺癌の診断を補助するために使用される、使用。
- ヒトに由来するサンプル中の上皮増殖因子受容体(EGFR)遺伝子内の突然変異を検出する方法であって、
(a)前記サンプル中の核酸を、配列番号1の中の2236_2248>ACCC、2237_2244>CGCCC、2252_2277>AC、及び2264C>Aからなるリストの中から選択した突然変異を含む標的核酸と選択的にハイブリダイズすることのできる突然変異特異的オリゴヌクレオチドと接触させるステップ、ここで、該突然変異特異的オリゴヌクレオチドは、
配列番号1の中の2252_2277>ACの突然変異を含む核酸に特異的にハイブリダイズする配列番号17〜20で表される核酸配列から選択され、配列番号1の中の2236_2248>ACCCの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号23〜25で表される核酸配列から選択され、配列番号1の中の2237_2244>CGCCCの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号28〜30で表される核酸配列から選択され、又は、配列番号1の中の2264C>Aの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号33〜35で表される核酸配列から選択される;
(b)前記標的核酸への前記オリゴヌクレオチドの選択的なハイブリダイゼーションが可能な条件下で前記サンプルをインキュベートするステップ;
(c)前記ハイブリダイゼーションを検出するステップ、
を含み、当該突然変異は肺癌の診断を補助するために使用される、方法。 - ステップ(b)の後に前記標的核酸を増幅する操作をさらに含んでいて、ステップ(b)のハイブリダイゼーションにより、選択的にハイブリダイズする前記オリゴヌクレオチドが伸長する、請求項2に記載の方法。
- ステップ(c)における検出を各回の増幅中に実施する、請求項2または3に記載の方法。
- ステップ(c)における検出を最終回の増幅の後に実施する、請求項2または3に記載の方法。
- 配列番号1の中の2252_2277>ACの突然変異を含む核酸に特異的にハイブリダイズする配列番号17〜20で表される核酸配列から選択されるオリゴヌクレオチド、配列番号1の中の2236_2248>ACCCの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号23〜25で表される核酸配列から選択されるオリゴヌクレオチド、配列番号1の中の2237_2244>CGCCCの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号28〜30で表される核酸配列から選択されるオリゴヌクレオチド、又は、配列番号1の中の2264C>Aの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号33〜35で表される核酸配列から選択されるオリゴヌクレオチドを含み、肺癌の診断を補助するために使用される、ヒトEGFR遺伝子内の突然変異を検出するためのキットであって、場合により核酸前駆体、核酸ポリメラーゼ、及び核酸ポリメラーゼの活性をサポートするのに必要な試薬及び溶液をさらに含む、キット。
- 配列番号1の中の2252_2277>ACの突然変異を含む核酸に特異的にハイブリダイズする配列番号17〜20で表される核酸配列から選択されるオリゴヌクレオチド、配列番号1の中の2236_2248>ACCCの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号23〜25で表される核酸配列から選択されるオリゴヌクレオチド、配列番号1の中の2237_2244>CGCCCの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号28〜30で表される核酸配列から選択されるオリゴヌクレオチド、又は、配列番号1の中の2264C>Aの突然変異を含む標的核酸に特異的にハイブリダイズする配列番号33〜35で表される核酸配列から選択されるオリゴヌクレオチドを含み、場合により核酸前駆体、核酸ポリメラーゼ、及び核酸ポリメラーゼの活性をサポートするのに必要な試薬及び溶液をさらに含む、反応混合物であって、当該突然変異は肺癌の診断を補助するために使用される、反応混合物。
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