JP2004531228A - Estrogen receptor alpha mutant and method for detecting the same - Google Patents

Abstract

The present invention is based on the genomic DNA sequence from human chromosome 6 and cDNA, which defines the genomic structure of the estrogen receptor alpha gene and the novel polymorphism / haplotype of the estrogen receptor gene / protein. Such polymorphisms / haplotypes can cause a variety of abnormalities mediated / modulated by mutant estrogen receptors, such as susceptibility to cancer, osteoporosis, cardiovascular disease, and the like. Based on this sequence approach, the present invention provides genomic nucleotide, cDNA, amino acid, and polymorphism / haplotype sequences for the estrogen alpha gene, methods for detecting these sequences / polymorphisms / haplotypes in samples, mutant estrogen Methods are provided for determining the risk of having or developing a receptor-mediated disorder, and for screening compounds used to treat a mutant estrogen receptor-mediated disorder.

Description

【０２１１】
【表２】
臨床サンプルにおける異型接合体の変化の要約：ＳＮＰは頻度２以上、特性スコア２０以上であった（図８参照）。

【０２１２】
図２（ｃ）は、リバプールコントロール群対血液又は腫瘍群における分析ＳＮＰを含んだ。
図５は、ＥＳＲ１タンパクのドメイン構造及びここに開示された多くのＳＮＰ位置を示す。図６は、被験サンプル中のＳＮＰ及び発生頻度のグラフ図である。
図８（ａ）（１）は、北欧州人、中国人、インド−パキスタン人、アフリカ系アメリカ人、及び南西部ネイティブアメリカ人の民族グループから集めたコリエールサンプル中のＳＮＰ及び発生頻度を示す。３’フランキングエキソン８において、ＳＮＰの位置は、遺伝子バンクにあるＡＬ０７８５８２の配列に基づく。結果は、特定の民族グループ間での検出のために使用可能である。図８（ａ）（２）は、コリエールパネル中の部位１６７９８９（イントロン１Ｄ，＃９）のＳＮＰのゲノタイピングを示す。このパネルは、１００名のコーカサス人を含み、５１名は男性で、４９名は女性である。結果は、マイナー対立遺伝子の頻度が９％であることを示す。
【０２１３】
図８（ｂ）（１）は、北欧人からの血液サンプル及び乳ガンサンプルのグループから選択したリバプールサンプルにおけるＳＮＰ及び発生頻度を示す。
図８（ｂ）（２）は、追加のリバプール患者６０名中のＳＮＰ部位１６７，９８９（イントロン１Ｄ，＃９）のタックヒトゲノタイピング（Ｔａｑ Ｍａｎ ｇｅｎｏｔｙｐｉｎｇ）の結果を示す。この結果は、マイナー対立遺伝子（Ｇ）の頻度が血液中１４％、腫瘍中１０％であったことを示す。これは、図８（ｂ）（１）に示されるコリエールサンプルのＳＮＰゲノタイピングと同様であり、マイナー対立遺伝子での頻度は血液中１６％、腫瘍中１７％であった。リバプールコントロール群（９５のケース）は、マイナー対立遺伝子での頻度は１０．５％であったことを示す（図２（ｃ））。
【０２１４】
２．ハプロタイプ分析
ＳＮＰ発見のために開発された方法は、ハプロタイプデータを得るよう設計された。ＳＮＰは、特定のハプロタイプに関連させることが可能であった。サンプルｃＤＮＡは、未知の比率で存在するランダム群から得た。特定のクローンからのＳＮＰはハプロタイプ中にクラスター化され、作られた。
【０２１５】
データは、２つのサンプルタイプからなる（図４（ａ）及び（ｂ））。リバプールサンプルは、４８名の患者からのものであり、各患者は腫瘍及び対応する血液サンプルを有した。コリエールサンプルは、コントロールであったが、それらは対応するコントロールではなかった。むしろ、それらは、欧州人、中国人、インド−パキスタン人、及びアフリカ系アメリカ人の混合に含まれた。ＥＳＲ１における４６のＳＮＰは、リバプールサンプルにおいて記録された。同じ４６のＳＮＰと、ＲＳＲ１遺伝子の３’の追加の６のＳＮＰが、コリエールサンプルにおいて記録された。
【０２１６】
これらのデータは個体の最も適当なハプロタイプフェーズを推測するための分析に供した。その結果は、図４（ａ）に示されているが、各ハプロタイプはナンバーを有し、ダッシュのついたナンバーはそのハプロタイプのカウント（又は頻度）である。
図４（ｂ）は、近接接合樹形図に適合させた非単独ハプロタイプデータである。樹形図が矢印のところで切断されている場合には、３Ｌ， １０−４， …７３Ｌを含む分岐は樹形図の残部から「分岐Ｘ」として区画される。下記に分岐Ｘ対樹形図残部のハプロタイプにおける腫瘍発生率の違いを示す。各ハプロタイプの発生率は、まずダッシュのついたナンバーを足すことによりカウントした。Ｌは腫瘍性リバプールサンプルを示し、Ｌはコリエールコントロールを示す。
【０２１７】

Ｘ^２は、２×２の表に基づき０．０００１より小さな確率を有する自由度で、２１．２９と計算された。従って、ＥＲ１遺伝子の分岐Ｘは、腫瘍に関連するより多くの機会を有する。この全分岐は、世界の他の場所では大変まれである。欧州人の間でさえ、２０のハプロタイプからたった１つしか存在しなかった。
【０２１８】
コリエールコントロール、リバプールコントロール、リバプール腫瘍サンプルの間の大きな頻度差で分岐を特定した部位は以下の通り。

【０２１９】
３ ． エストロゲン受容体１のプロモーター領域及びＳＮＰ
ＣＡＡＴ及びＴＡＴＡのボックスは配列の最初の２００ ｂ．ｐ．にあり、これらの距離は、基礎プロモーターとして機能的であるかもしれないと考えられる。
ＣＡＡＴ−ＴＡＴＡ部位間の距離及び実際に特定された転写の開始は、約３００ ｂ．ｐ．であり（通常は約２０−４０ ｂ．ｐ．）、この領域はＮＦＡＴ， ＮＦＫＢ， ＳＰ１−ファミリー， ＣＥＢＰ 及び ＥＢＯＸ等のマルチプルＴＦ領域を含む。これらの大部分は特異の（組織、細胞型）の発現の調節に関係した。
【０２２０】
Ｔ（ＳＮＰ）の領域は、興味深い性質を有する。それは維持（ＴＣ）６−繰り返しを有するだけでなく、５−プライムに（ＣＡ）６も有する。維持モチーフは同じ領域のＣＡＣＡＹＴＣＴＣである。公知のＴＦ部位からこの領域に顕著に合うものはなく、それは新規なＴＦ部位のようである。Ｔ（ＳＮＰ）に大変近いのはアリール炭化水素／Ａｒｎｔヘテロ二量体に関するＡＨＲＡＲＮＴ＿０２と呼ばれるＴＦ部位である。ＣＡＣＡＹＴＣＴＣ部位は、コファクターの結合点か、Ａｒｎｔ複合体を正しく配置するのを助けるものである。ＴＦ結合部位における単独の突然変異は、タンパク結合の親和性を数倍低下させ、疾患経路に導くかもしれない。
【０２２１】
本発明において、ＳＮＰはちょうど、短いＧＡの繰り返し（ＧＡＧＡＧＡＧＡ）中のエキソン１Ｃの１３ｂｐ上流に起こる。分岐（図４）中の関連ＳＮＰのうち、３つはサイレント突然変異であり、１つは３’ＵＴＲ中にあり、残りはイントロン領域中にある。
重要なＴからＧのＳＮＰ（＃９）は、プロモーター領域のサイト１６７９８９（イントロン１Ｄ）中にある（図２（ａ），（ｂ），（ｃ））。遺伝子コピーの欠損は、ガンの危険性に関連する。遺伝子発現を減じるプロモーター突然変異は、同様の影響を及ぼす。
【０２２２】
ＧからＴのトランスバージョンは、交互のプロモーター部位中にある。エストロゲン生理学、おそらくは核でのシグナル伝達の全体的なレベルに対してある効果がある。関連した分岐（及び分岐のハプロタイプ）（図４）の「効果」は、全体的なエストロゲン受容体感受性及び反応性を増加させること、又はそのサイクルが非調節（非生理学的）成長シグナルの方向である別の成長過程の方に誘導可能なことであると予測される。エストロゲンの露出は、事実すべての疫学的研究において乳ガンの原因に重大な役割を果たす。
【０２２３】
さらに、ＴＦ結合部位とＳＮＰは、下記のように、５のＳＮＰが転写結合部位に生じたＥＳＲ１遺伝子中に検出された。

【０２２４】
４ ． ＥＳＲ１ ゲノム配列決定−エストロゲン受容体アルファの完全ゲノム構造
エストロゲン受容体（ＥＲ）は、核ホルモン受容体遺伝子スーパーファミリーの１つである。この遺伝子のファミリーは、３つの別個のドメイン、すなわち、変異（Ｎ）−末端ドメイン、高度維持ＤＮＡ結合ドメイン及び維持（Ｃ）−末端ドメインのモジュラー構造を特徴とする（参考文献１，２参照）。機能的には、（Ｎ）−末端ドメインはトランス活性化を、ＤＮＡ結合ドメインは二量体化及びＤＮＡ結合を、（Ｃ）−末端ドメインはトランス活性化、二量化、リガンド結合、核トランスロケーション、サイレンシング、及び熱ショックタンパク結合を、それぞれ調節する。核ホルモン遺伝子スーパーファミリーの各ドメインの機能は、それらが見つかる受容体に無関係で、ドメインは異なる異型タンパク中に位置する場合であっても、その機能を保有する（参考文献３、４、５参照）。これら遺伝子の主なサブファミリーは、進化の初期に単純な遺伝子複製を通じて進化したので、核ホルモン受容体遺伝子スーパーファミリーのドメインモジュール性が存在する（参考文献６）。核ホルモン受容体遺伝子ファミリーは、２つの異なる分類スキームに従って分離することができる。一方はホルモン結合に基づき、他方は二量化と、受容体のＤＮＡ応答エレメントへの結合方法に基づく（参考文献２参照）。
【０２２５】
ＥＲαのｃＤＮＡは、まず、ＭＣＦ−７乳ガン細胞系列からクローン化及びシークエンスし、ｖ−ｅｒｂ−Ａ 遺伝子に対して２７％の同一性と４１％の保存性を有することが判明した（参考文献７）。ＥＲαは、蛍光ｉｎ ｓｉｔｕハイブリダイゼーション（ＦＩＳＨ）及び染色体結合を用いて染色体６ｑ２５．１にマップした（参考文献８）。１９９６年に、新規なエストロゲン受容体（ＥＲβ）が縮重ＰＣＲにより特定され（参考文献９）、ＦＩＳＨにより１４ｑ２２−２４にマップされた（参考文献１０）。ＥＲα及びＥＲβは、ＤＮＡ結合ドメイン中９６％の配列同一性と、リガンド結合ドメイン中５８％の同一性を有し、ヒンジ（ｈｉｎｇｅ）（ドメインＤ）と同様に５’及び３’末端においては同一性が低いことが示された。様々なＥＲα及びＥＲβ変異体は、これまで単一及び複数のエキソン欠損、切断転写、及び挿入を含む転写物を含め記載されてきた（参考文献１１，１２，１３）。これらの変異体は、正常組織、腫瘍組織及び細胞系列を含む様々な起源から単離された。乳ガン患者の腫瘍のＥＲ状態は、内分泌療法に対する応答のインジケータとして用いられ（参考文献１４，１５）、多くの研究により乳ガン腫瘍の進行、ＥＲ−ネガティブ状態、及びホルモンアンタゴニスト抵抗性におけるＥＲの役割が検討されてきた（参考文献１６参照）。
【０２２６】
ＥＲ遺伝子の重要性から、我々はその全体をクローン化し、その完全な構造を決定しようとした。初めに、標準細菌人工染色体（ＢＡＣ）配列決定を用いて遺伝子のコーディング領域の配列情報を発生させた。セレーラのヒトゲノム配列決定が進行したとき、ＥＲの残存領域はセレーラレジオンアセンブリを用いて埋められた。２５ｋｂより小さな領域は、公知のＢＡＣ（Ａｌ３５３６１１．６， ｐｏｓｉｔｉｏｎｓ １，４９７−２５，９４１）を用いてＥＲα上に埋められた。
【０２２７】
材料及び方法
１）ＢＡＣスクリーニング
適当なマーカーがＥＲα及びＥＲβエキソンに設計され、リサーチジェネティクス（Ｈｕｎｔｓｖｉｌｌｅ， ＡＬ）から市販のＢＡＣクローンを得るために用いられた。いくつかのポジティブＢＡＣを選び、それぞれのクローンを確認のために再スクリーニングした。
【０２２８】
２）ＤＮＡの単離及びライブラリーの作製
ＢＡＣのＤＮＡは、ＱＩＡＧＥＮカラム（ＱＩＡＧＥＮ， Ｉｎｃ．， Ｖａｌｅｎｃｉａ， ＣＡ） を用い、メーカー仕様書に従って、確認したクローンから単離した。ショットガンライブラリーは、標準プロトコルに従い作製した（参考文献１７）。簡単に説明すると、単離したＢＡＣのＤＮＡを音波処理、ポリッシュ、及びサイズ分画した。サイズを選択したＤＮＡフラグメントは、その後標準の連結反応技術を用いてｐＵＣ１９中にサブクローン化した。連結したＤＮＡは、エレクトロコンピテント細胞（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｒｏｃｋｖｉｌｌｅ， ＭＤ）中にトランスフォームし、一晩成長させた。
【０２２９】
３）ＤＮＡ配列決定及び注釈
配列決定反応は、ビッグダイターミネーターケミストリー（Ｂｉｇ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ ｃｈｅｍｉｓｔｒｙ， Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ， Ｆｏｓｔｅｒ Ｃｉｔｙ， ＣＡ）を用いて実行し、ＡＢＩ ＰＲＩＳＭ ３７００ ＤＮＡアナライザー （Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）で行った。Ｐｈｒｅｄ （参考文献１８）， Ｐｈｒａｐ 及び Ｃｏｎｓｅｄ （参考文献１９）は塩基呼び出し、アセンブリ及びフィニシングにぞれぞれ使用した。エキソン位置は、クロスマッチを用いて決定し、公表された遺伝子配列をゲノムのコンティグと比較した。
【０２３０】
結果
１．エストロゲン受容体アルファ
ＥＲαのゲノム配列及びＥＲαの公表されたｍＲＮＡ配列のアライメントは、その遺伝子が１４エキソンからなり、４４６，２９６ ｂｐのゲノム配列をカバーすることを示す （図７，表３）。
２． エストロゲン受容体ベータ
ＥＲβのゲノム配列及びＥＲβの公表されたｍＲＮＡ配列のアライメントは、その遺伝子が１７エキソンからなり、２５３，７４８ ｂｐのゲノム配列をカバーすることを示す（図２，表１）。セレーラゲノムブラウザでの分析により、遺伝子、ヒトシナプス核発現遺伝子２（ｓｙｎｅ−２， ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ ＮＭ＿０１５１８０．１）、を特定することができた。それは、反対の鎖上でＥＲβのイントロン９内に完全に含まれる。ｓｙｎｅ−２遺伝子のさらなる分析により、それは２１エキソンからなり、５１，４７１ ｂｐのゲノム配列をカバーすることが示された。
【０２３１】
考察
完全なＥＲαゲノム配列と様々なＥＲβ転写物のアライメントは、遺伝子が４４６，２９６ ｂｐのゲノム配列をカバーし、１４エキソンからなることを示す。エキソン１Ｅ（ＡＪ００２５６１） （参考文献２０）の公表配列とＥＲαゲノム配列のアライメントは、エキソン１Ｅが実際には２つの別のエキソンからなることを明らかにした。新しく示されたエキソンは、これまでに確立された命名規約に従い、ここではエキソン１Ｇという。エキソン１Ｇは、エキソン１Ｅの約４５ ｋｂ上流に位置し、ＧＴ／ＡＧ スプラス部位共通配列に一致する（図１，表１）。
【０２３２】
完全なＥＲβゲノム配列に対する様々なＥＲβ転写物のアライメントは、これまで受け入れられていたよりも複雑な体制を示す（参考文献１３）。ＥＲβｃｘ変異体（ＡＢ００６５８９）の５’ＵＴＲは実際には７つの非翻訳エキソン（ここではエキソン−１〜−７という）からなり、これらは全て、ＧＴ／ＡＧスプライス部位共通配列に一致する（図２，表１）。ＥＲβ変異体 ＡＦ０６１０５５及びＡＦ０６１０５４（参考文献１２）の配列アライメントは、これらの転写物が両方ともイントロン配列を含み、おそらく部分的に成熟した転写物であることを示した。これらの部分的成熟転写物は両方ともエキソン７と、エキソン９の一部を含むが、イントロン配列が存在する部位のスプライス部位共通配列には一致していない。
【０２３３】
セレーラゲノムブラウザを用いてＥＲゲノム配列を調べることにより、ＥＲβのイントロン９内に完全に含まれた分離遺伝子を特定することができた。この遺伝子は、ヒトシナプス核発現遺伝子２（ｓｙｎｅ−２）であり、５０Ｋｂのゲノム配列をカバーし、２１のエキソンからなることが示された。これらは全てＧＴ／ＡＧスプライス共通配列に一致する（表４）。ｓｙｎｅ−２遺伝子は、ＥＲβのアンチセンス鎖上に位置する。
ＥＲα及びＥＲβの配列及び構造の完成は、これらの重要な受容体のさらなる理解と特徴づけに寄与する。
【０２３４】
【表３】
ヒトエストロゲン受容体におけるエキソン−イントロン境界及び位置：
エキソン配列は大文字、イントロン配列は小文字、スプライス部位は太字で示す。

【０２３５】
【表４】
ヒトシナプス核発現遺伝子２におけるエキソン−イントロン境界及び位置：
エキソン配列は大文字、イントロン配列は小文字、スプライス部位は太字で示す。

【０２３６】
参考文献
１． Ｒｉｂｅｉｒｏ ＲＣ， Ｋｕｓｈｎｅｒ ＰＪ， Ｂａｘｔｅｒ ＪＤ， Ｔｅｎｂａｕｍ Ｓ， Ｂａｎｉａｈｍａｄ Ａ． Ｔｈｅ ｎｕｃｌｅａｒ ｈｏｒｍｏｎｅ ｒｅｃｅｐｔｏｒ ｇｅｎｅ ｓｕｐｅｒｆａｍｉｌｙ． Ａｎｎｕ Ｒｅｖ Ｍｅｄ １９９５；４６：４４３−５３．
２． Ｔｅｎｂａｕｍ Ｓ， Ｂａｎｉａｈｍａｄ Ａ． Ｎｕｃｌｅａｒ ｒｅｃｅｐｔｏｒｓ： ｓｔｒｕｃｔｕｒｅ， ｆｕｎｃｔｉｏｎ ａｎｄ ｉｎｖｏｌｖｅｍｅｎｔ ｉｎ ｄｉｓｅａｓｅ． Ｉｎｔ Ｊ Ｂｉｏｃｈｅｍ Ｃｅｌｌ Ｂｉｏｌ． １９９７ Ｄｅｃ；２９（１２）：１３２５−４１．
３． Ｂａｎｉａｈｍａｄ Ｃ， Ｂａｎｉａｈｍａｄ Ａ， Ｏ’Ｍａｌｌｅｙ ＢＷ． Ａ ｒａｐｉｄ ｍｅｔｈｏｄ ｃｏｍｂｉｎｉｎｇ ａ ｆｕｎｃｔｉｏｎａｌ ｔｅｓｔ ｏｆ ｆｕｓｉｏｎ ｐｒｏｔｅｉｎｓ ｉｎ ｖｉｖｏ ａｎｄ ｔｈｅｉｒ ｐｕｒｉｆｉｃａｔｉｏｎ． Ｂｉｏｔｅｃｈｎｉｑｕｅｓ． １９９４ Ｆｅｂ；１６（２）：１９４−６．
４． Ｐａｒｋｅｒ ＭＧ， Ｗｈｉｔｅ Ｒ． Ｎｕｃｌｅａｒ ｒｅｃｅｐｔｏｒｓ ｓｐｒｉｎｇ ｉｎｔｏ ａｃｔｉｏｎ． Ｎａｔ Ｓｔｒｕｃｔ Ｂｉｏｌ． １９９６ Ｆｅｂ；３（２）：１１３−５．
５． Ｓｃｈｗａｂｅ ＪＷ． Ｔｒａｎｓｃｒｉｐｔｉｏｎａｌ ｃｏｎｔｒｏｌ： ｈｏｗ ｎｕｃｌｅａｒ ｒｅｃｅｐｔｏｒｓ ｇｅｔ ｔｕｒｎｅｄ ｏｎ． Ｃｕｒｒ Ｂｉｏｌ． １９９６ Ａｐｒ １；６（４）：３７２−４．
【０２３７】
６． Ｌａｕｄｅｔ Ｖ， Ｈａｎｎｉ Ｃ， Ｃｏｌｌ Ｊ， Ｃａｔｚｅｆｌｉｓ Ｆ， Ｓｔｅｈｅｌｉｎ Ｄ． Ｅｖｏｌｕｔｉｏｎ ｏｆ ｔｈｅ ｎｕｃｌｅａｒ ｒｅｃｅｐｔｏｒ ｇｅｎｅ ｓｕｐｅｒｆａｍｉｌｙ． ＥＭＢＯ Ｊ． １９９２ Ｍａｒ；１１（３）：１００３−１３．
７． Ｇｒｅｅｎ Ｓ， Ｗａｌｔｅｒ Ｐ， Ｋｕｍａｒ Ｖ， Ｋｒｕｓｔ Ａ， Ｂｏｒｎｅｒｔ ＪＭ， Ａｒｇｏｓ Ｐ， Ｃｈａｍｂｏｎ Ｐ． Ｈｕｍａｎ ｏｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ ｃＤＮＡ： ｓｅｑｕｅｎｃｅ， ｅｘｐｒｅｓｓｉｏｎ ａｎｄ ｈｏｍｏｌｏｇｙ ｔｏ ｖ−ｅｒｂ−Ａ． Ｎａｔｕｒｅ． １９８６ Ｍａｒ １３−１９；３２０（６０５８）：１３４−９．
８． Ｍｅｎａｓｃｅ ＬＰ， Ｗｈｉｔｅ ＧＲ， Ｈａｒｒｉｓｏｎ ＣＪ， Ｂｏｙｌｅ ＪＭ． Ｌｏｃａｌｉｚａｔｉｏｎ ｏｆ ｔｈｅ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ ｌｏｃｕｓ （ＥＳＲ） ｔｏ ｃｈｒｏｍｏｓｏｍｅ ６ｑ２５．１ ｂｙ ＦＩＳＨ ａｎｄ ａ ｓｉｍｐｌｅ ｐｏｓｔ−ＦＩＳＨ ｂａｎｄｉｎｇ ｔｅｃｈｎｉｑｕｅ． Ｇｅｎｏｍｉｃｓ １９９３ Ｊｕｌ；１７（１）：２６３−５．
９． Ｍｏｓｓｅｌｍａｎ Ｓ， Ｐｏｌｍａｎ Ｊ， Ｄｉｊｋｅｍａ Ｒ． ＥＲ ｂｅｔａ： ｉｄｅｎｔｉｆｉｃａｔｉｏｎ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｎｏｖｅｌ ｈｕｍａｎ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ． ＦＥＢＳ Ｌｅｔｔ． １９９６ Ａｕｇ １９；３９２（１）：４９−５３．
１０． Ｅｎｍａｒｋ Ｅ， Ｐｅｌｔｏ−Ｈｕｉｋｋｏ Ｍ， Ｇｒａｎｄｉｅｎ Ｋ， Ｌａｇｅｒｃｒａｎｔｚ Ｓ， Ｌａｇｅｒｃｒａｎｔｚ Ｊ， Ｆｒｉｅｄ Ｇ， Ｎｏｒｄｅｎｓｋｊｏｌｄ Ｍ， Ｇｕｓｔａｆｓｓｏｎ ＪＡ． Ｈｕｍａｎ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ ｂｅｔａ−ｇｅｎｅ ｓｔｒｕｃｔｕｒｅ， ｃｈｒｏｍｏｓｏｍａｌ ｌｏｃａｌｉｚａｔｉｏｎ， ａｎｄ ｅｘｐｒｅｓｓｉｏｎ ｐａｔｔｅｒｎ． Ｊ Ｃｌｉｎ Ｅｎｄｏｃｒｉｎｏｌ Ｍｅｔａｂ． １９９７ Ｄｅｃ；８２（１２）：４２５８−６５．
【０２３８】
１１． Ｍｕｒｐｈｙ ＬＣ， Ｄｏｔｚｌａｗ Ｈ， Ｌｅｙｇｕｅ Ｅ， Ｄｏｕｇｌａｓ Ｄ， Ｃｏｕｔｔｓ Ａ， Ｗａｔｓｏｎ ＰＨ． Ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ ｖａｒｉａｎｔｓ ａｎｄ ｍｕｔａｔｉｏｎｓ． Ｊ Ｓｔｅｒｏｉｄ Ｂｉｏｃｈｅｍ Ｍｏｌ Ｂｉｏｌ． １９９７ Ａｕｇ；６２（５−６）：３６３−７２．
１２． Ｍｏｏｒｅ ＪＴ， ＭｃＫｅｅ ＤＤ， Ｓｌｅｎｔｚ−Ｋｅｓｌｅｒ Ｋ， Ｍｏｏｒｅ ＬＢ， Ｊｏｎｅｓ ＳＡ， Ｈｏｒｎｅ ＥＬ， Ｓｕ ＪＬ， Ｋｌｉｅｗｅｒ ＳＡ， Ｌｅｈｍａｎｎ ＪＭ， Ｗｉｌｌｓｏｎ ＴＭ． Ｃｌｏｎｉｎｇ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｈｕｍａｎ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ ｂｅｔａ ｉｓｏｆｏｒｍｓ． Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ． １９９８ Ｊｕｎ ９；２４７（１）：７５−８．
１３． Ｏｇａｗａ Ｓ， Ｉｎｏｕｅ Ｓ， Ｗａｔａｎａｂｅ Ｔ， Ｏｒｉｍｏ Ａ， Ｈｏｓｏｉ Ｔ， Ｏｕｃｈｉ Ｙ， Ｍｕｒａｍａｔｓｕ Ｍ． Ｍｏｌｅｃｕｌａｒ ｃｌｏｎｉｎｇ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｈｕｍａｎ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ ｂｅｔａｃｘ： ａ ｐｏｔｅｎｔｉａｌ ｉｎｈｉｂｉｔｏｒ ｏｆ ｅｓｔｒｏｇｅｎ ａｃｔｉｏｎ ｉｎ ｈｕｍａｎ． Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １９９８ Ａｕｇ １；２６（１５）：３５０５−１２．
１４． Ｏｓｂｏｒｎｅ ＣＫ， Ｙｏｃｈｍｏｗｉｔｚ ＭＧ， Ｋｎｉｇｈｔ ＷＡ ３ｄ， ＭｃＧｕｉｒｅ ＷＬ． Ｔｈｅ ｖａｌｕｅ ｏｆ ｅｓｔｒｏｇｅｎ ａｎｄ ｐｒｏｇｅｓｔｅｒｏｎｅ ｒｅｃｅｐｔｏｒｓ ｉｎ ｔｈｅ ｔｒｅａｔｍｅｎｔ ｏｆ ｂｒｅａｓｔ ｃａｎｃｅｒ． Ｃａｎｃｅｒ １９８０ Ｄｅｃ １５；４６（１２ Ｓｕｐｐｌ）：２８８４−８．
１５． ＤｅＳｏｍｂｒｅ ＥＲ， Ｃａｒｂｏｎｅ ＰＰ， Ｊｅｎｓｅｎ ＥＶ， ＭｃＧｕｉｒｅ ＷＬ， Ｗｅｌｌｓ ＳＡ Ｊｒ， Ｗｉｔｔｌｉｆｆ ＪＬ， Ｌｉｐｓｅｔｔ Ｍ Ｓｐｅｃｉａｌ ｒｅｐｏｒｔ． Ｓｔｅｒｉｏｄ ｒｅｃｅｐｔｏｒｓ ｉｎ ｂｒｅａｓｔ ｃａｎｃｅｒ． Ｎ Ｅｎｇｌ Ｊ Ｍｅｄ １９７９ Ｎｏｖ １；３０１（１８）：１０１１−２．
【０２３９】
１６． Ｐａｒｌ， Ｆｒｉｔｚ Ｆ．， Ｅｓｔｒｏｇｅｎｓ， Ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ， ａｎｄ Ｂｒｅａｓｔ Ｃａｎｃｅｒ． ＩＯＳ Ｐｒｅｓｓ， Ａｍｓｔｅｒｄａｍ， Ｎｅｔｈｅｒｌａｎｄｓ， ２０００．
１７． Ｂｉｒｒｅｎ Ｂ， Ｇｒｅｅｎ ＥＤ， Ｋｌａｐｈｏｌｚ Ｓ， Ｍｙｅｒｓ， Ｒ， Ｒｉｅｔｈｍａｎ Ｈ， Ｒｏｓｋａｍｓ Ｊ， （ｅｄｓ．） （１９９７）， Ｇｅｎｏｍｅ Ａｎａｌｙｓｉｓ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ． Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ， ＮＹ．
１８． Ｅｗｉｎｇ Ｂ， Ｈｉｌｌｉｅｒ Ｌ， Ｗｅｎｄｌ ＭＣ， Ｇｒｅｅｎ Ｐ． Ｂａｓｅ−ｃａｌｌｉｎｇ ｏｆ ａｕｔｏｍａｔｅｄ ｓｅｑｕｅｎｃｅｒ ｔｒａｃｅｓ ｕｓｉｎｇ ｐｈｒｅｄ． Ｉ． Ａｃｃｕｒａｃｙ ａｓｓｅｓｓｍｅｎｔ． Ｇｅｎｏｍｅ Ｒｅｓ １９９８ Ｍａｒ；８（３）：１７５−８５．
１９． Ｇｏｒｄｏｎ Ｄ， Ａｂａｊｉａｎ Ｃ， Ｇｒｅｅｎ Ｐ． Ｃｏｎｓｅｄ： ａ ｇｒａｐｈｉｃａｌ ｔｏｏｌ ｆｏｒ ｓｅｑｕｅｎｃｅ ｆｉｎｉｓｈｉｎｇ． Ｇｅｎｏｍｅ Ｒｅｓ １９９８ Ｍａｒ；８（３）：１９５−２０２．
２０． Ｆｌｏｕｒｉｏｔ Ｇ， Ｇｒｉｆｆｉｎ Ｃ， Ｋｅｎｅａｌｙ Ｍ， Ｓｏｎｎｔａｇ−Ｂｕｃｋ Ｖ， Ｇａｎｎｏｎ Ｆ． Ｄｉｆｆｅｒｅｎｔｉａｌｌｙ ｅｘｐｒｅｓｓｅｄ ｍｅｓｓｅｎｇｅｒ ＲＮＡ ｉｓｏｆｏｒｍｓ ｏｆ ｔｈｅ ｈｕｍａｎ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ−ａｌｐｈａ ｇｅｎｅ ａｒｅ ｇｅｎｅｒａｔｅｄ ｂｙ ａｌｔｅｒｎａｔｉｖｅ ｓｐｌｉｃｉｎｇ ａｎｄ ｐｒｏｍｏｔｅｒ ｕｓａｇｅ． Ｍｏｌ Ｅｎｄｏｃｒｉｎｏｌ １９９８ Ｄｅｃ；１２（１２）：１９３９−５４．
【０２４０】
上記明細書に記載の全ての出版物及び特許はここに織り込まれるものである。本発明に記載の方法及びシステムの様々な修飾及び変更は本発明の範囲ならびに精神を外れない範囲で当業者に明らかである。本発明は特定の好ましい実施例に関連して記載しているが、クレームされた本発明はこのような特定の実施例に不当に限定されないことが理解されるべきである。実際に、分子生物学及び関連する分野の当業者に自明な、前記本発明の実施態様の様々な修飾はクレームの範囲内である。
【配列表】

【図面の簡単な説明】
【図１】エストロゲン受容体アルファ遺伝子の完全ゲノム配列。
【図２】エストロゲン受容体アルファゲノムＤＮＡに見出される配列多型（ヌクレオチドの位置は図１で与えられる配列に基づく）。
（ａ）リバプール臨床組織サンプル中のＳＮＰ
（ｂ）コリエール多様性パネル中のＳＮＰ
（ｃ）リバプールコントロール母集団中のＳＮＰ
（ｄ）ＰＣＲプライマー
（ｅ）シークエンシングプライマー
【図３】エストロゲン受容体アルファタンパクのアミノ酸配列。
【図４】エストロゲン受容体ハプロタイプ（ハプロタイプの項参照）。
（ａ）４８人の患者からのリバプールサンプル、各患者は、腫瘍と対応する血液サンプルを有する。コリエールサンプルはコントロールである。
（ｂ）隣接樹に適する非単独ハプロタイプデータ（Ｌはリバプールサンプル）。
【図５】ＥＳＲ１タンパクのドメイン構造とここに開示されるＳＮＰ位置。
【図６】本発明に係る多くのＳＮＰの分布及び頻度。
【図７】ヒトＥＳＲ１遺伝子座のグラフ表現。
（ａ）ヒトエストロゲン受容体アルファ（ＥＲα）の完全構造。エキソンは塗りつぶした四角で、イントロンは水平線で表す。
（ｂ）ゲノム配列を完成するために使用されたコンティグの順番と名前。ＧＡナンバーはセレーラコンティグナンバーを表す。リサーチジェネティクスＢＡＣクローンは基準プレート及び詳細番号で表される。
【図８】ＥＳＲアルファＳＮＰ：ａ）コリエールサンプル中、ｂ）リバプールサンプル中（Ｔ＝腫瘍サンプル、Ｂ＝血液サンプル、ＬＣ＝リバプールコントロール）、ｃ）リバプールコントロールサンプル中。
【図９】ＳＮＰを有するＥＳＲアルファエキソン（「Ｎ」、「Ｃ」、「Ｉ」、「Ａ」、「Ｓ」については図２を参照）。下線の配列はプライマー配列を表す。[0001]
Related application
This application is related to U.S. Patent Application Ser. S. Serial No. 09 / 692,414 (Atty. Docket CL000258), filed on Jan. 24, 2001. S. Serial No. 09 / 768,184 (Atty. Docket CL000258CIP), filed on March 13, 2001, U.S. Pat. S. Serial No. 09 / 804,076 (Atty. Docket CL000258CI2), and U.S. Patent Application Ser. S. Serial No. 09 / 826,314 (Atty. Docket CL000258CI3).
[0002]
Technical field
The present invention relates to methods for detecting and treating diseases. In particular, the present invention relates to the development of diagnostic and therapeutic methods for previously unknown nucleic acid / amino acid polymorphisms in the estrogen receptor alpha gene (ESR-α) and diseases and disorders mediated / modulated by the estrogen receptor. , The gene sequence of the gene.
[0003]
Background art
Estrogen receptor
Human estrogen receptor alpha belongs to the family of nuclear hormone receptors. Nuclear hormone receptors are a family of hormonally active transcription factors capable of initiating or increasing the transcription of genes, including certain hormonal response factors.
The estrogen receptor protein consists of 595 amino acids with a molecular weight of 66 kDa, has eight translated exons, and six domains with different functions. In two of these domains, the early sequences of the nuclear hormone receptor superfamily are highly conserved. One of these domains, the DNA binding domain (DBD), contains two zinc fingers that mediate the binding of the receptor to hormone responsive elements in the promoter of the hormone responsive gene. In the C-terminal region, the hormone binding domain (HBD) contains two regions with sequence homology to other hormone receptors, conferring hormonal properties and selectivity. The human estrogen receptor alpha gene is located on chromosome 6q. 25.1.
[0004]
Estrogen receptors, and other similar steroid receptors, are transcription factors that are activated by binding to steroids (estradiol) or steroid analogs such as tamoxifen. Upon activation, the receptor binds to the estrogen receptor component (ERE) located in the promoter region of the estrogen activating gene to form a homodimer or heterodimer, which interacts with the host coactivator. Adjust the transfer by action.
[0005]
The role of estrogen in cardiovascular disease
Heart disease is the leading cause of death in women, a fact recognized by women and doctors. One in nine women between the ages of 45 and 65 has some cardiovascular disease, increasing to one in three for those aged 65 and older. Every year, 240,000 people die from heart disease in the United States, of which about 90,000 die from stroke. In addition, about 44% of people who have had a heart attack within one year die. In comparison, it is 26% for males (Warren MP and Kulac J Clin Obs Gyn 1998 1 (4): 976-187).
[0006]
Estrogen has a wide range of physiological effects on a wide variety of cell types. For example, it regulates a myriad of functions related to cell growth and apoptosis, and replication. There are two types of estrogen receptors, α and β. Blood vessels and bones contain β receptors, and liver contains α receptors. And in the central nervous system, both α and β receptors are found. The interaction of these different receptor sites affects the biological effects of estrogen and selective estrogen receptor modulators (SERMs) such as, for example, raloxifene. The binding pattern directs the estrogen or SERM to act as an estrogen agonist or antagonist (Mendelsohn ME and Karas RH New Engl J Med 1999, 340 (23): 1801-1181; 4: 71-92). There is a tissue-specific relationship between SERMs and receptor binding sites. Estrogen increases HDL cholesterol levels, lowers LDL cholesterol levels, and decreases plasminogen activity inhibitor (Meisler JG Jour Woman's Health 1999, 8 (1): 51-57). All estrogens require a cell receptor for expression. In general, the estrogen receptor is a ligand-inducible transcription factor, and regulates the expression of a target gene after binding to a hormone (Faustini-Fustini et al. Eur J Endocrin 1999, 140: 111-129). Estrogen probably also has important effects on the vascular wall. Estradiol and progesterone receptors have been identified in arterial endothelium and smooth muscle cells (Campis D et al. Int J Tissue React 1987, IX (5): 393-398). Estrogen acts on the arterial wall, relaxing vascular smooth muscle and reducing vascular resistance. This mechanism appears to be due to stimulation of endothelium-derived relaxing factors and endogenous nitrates (Warren MP and Kulac J Clin Obs Gyn 1998 41 (4): 976-187). Alleviation by 17B-estradiol plays an important role in regulating cardiac status and reduces the risk of heart disease in postmenopausal women. Nitric oxide production is mediated by estrogen receptors. This is because nitric oxide is suppressed when the receptor is blocked by antiestrogens.
[0007]
Several studies have shown that estrogen therapy reduces the risk of heart disease by up to 50% (most recently reviewed by Mendelshon ME and Karas RH New Engl J Med 1999, 340 (23): 1801-1181; Rich-Edwards JW N Engl J Med, 1995, 332: 1758-1765; Gerhard M, Ganz P, Circulation, 1995, 92: 5-8; rodstein F, et al. 1769-75; Chasen-Taber Land and Stampfer MJ Ann of Int Med, 1998, 128: 467-474; Warren M and Kulak J Clin Obstet Gyn 1998, 41 (4): 976-987). Estrogen depletion may be one of the most important factors in the development of cardiovascular disease in women.
[0008]
While there is no direct evidence that estrogen prevents atherogenesis, there is much epidemiological evidence suggesting that estrogen is effective in reducing cardiovascular disease; (1) All ages Women have a lower incidence of cardiovascular disease than men; (2) women who have early menopause and do not use estrogen have twice as many cardiovascular diseases as pre-menopausal women than men of the same age. (3) postmenopausal women using estrogen have a significantly lower incidence of cardiovascular disease compared to women not using it; (4) women with cardiovascular disease detected by angiography When using estrogen, the survival rate is high.
[0009]
In recent years, the use of estrogen in postmenopausal women has gained widespread attention as good results on cardiovascular disease morbidity and mortality from estrogen therapy have been reported (Meinertz T Herz 1997, 22: 151-157). Guidelines for the treatment of estrogen, published by the United States Medical College, state that "hormonal therapy is effective in women with coronary heart disease."
[0010]
More than 30 prospective and 13 controlled studies have investigated the incidence or prevalence of cardiovascular disease and the effects of estrogen replacement therapy on all causes of death (Stampfer MJ et al. New. Engl J Med 1991, 325: 756-62; Grady D et al. Ann Intern Med 1992, 117: 1016-37). Most of these studies have shown that the incidence and mortality of coronary heart disease in postmenopausal estrogen users is lower than in non-users. In particular, coronary artery disease among estrogen users has been shown to be about 50% of non-users. Overall, most of this evidence strongly supports the protective effect of estrogen with a relative risk of 0.56 (95% confidence interval 0.50-0.61). However, there is a “trend of choice for healthy women” in these studies, which may potentially confuse the results (estrogens use better weight management compared to non-estrogen users) There is more exercise, less smoking). In addition, the tendency for estrogen users to be better educated and have higher incomes has also disrupted these epidemiological studies (Abrams J Clin Cardiol 1998, 21: 218-222).
[0011]
Earlier observations indicated that 25% of the 50% reduction in risk was trusted due to various methodological trends because it was not randomized (Grodstein F and Stampfer MJ Prog Cardiol Dis 1995, 38: 199). -210; Barrett-Conner E and Grady D 1998, Annu Rev Public Health 19: 55-72). Recently, two meta-analyses have estimated that estrogen use reduces coronary heart disease by 35-44%. Recent studies have investigated the use of estrogen, with reports of an increased risk of uterine cancer in women using estrogen alone with an intact uterus. New evidence suggests that the use of progestins (usually medroxyprogestin acetate) in addition to estrogens is not as protective of the heart as the use of estrogens alone (Hulley Set et al.). al 1998 JAMA 280: 605-613; Abrams J Clin Cardiol 1998, 21: 218-222).
[0012]
Estrogen reduction at menopause is associated with a 6% decrease in HDL cholesterol levels and a 5% increase in LDL cholesterol levels, which is higher in postmenopausal women than in premenopausal women It may be an explanation for having a cardiovascular disease incidence. The lower incidence of cardiovascular disease in postmenopausal women using estrogens may be partially explained by the results of a 15-19% decrease in LDL cholesterol and a 16-18% increase in HDL cholesterol. (JAMA 1995, 273: 199-208). In a randomized, double-blind, placebo-controlled trial of PEPI (Postmenopausal Estrogen / Progestin Intervention), HDL cholesterol levels in women using estrogen alone are significantly increased compared to women using estrogen in combination. (JAMA 1995, 273: 199-208). Recently, studies of non-human primates have corroborated these findings (Clarkson TB Lab An Sci 1998, 48 (6): 569-72). Statistical models for the effects of estrogen on the lipid profile have shown that 25-50% of apparent cardioprotection by estrogen is mediated by good changes in HDL-cholesterol (Bush TL et al. 1987 Circulation 75). Gruchow HW et al. 1988 Am Heart J 115: 954-63).
[0013]
Estrogen replacement therapy is not without danger. For many years, users of oral contraceptives have been shown to increase the risk of venous thromboembolism (VTE) by a factor of 3 to 4 compared to non-users (Weiss G Am J Obstet Gynecol 1999). 180: S295-301). One study has shown that intrinsic clotting factors play an important role in VTE associated with oral contraceptives (Vandenbrooke JP et al. Lancet 1994 344: 1453- 7; Rossing J et al. Br J Haematol). 1997, 97: 233-238). Leiden Mutant V increases the risk of VTE 5- to 10-fold in non-contraceptive users, but 30-fold in third-generation oral contraceptive users. The bound estrogen causes resistance to the body's natural anticoagulant system (APC). Heterozygotes due to Leiden Mutant V in oral contraceptive users develop equivalent APC resistance to homozygous women.
[0014]
Estrogen increases the risk of endometrial cancer about 6-fold, but with the addition of progestin this effect is almost eliminated (Barrett-Conner E and Grady D 1998, Ann Rev Public Health 19: 55-72). There is ongoing debate whether estrogen replacement increases the risk of breast cancer, but several studies have shown that the risk is increased by 30% (Greendale GA et al. Lancet 1999, 353: 571-80).
[0015]
Many randomized studies are underway to establish whether estrogen replacement therapy reduces the risk of cardiovascular disease in women and whether it increases the risk of breast cancer. One recently completed study, HERS (Heart and Estrogen / Progestin Replacement Study), showed that 2,700 women who already had heart disease were continuously prescribed estrogen and medroxyprogesterone acetate, with and without a control placebo. (Hully S et al. 1998 JAMA 280 (7): 605-13). Compared to controls, the intervention group had significantly more heart disease onset in the one year study, but significantly less in the four to five year study. In addition, studies of hormone-using women have shown a significant increase in the incidence of thromboembolism at an early stage. Based on these results, hormone replacement therapy cannot be recommended for secondary prevention of heart disease.
[0016]
Two other major clinical studies are currently underway for the primary prevention of cardiovascular disease using estrogens. A study called The Women's Health Initiative, slated to be completed in the UK in 2005, and a WIS-DOM, slated to be completed in 2010, indicate that estrogen has a new protective effect on cardiovascular disease. Will cast light (Meisler JG Jour Women's Health 1999, 8 (1): 51-5).
[0017]
In summary, ongoing research suggests that estrogen replacement therapy, particularly in connection with designer estrogen or SERMs, has recently been shown to be as effective in the cardiovascular system as bone, without adversely affecting breast and endometrial tissue. Has a significant effect. Nevertheless, careful observation is required. On average, women who take estrogen are better educated, healthier, have higher incomes, and are better at managing their health. Rather than estrogen, these differences may reduce the risk of heart disease.
[0018]
In postmenopausal women who are currently ill, estrogen replacement therapy appears to have a useful impact given the importance, consistency, and biological credibility of the data. For women who already have the disease, issues remain regarding the safety and effectiveness of exogenous estrogens as protective medicine against cardiovascular disease.
[0019]
Estrogen and autoimmune diseases
A. Systemic lupus erythematosus (SLE)
The view that estrogen is associated with systemic lupus erythematosus is widely known from the following:
1. Women who give birth to a child are nine times more likely to develop lupus erythematosus than men. Before puberty, the incidence of women is more than three times that of men, but in postmenopausal women, the incidence is similar to that of men of the same age. Numerous studies have shown that these gender differences are probably related to estrogen (Lahita RG, 1986: Springer Seminars in Immunopathology 9, 305-314; Krammer, GM and. Tsokos, GC, 1998 Clinical Immunology and Immunopathology 89: 192-195; Rider at al., 1998 Clinical Immunology and Immunopathology 180: 89.
[0020]
A clue to the role of estrogen in SLE came from studies that concluded that birth control pills had a detrimental effect on this condition (Buton, JP, 1996 Ann. Med. International, 147: 259-264). Julkunen, 1991: Scan. J. Rheumatol. 20: 427-433).
[0021]
2. A patient with Klinefilter syndrome (XXY) with SLE was reported (Stern et al., 1977: Arthritis and Rheumatism 20: 18-22).
[0022]
3. SLE patients have anti-estrogen antibodies (Feldman, 1987: Biochem. Biophys. Acta, 145: 1342-1348: Bucala et al., 1987: Clin. Exp. Immunol. 67: 167-175).
[0023]
In the past, birth control pills have been shown to cause SLE spontaneously, and up to the current belief that low doses are safe for SLE patients, the use of birth control pills in women with SLE has been agreed. (Julkunen HA Scand J Rheumatol 1991; 20 (6): 427-33). Similarly, hormone replacement therapy is also considered safe for SLE patients (Mok et al., Scand J Rheumatol 1998; 27 (5): 342-6: Kreidstein et al., 1997, J Rheumatol 1997 Nov). 24 (11): 2149-52).
[0024]
4. Tamoxifen, an estrogen antagonist, appears to improve disease progression (Sthoeger, 1997, Ann NY Acad Sci 1997 Apr 5; 815: 367-8: Sthoeger, 1994, J Rheumatol 1994: 12 (12); 2231-8).
[0025]
B. Estrogen, rheumatoid arthritis (RA) and osteoarthritis
The literature relating to estrogen in rheumatoid arthritis is less clear than osteoarthritis. In epidemiological studies, oral contraceptives delay the onset of RA, but estrogen alone does not reduce RA symptoms, suggesting that RA is affected by female hormones (Bijlsma Am J Reprod). Immunol 1992 Oct-Dec; 28 (3-4): 231-4). Estrogen adjuvant prescriptions increase bone density in postmenopausal women with RA and may provide protection against osteoporosis, which can often accompany RA (van den Brink: Ann Rheum Dis 1993 Apr; 52 (4): 302-5). The above study showed that estrogen did not reduce the symptoms of RA, and other studies concluded that even estrogen adjuvant therapy did not improve the symptoms. One polymorphism at the estrogen receptor that was thought to be associated with the age of RA onset was reported (Ushiyama Ann Rheum Dis 1999 Jan; 58 (1): 7-10).
[0026]
On the other hand, the fact that osteoarthritis is less likely to occur in postmenopausal women receiving estrogen replacement therapy (ERT) (Felson Curr Opin Rheumatol 1998 May; 10 (3): 269-72), suggests that ERT prevents osteoarthritis. Suggests that it might be useful.
[0027]
C. Estrogen and osteoporosis
Osteoporosis is a bone disease that causes the bones to become brittle and at risk of fracture. Treatment of postmenopausal women with osteoporosis involves the replacement of hormones, especially estrogens. Estrogen reduces bone loss and the occurrence of fractures (Weiss et al., N Engl J Med 1980 Nov 20; 303 (21): 1195-8: Paganini-Hill et al., Ann Intern Med 1981 Jul; 95 ( 1): 28-31: Ettinger et al., Ann Intern Med 1985 Mar; 102 (3): 319-24).
[0028]
In addition, polymorphisms at the estrogen receptor have been associated with bone loss in both humans and mice. (Kobayashi J Bone Miner Res 1996 Mar; 11 (3): 306-11: Kurabayashi Am J Obstet Gynecol 1999 May; 180 (5): 11115-20; Deng Hum Genet 1998-85; 103; .
[0029]
Estrogen and cognitive function
Women are at higher risk for Alzheimer's disease than men. Several studies have shown that estrogen use can reduce the incidence of Alzheimer's disease in postmenopausal women. Even in women with Alzheimer's disease, the use of estrogens reduces serious symptoms and slows the decline in intelligence. The risk is lowest for women who use it for a long time (10 years or more). However, the effect was also obtained in women who used it for a short time.
[0030]
Estrogen and breast cancer
The major risk factors for developing breast cancer are sex, age, family breast cancer history, age at menarche, age at first term pregnancy, and age at menopause. All of these factors (except family history) have been shown to be directly related to estrogen secretory lifespan, and increased hormone secretion is associated with increased risk of breast cancer. The increased risk of cancer has been attributed to estrogen receptor-mediated proliferative responses in breast epithelial cells.
[0031]
Tamoxifen, an estrogen receptor antagonist, has been shown to be a drug that is effective in both preventing and treating breast cancer. By using immunohistochemical methods, breast tumors can be classified as estrogen receptor positive or negative according to the amount of estrogen receptor protein expressed in the tissue. Estrogen receptor positive tumors respond better to treatment with tamoxifen than estrogen receptor negative tumors. Premenopausal women develop estrogen receptor-negative breast cancer more often than postmenopausal women.
[0032]
Mutations in the structure and function of the estrogen receptor have been described in early breast tumor or breast cancer cell lines. However, it is not clear whether these changes are primary (and related to the process leading to carcinogenesis) or secondary (and the consequence of gene instability in cancer tissue). Furthermore, in addition to these body mutations, several studies have pointed to a possible relationship between genetic DNA sequence alterations and breast cancer development, but these are also controversial.
[0033]
And, more recently, as evidence for the role of estrogen receptors in breast cancer, it has been discovered that the gene BRCA1, inherited in a mutant that predisposes to breast cancer development, suppresses estrogen receptor signaling.
[0034]
Estrogen and endometrial cancer
Endometrial cancer is the most common pelvic malignancy in women, but can usually be cured by hysterectomy, as about 75% are found in the uterus at the time of diagnosis. Excessive exposure of endometrial cells to estrogen dramatically increases the chance of developing uterine cancer cells, so hormone replacement therapy with estrogen alone should not be given to women with an intact uterus. Periodic or continuous administration of progesterone helps prevent excessive proliferation of endometrial cells and may increase the risk of endometrial cancer in postmenopausal women who are prescribed estrogen as part of hormone replacement therapy. Reduce.
[0035]
In many cases of endometrial cancer, the estrogen receptor is expressed, and in general, estrogen-responsive tumors can be suitably predicted. Acquired (body) mutations are said to be up to 8.5%, but the role of the mutation in the development and progression of endometrial cancer is currently unclear.
[0036]
Although somewhat controversial, studies suggest that the use of tamoxifen has increased the chances of developing endometrial cancer. This indicates that, in addition to the role of estrogen receptor blockade, tamoxifen has partial receptor agonist activity and down-regulates estrogen responsive genes that induce endometrial proliferation.
[0037]
It is important to gain the development of estrogen receptors in mediating / modulating various diseases, identify sequence polymorphisms in estrogen receptors, and correlate them with disease states, therapeutic effects, and the like. The present invention advances these techniques by providing various previously unknown ESR-alpha protein polymorphisms.
[0038]
SNP
The genomes of all organisms undergo spontaneous mutations during the evolution process, producing mutated forms of ancestral sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). Mutant forms may be advantageous or inconvenient in terms of evolution compared to ancestral forms, or may not be relevant. For example, if a mutant suffers a fatal inconvenience, it will not be passed on to later generations. If, on the other hand, the mutant form is favorable to the evolution of the species, it will be incorporated into much or most of the DNA of that species, effectively becoming the ancestral form. In addition, the effects of the variant may be both beneficial and deleterious, depending on the situation. For example, heterozygous sickle cell mutations have resistance to malaria, whereas homozygous sickle cell mutations are usually fatal. In many instances, ancestral and mutant forms coexist in species. The coexistence of multiple forms of the sequence causes polymorphisms such as SNPs.
[0039]
The reference allelic form is arbitrarily indicated, for example, the most prevalent form or the first specified allelic form, while the other allelic form is indicated as an alternative, mutated, polymorphic allele. The most frequently occurring allelic form in the chosen population is called the "wild-type" form.
[0040]
About 90% of polymorphisms in the human genome are single nucleotide polymorphisms (SNPs). The location or location of a SNP usually follows a highly protected allele (a sequence that varies by 1/100 or 1/1000 or less of the population). The allele at each SNP position in an individual may be homozygous or heterozygous. As identified in the present invention, the least frequent allele at the SNP location is less frequent than the more frequent allele and less than 1%. In some examples, the SNP is referred to as "cSNP", meaning that the nucleotide sequence containing the SNP is an amino acid coding sequence.
[0041]
SNPs occur when one nucleotide at a polymorphic site is replaced by another. Substitution occurs by transposition or conversion. Transfer is the exchange of a purine nucleotide for another purine nucleotide or for a pyrimidine nucleotide. Conversion is the conversion of a purine into a pyrimidine or vice versa. SNPs may also be in a mutated form (called indel) with one base inserted or deleted. Substitution of an amino acid for another amino acid in codon coding is called a non-synonymous codon mutation or missense mutation. Synonymous codon mutations or silent mutations do not result in amino acid mutations due to the degeneracy of the genetic code. Nonsense mutations are non-synonymous codon mutations that form a stop codon, leading to premature termination of the polypeptide chain and incomplete protein.
[0042]
SNPs can in principle be bi-, tri-, tetra- alleles. However, tri- and tetra- are extremely rare and almost nonexistent (Brookes, Gene 234 (1999) 177-186). For this reason, SNPs are often referred to as “bi-allele markers” or “di-allele markers”.
[0043]
A causal SNP is a SNP that causes a change in gene expression or functional expression of a gene product, and in most cases, can predict a clinical phenotype. These include SNPs that enter the gene region encoding the polypeptide product, ie, cSNPs. These SNPs result in alterations in the amino acid sequence of the polypeptide product (ie, non-synonymous codon mutations) and may cause deletion or expression of other mutated proteins. Furthermore, in the case of nonsense mutations, SNPs may lead to premature termination of the polypeptide product. Such mutated products can result in a disease state, for example, a genetic disease. Examples of genes having polymorphisms in the coding sequence cause genetic diseases including sickle cell anemia and cystic fibrosis. The causal SNP need not necessarily occur in the coding region, but can occur in any region that ultimately affects the expression and / or activity of the protein encoded by the nucleic acid. These gene regions include gene regions related to transcription of SNPs and the like in the promoter region; SNPs in the intron-exon boundary that cause defective pricing, and SNPs and the like in mRNAs that process signal sequences such as polyadenylation signal regions. Contains related gene regions. For example, SNPs suppress splicing of introns, causing mRNAs containing premature stop codons, which may lead to defective proteins. Therefore, the SNP in the regulatory region greatly affects the expression.
[0044]
Some SNPs that are not causative SNPs are still closely related and segregated with the disease-causing sequence. In this case, the presence of the SNP correlates with the presence or susceptibility of the disease. These SNPs are very useful for diagnostics and screening for susceptibility to disease.
[0045]
Clinical trials have shown that patients' responses to treatment with drugs are often heterogeneous. Therefore, it may be necessary to improve the design and treatment of medicines. SNPs can be used to determine the particular drug that is most appropriate for treating a patient (this is often referred to as "pharmacogenomics"). Pharmacogenomics can also be used in the drug selection process in pharmaceutical research. (Linder et al. (1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; , 16, 3)
[0046]
Group study
Population genetics is a study of how Mendel's laws and other genetic principles are applied across populations. Such research is essential for a proper understanding of evolution. This is because evolution is basically the result of innovative changes in the genetic makeup of a population. Therefore, population genetics seeks to understand and predict the effects of genetic phenomena, such as segregation, recombination, and mutation, while at the same time, population size, mating patterns, individual geographic distribution, migration, And ecological and evolutionary factors such as natural selection must be considered.
[0047]
Ideally, how to discuss population gene types and frequencies in order to understand how population genes are organized and to predict population changes resulting from natural or artificial selection. It is desired to understand.
[0048]
To explain many of these issues, it is important to understand the relationships (linkages) that exist between loci.
[0049]
Linkage is a co-inheritance of two or more non-allelic genes because the loci are in close proximity on the same chromosome, and more than 50% of the predicted non-linked genes are associated after meiosis . It is clear that there is a physical crossover during meiosis and that there is a physical exchange of maternal and paternal genetic contributions between individual chromatids during germ cell generation. This exchange necessarily resulted in the separation of genes in the chromosomal regions that were adjacent to each parent and mixing with the remaining sequences, resulting in “recombination”. The process of generation of recombination by meiotic crossover is an important feature in the rearrangement of genetic characteristics and is central to understanding gene transfer.
[0050]
Recombination generally occurs between large segments of DNA. This means that the adjacent region of DNA and the gene are linked. Conversely, regions of DNA far apart from each other on a given chromosome also appear to be apart during the process of crossover.
[0051]
Molecular markers can be used to reveal recombination events that occur during meiosis. Different length (CA)_nSome markers that are repeats of are ubiquitous in human DNA, have a slight selection pressure on their length, and are used as position markers and chromosomal localization features. These markers can be used to distinguish paternally derived genes from maternally derived gene regions.
[0052]
Other markers are single nucleotide polymorphisms (SNPs), which are bi-allelic markers, which can also be used to analyze the transmission of these markers to progeny.
[0053]
The pattern of a set of markers along a chromosome is called a "haplotype." And alleles on the same small chromosomal segment tend to be transmitted as blocks throughout the pedigree. By analyzing haplotypes in offspring of a parent whose haplotype is known, it is possible to confirm which parental segment in which chromosome was transmitted to which child. If not broken by recombination, the haplotype can be considered as an allele of a single highly polymorphic locus for mapping purposes.
[0054]
The preferential generation of a virulence gene associated with a particular allele of a linkage marker is called "linkage disequilibrium" (LD). This type of imbalance generally means that most pathogenic chromosomes carry the same mutation, and that the marker tested is very close to the virulence gene. For example, there is a lot of linkage disequilibrium throughout the HLA locus. The A3 allele is in LD along with the B7 and B14 alleles, so that B7 and B14 are highly associated with hemoglobinosis. Thus, the HLA classification alone can significantly alter the estimate of hemoglobin disease risk, even if other family members are not available for formal linkage analysis. As a result, haplotype determinations can be made with or without affecting families by using a combination of several markers around the putative gene location.
[0055]
Association analysis and linkage disequilibrium mapping based on SNP
SNPs are useful in association studies to identify specific SNPs or other polymorphisms associated with a pathological condition, such as breast cancer. Association studies may be performed within a general population and are not limited to studies performed on individuals in affected families (linkage studies). Association studies with SNPs involve determining the frequency of SNP alleles in many patients with significant disorders (eg, breast cancer), as well as controls of similar age and history. Proper selection of patients and controls is critical to the success of SNP association studies. Therefore, a set of individuals with a well-characterized phenotype is highly desirable. For example, to find an association between a particular SNP genotype and a known phenotype, blood pressure and heart rate can be correlated with the SNP pattern of a hypertensive person whose physiological parameters are known. . Significant associations between a particular SNP or SNP haplotype and phenotypic traits can be determined by standard statistical methods. Association analysis can be performed directly or based on LD. In direct association analysis, a causative SNP that is a candidate for a pathogenic sequence is tested.
[0056]
In LD-based SNP association analysis, random SNPs are tested in large genomic regions (or the entire genome) to find SNPs in LD, with either true pathogenic sequences or pathogenic SNPs. For this study, a high-density SNP map was used to detect associations, such that the random SNPs were located close enough to the unknown virulence loci to be in linkage disequilibrium by that locus. is necessary. SNPs tend to occur frequently and are evenly spaced throughout the genome. The frequency and homogeneity of SNPs means that they are more likely to be found in close proximity to the pathogenic gene location compared to other types of polymorphisms, such as tandem repeat polymorphisms. SNPs are also more mutationally stable than tandemly repeated polymorphisms such as VNTR. With LD-based association studies, it is possible to discover genes susceptible to disease without prior knowledge of what genes are and where.
[0057]
However, currently there is no feasible SNP association study in all human genomes, and candidate genes associated with breast cancer are targeted for SNP identification and association analysis. In studying candidate genes, a priori knowledge of the cause of the disease is used to identify genes that are assumed to directly affect disease development. Studies of candidate genes may focus on genes that are directly targeted by drugs used to treat the disease. To find SNPs associated with increased infectivity of breast cancer, candidate genes are selected from systems that are physiologically involved in the disease pathway. SNPs found in these genes are tested for a statistical association with the disease in sick individuals, as compared to appropriate controls. Studying candidate genes significantly reduces the number of candidate SNPs and the number of individuals that need to be categorized and compared to LD-based association studies of random SNPs over a large (or complete genomic region) Has the advantage of being able to. Furthermore, candidate gene studies do not predict the LD range of specific regions of the genome.
[0058]
Association studies (including linkage disequilibrium-based genome-wide association), combined with the use of densely spaced maps of well-spaced and well-informed SNP markers, have led to the identification of most diseases associated with complex diseases such as breast cancer. Can be identified. This improves the selectivity of candidate genes, including the causal SNP associated with a particular disease. All of the SNPs disclosed in the present invention can be used for part of genome wide association studies or candidate gene association studies.
[0059]
The present invention advances the state of the art and provides commercially useful examples by providing previously unknown SNPs in the estrogen receptor gene.
[0060]
Summary of the Invention
The present invention is based on the sequencing of genomic DNA and cDNA from human chromosome 6, defining the genomic structure of the estrogen receptor alpha gene, a novel polymorphism of the estrogen receptor gene / protein, and an unknown haplotype. Such polymorphisms / haplotypes can lead to various diseases mediated / modulated by mutant estrogen receptors, such as susceptibility to cancer, osteoporosis, cardiovascular disease and the like. Based on this sequencing approach, the present invention detects genomic nucleotide sequences, cDNA sequences, amino acid sequences, sequence polymorphisms in the ESR-alpha gene, haplotypes of these polymorphisms, and these sequences / polymorphisms in a sample. Methods of determining the risk of developing or developing a disease mediated by a mutant estrogen receptor and methods of screening compounds used to treat a disease mediated by a mutant estrogen receptor .
[0061]
DETAILED DESCRIPTION OF THE INVENTION
General description
The present invention is based on the sequencing of genomic DNA from human chromosome 6 and cDNA to define the genomic structure of the estrogen receptor alpha gene, novel polymorphisms of the estrogen receptor gene / protein, and unknown haplotypes. . Such polymorphisms / haplotypes can lead to various diseases, such as cancer, osteoporosis, and susceptibility to cardiovascular disease, which are mediated / modulated by mutant estrogen receptors. Based on this sequencing approach, the present invention detects genomic nucleotide sequences, cDNA sequences, amino acid sequences, sequence polymorphisms in the ESR-alpha gene, haplotypes of these polymorphisms, and these sequences / polymorphisms in a sample. Methods of determining the risk of developing or developing a disease mediated by a mutant estrogen receptor and methods of screening compounds used to treat a disease mediated by a mutant estrogen receptor .
[0062]
Isolated SNP-containing nucleic acid molecule
The present invention provides an isolated nucleic acid molecule comprising one or more SNPs disclosed by the present invention. The invention further provides an isolated nucleic acid molecule that encodes the mutant protein. Such a nucleic acid molecule consists, consists essentially of, or comprises one or more SNPs according to the invention. Nucleic acid molecules can have additional nucleic acid residues. Such nucleic acid residues are, for example, those naturally associated with the nucleic acid molecule or a heterologous nucleotide sequence.
[0063]
Here, an "isolated" SNP-containing nucleic acid molecule includes a SNP of the present invention and is separated from other nucleic acids present in the natural source of the nucleic acid. Generally, an isolated SNP-containing nucleic acid as used herein comprises one or more of the SNP positions disclosed by the present invention with flanking nucleotide sequences on either side of the SNP. The flanking sequence, if used for a detection reagent, is preferably up to about 300 bases, up to 100 bases, up to 50 bases, up to 30 bases, up to 15 bases, up to 4 bases, or up to 4 bases, or If used to generate proteins containing the coding variants disclosed in the figures, the length of the entire protein coding sequence will be on either side of the SNP position. The important point is that the nucleic acid is isolated from distant and insignificant flanking sequences, and that recombinant expression, preparation of probes and primers for SNP positions, and other features specific to SNP-containing nucleic acid sequences It is of an appropriate length to allow for the particular operations or uses disclosed herein, such as use.
[0064]
In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule containing a SNP position of the invention, may be produced by recombinant techniques, chemical precursors, or other chemicals when chemically synthesized. It may be substantially free of cellular material or culture media. However, the nucleic acid molecule can be fused to other coding or regulatory sequences, and still be considered isolated. For example, a recombinant DNA molecule contained in a vector is considered isolated. Further examples of an isolated DNA molecule include a recombinant DNA molecule retained in an atypical host cell, or a purified (partially or substantially) DNA molecule in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated SNP-containing DNA molecules of the invention. Isolated nucleic acid molecules according to the present invention further include molecules that are synthetically produced.
[0065]
An isolated SNP-containing nucleic acid molecule can be in the form of RNA, such as mRNA, or in the form of DNA. This DNA includes cDNA and genomic DNA obtained by cloning, produced by chemical synthesis techniques, or produced by a combination thereof. Nucleic acids, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acids can be the coding strand (sense strand) or the non-coding strand (antisense strand).
[0066]
The invention further provides related nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecules disclosed herein. As used herein, “hybridization under stringent conditions” means that the nucleotide sequences encoding peptides having at least 60 to 70% homology to each other generally hybridize and remain hybridized to each other. It means the condition for performing the washing. The conditions may be such that sequences that are at least 60%, 70%, 80%, 90% or more homologous to each other are usually still hybridizing to each other. Such stringent conditions are known to those skilled in the art, and are described in Current Protocols in Molecular Biology, John Wiley & Sons, NJ. Y. (1989), 6.3.1-6.3.6. One example of hybridization under stringent conditions is hybridization at about 45 ° C. with 6 × sodium chloride / sodium citrate (SSC), followed by 50 × 65 ° C. with 0.2 × SSC, 0.1% SDS. The condition is that washing is performed at least once. Examples of moderating to low stringent hybridization conditions are well known in the art.
[0067]
Specific examples
Peptide molecule
[0068]
The present invention provides nucleic acid sequences encoding variants of the estrogen receptor. These variant molecules / sequences are referred to herein as estrogen receptor variants of the invention, estrogen receptor proteins of the invention, or peptides / proteins of the invention.
[0069]
The present invention provides an isolated estrogen receptor protein molecule consisting of, consisting essentially of, or comprising the amino acid sequence of an estrogen receptor mutant protein disclosed herein.
[0070]
Here, a protein or peptide is said to be "isolated" or "purified" if it is substantially free of cellular material or substantially free of chemical precursors or other chemicals. The peptides of the invention can be purified to homogeneity or to some degree of purity. The level of purification is based on the intended use. An important feature is that the preparation can perform the desired function of the peptide, even in the presence of significant amounts of other components.
[0071]
As used herein, "substantially free of cellular material" refers to at least about 30% (dry weight) or less of other proteins (ie, contaminated proteins), 20% or less of other proteins, and 10% or less of other proteins. Or the preparation of peptides having 5% or less of other proteins. If the peptide is produced recombinantly, it is substantially free of culture medium, ie, the culture medium is less than about 20% of the protein preparation.
[0072]
"Substantially free of chemical precursors or other chemicals" includes the preparation of peptides in which the peptide is isolated from the chemical precursors or other chemicals involved in its synthesis. In certain examples, “substantially free of chemical precursors or other chemicals” refers to at least about 30% or less (dry weight) or less of chemical precursors or other chemicals, 20% or less of chemical precursors or Including the preparation of estrogen receptor proteins with less than 10% of other chemicals or less than or equal to 5% of chemical precursors or other chemicals.
[0073]
The isolated estrogen receptor protein can be purified from cells that naturally express it, can be purified from cells mutated to express it (recombinant), or known protein synthesis techniques. Can be used for synthesis. For example, a nucleic acid molecule encoding an estrogen receptor protein is cloned into an expression vector, the expression vector is introduced into a host cell, and the protein is expressed in the host cell. The protein can then be isolated from the cells by a suitable purification process using standard protein purification techniques. Many of these techniques are described below.
[0074]
Accordingly, the present invention provides a protein comprising one or more of the sequence polymorphisms given in FIG. 2 and comprising the amino acid sequence outlined in FIG. If the amino acid sequence is the final amino acid sequence of the protein, the protein consists of the amino acid sequence.
[0075]
The invention further provides a protein comprising one or more of the sequence polymorphisms given in FIG. 2 and consisting essentially of the amino acid sequence outlined in FIG. If the amino acid sequence is, for example, only a small amount of additional amino acid residues present in the final protein, the protein consists essentially of that amino acid sequence.
[0076]
Further, the present invention provides a protein comprising the amino acid sequence of FIG. 1 comprising one or more of the sequence polymorphisms given in FIG. If the amino acid sequence is at least part of the final amino acid sequence of the protein, the protein comprises the amino acid sequence. Thus, the protein may be the peptide alone, or additional amino acid sequences, such as amino acid residues that are naturally linked to the protein or that are atypical amino acid residues / peptide sequences (adjacent coding sequences). Can have. Such proteins can have several additional amino acid residues, or can include hundreds or more additional amino acids. The method for preparing / isolating these various proteins is briefly described below.
[0077]
The estrogen receptor proteins of the present invention may be conjugated to heterologous sequences to form chimeric or fusion proteins. Such chimeric or fusion proteins include an estrogen receptor protein operably linked to a variant protein having an amino acid sequence that is not substantially homologous to the estrogen receptor protein. "Operably linked" indicates that the estrogen receptor protein and the atypical protein fuse in frame. Variant proteins can be fused to the N-terminus or C-terminus of the estrogen receptor protein.
[0078]
Here, the fusion protein has essentially no effect on the activity of the estrogen receptor protein. For example, fusion proteins include, but are not limited to, enzyme fusion proteins, such as beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC labels, HI labels, Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate purification of the recombinant estrogen receptor protein. In certain host cells (eg, mammalian host cells), protein expression and / or secretion can be increased through the use of atypical signal sequences.
[0079]
Chimeric or fusion proteins can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are framed together by conventional techniques. In another example, the fusion gene can be synthesized by conventional techniques, including automatic DNA synthesizers. Alternatively, a PCR amplification of gene fragments can be performed using an anchor primer that produces a complementary overhang between two consecutive gene fragments, followed by annealing and reamplification to produce a chimeric gene sequence (Ausubel et al. al., Current Protocols in Molecular Biology, 1992). In addition, many expression vectors are commercially available that already encode a fusion moiety (including, for example, GST protein). A nucleic acid encoding an estrogen receptor protein can be cloned into an expression vector or the like by linking the fusion moiety to the estrogen receptor protein in frame.
[0080]
Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. In addition, many amino acids, including terminal amino acids, are modified by natural processes, processing or post-translational modifications, or chemical modification techniques well known in the art. Typical modifications that occur naturally in polypeptides are described in basic books, detailed major dissertations, and research literature, and are well known to those skilled in the art. Thus, the polypeptide is a derivative or analog in which the substituted amino acid residue is not encoded by the genetic code; a derivative or analog containing the substituent; the mature polypeptide increases the half-life of the polypeptide Derivatives or analogs fused to other compounds (eg, polyethylene glycol), such as compounds; additional amino acids, such as leader or secretory sequences, or sequences for purification of mature transporter peptides or proproteins, Derivatives or analogs fused to the peptide are included.
[0081]
Known modifications include acetylation, acylation, ADP-ribosylation, covalent attachment of flavin, covalent attachment of half of heme, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphotidylinositol Bond, crosslink, cyclization, disulfide bond formation, dimethylation, formation of covalent crosslink, formation of cystine, formation of pyroglutamine, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydration, iodine Transfer RNA-mediated addition of amino acids to proteins, including acetylation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation, arginylation, and ubiquitination. It is not limited to.
[0082]
Such modifications are well known to those skilled in the art and have been described in great detail in the scientific literature. Some commonly used modifications, such as glycosylation, lipid binding, sulfation, gamma carboxylation of glutamic acid residues, hydration, and ADP ribosylation are described in most textbooks. Such a basic book includes Proteins-Structure AND Molecular Properties, 2nd Ed. , T .; E. FIG. Creighton, W.C. H. Freeman AND Company, New York (1993). Many detailed commentaries on this subject are available, see for example, Wold, F .; B., Posttranslational Covalent Modification of Proteins, B .; C. Johnston, Ed. , Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) AND Rattan et al. (Ann. NY Acad. Sci. 663: 48-62 (1992)).
[0083]
The present invention further provides fragments of the estrogen receptor proteins of the present invention, as well as proteins and peptides comprising and consisting of these fragments. However, the fragments of the present invention do not include the fragments published prior to the present invention.
[0084]
Here, the fragment contains eight or more adjacent amino acid residues from the estrogen receptor protein. The fragments may be selected based on their ability to retain one or more biological activities of the estrogen receptor protein, or may be selected for their ability to perform a function (eg, acting as an immune antigen). . Particularly important fragments are biologically active fragments, peptides, which are, for example, about 8 or more amino acids in length and include mutated amino acid residues (FIG. 2). Such fragments usually comprise a domain or motif of an estrogen receptor protein of the invention, such as an active site, a ligand binding domain, or a DNA binding domain. Further possible fragments include, but are not limited to, fragments containing domains or motifs, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites can be readily identified by computer programs known to the skilled artisan and readily available (eg, PROSITE analysis).
[0085]
Use of proteins / peptides
The proteins of the invention may be used to increase antibodies or elicit other immune responses; in assays designed to quantitatively determine the level of protein (or its binding partner or receptor) in biological fluids. As reagents (including labeling reagents); as markers for tissues in which the corresponding protein is preferentially expressed (formally or at a particular stage of tissue differentiation or development, or in disease states); high-throughput screening To determine the biological activity of a protein, including a large number of protein panels. Any or all of these research applications can be developed into reagent grade or kit formats for commercialization as research products. Methods for achieving the above uses are known to those skilled in the art. References disclosing such a method include "Molecular Cloning: A Laboratory Manual", 2d ed. , Cold Spring Harbor Laboratory Press, Sambrook, J. et al. , E .; F. Fritsch AND T. Maniatis eds. 1989, and "Methods in Enzymology: Guide to Molecular Cloning Technologies", Academic Press, Berger, S.M. L. AND A. R. Kimmels eds. , 1987.
[0086]
The estrogen receptor proteins of the present invention are useful in biological assays. The assay also involves properties or activities of known estrogen receptors, or properties that are useful for diagnosing and treating conditions associated with estrogen receptors.
[0087]
The estrogen receptor proteins of the present invention are also useful in drug screening assays in cell-based or cell-free systems. Cell-based systems may be native, ie, cells that normally express the receptor protein, may be biopsied, or may be expanded into cell culture. However, in certain instances, cell-based assays involve recombinant host cells that express the receptor protein.
[0088]
The estrogen receptor proteins of the present invention can be used to identify compounds that modulate receptor activity. Both the estrogen receptor proteins and appropriate fragments of the invention can be used as high-throughput screens to test candidate compounds for their ability to bind and / or modulate the activity of the receptor. These compounds can be further screened against a functional receptor to determine the effect of the compound on receptor activity. In addition, these compounds can be tested on animal or invertebrate systems to determine activity / effect. Compounds that activate (agonist) or inactivate (antagonist) the receptor to the desired degree are identified. Such compounds are selected for their ability to act on one or more of the mutant estrogen receptor proteins of the present invention.
[0089]
In addition, receptor polypeptides can be used to screen compounds for their ability to stimulate or inhibit the interaction between a receptor protein and a molecule of interest that normally interacts with the receptor protein (eg, estrogen). . The subject can be a ligand or binding partner (eg, an estrogen ligand or DNA sequence) with which the receptor protein normally interacts. Such assays typically bind the receptor protein to a candidate compound under conditions in which the receptor protein or fragment interacts with the molecule of interest, and detect the formation of a complex between the protein and the subject, or Or detecting the biological consequences of the interaction between the protein and the subject.
[0090]
The candidate compounds include, for example, the following. 1) Ig-tail fusion peptides, and members of random peptide libraries (see, for example, Lam et al., Nature 354: 82-84 (1991); Houghten et al., Nature 354: 84-86 (1991)) and D Peptides, such as soluble peptides, including members of a chemically-derived molecular library made from type and / or L-type amino acids; 2) phosphopeptides (eg, random and partially degenerate members of a phosphopeptide library (eg, Songyang et al.). , Cell 72: 767-778 (1993)); 3) Antibodies (e.g., Fab, F (ab ') 2, Fab expression library fragments, epitope binding fragments of antibodies as well as polyclonal, monoclonal, Humanized, anti-idiopic, chimeric, and single-chain antibodies); 4) small organic, inorganic molecules (eg, molecules obtained from combinatorial and natural product libraries).
[0091]
Certain candidate compounds include soluble fragments of the receptor that compete for ligand binding. Other candidate compounds include mutant receptors or suitable fragments containing mutations that affect receptor function, which result in competition with the ligand. Thus, for example, fragments that compete with the ligand with high affinity, or that bind to and remain with the ligand are included in the invention.
[0092]
The invention further includes terminal assays to identify compounds that modulate (stimulate or inhibit) the activity of the receptor. This assay usually involves an assay of behavior in a signal transduction pathway indicative of receptor activity. For example, the expression of a gene that is up or down regulated in response to a signal cascade that depends on the receptor protein can be assayed. In certain instances, the regulatory region of such a gene can be operatively linked to a readily detectable marker such as luciferase. Alternatively, phosphorylation of the receptor protein or receptor protein subject can also be measured. Any biological or biochemical function mediated by the receptor can be used as a terminal assay. These include all biochemical or biochemical / biological phenomena, and other functions known to those skilled in the art, incorporated herein by reference to these terminal assays, as described herein.
[0093]
Receptor polypeptides are also useful as competitive binding assays in methods designed to discover compounds that interact with the receptor. The compound is exposed to the receptor polypeptide under conditions that cause the compound to bind or otherwise interact with the polypeptide. A ligand for the receptor is also added to the mixture. When the test compound interacts with the receptor or ligand, it reduces the amount of complex formed or activity from the receptor subject. This type of assay is particularly useful when looking for compounds that interact with specific regions of the receptor.
[0094]
To perform a cell-free drug screening assay, a receptor protein, fragment, or peptide is used to facilitate the automation of the assay while facilitating separation of the complex from the uncomplexed form of one or both proteins. It may be desirable to immobilize the molecule of interest.
[0095]
Techniques for immobilizing proteins on matrices can be used in drug screening assays. In one example, a fusion protein is provided that adds a domain where the protein is bound to a matrix. For example, the glutathione-S-transferase / 15625 fusion protein can be adsorbed to glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates. This is followed by a cell lysate (eg,³⁵S) and the candidate compound, and the mixture is cultured under conditions that lead to complex formation (eg, physiological conditions of salt and pH). Following incubation, the beads are washed to remove unbound label, matrix immobilization is performed, and the radiolabel is determined either directly or in the supernatant after dissociation of the complex. Alternatively, the complex can be dissociated from the matrix, separated by SDS-PAGE, and the level of receptor binding protein found in the bead fraction quantified from the gel using standard electrophoresis techniques. For example, the polypeptide or its target molecule can be immobilized using conjugation of biotin and steptavidin using a conventionally well-known technique. Alternatively, an antibody that is reactive with the protein but does not interfere with the binding of the protein to the molecule of interest is derivatized into the wells of the plate, and the protein is trapped in the wells by conjugation of the antibody. Preparations of the receptor binding protein and the candidate compound are cultured in the cavity in which the receptor protein is present, and the complexes trapped in the cavity can be quantified. Methods for detecting such complexes include those described above for GST-immobilized complexes, as well as enzyme-linked assays by detecting enzyme activity associated with the molecule of interest, as well as receptor protein targets. Includes immunodetection of the conjugate using an antibody that is reactive with the molecule or with the receptor protein and competes with the molecule of interest.
[0096]
The protein-modulating reagents of the present invention can be identified by using one or more of the above assays alone or in combination. It is generally preferred to use a cell-based or cell-free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can be used immediately in this circumstance. Receptor protein modulators identified by these drug screening assays can be used to treat patients with a receptor mediated disease by treating cells that express the estrogen receptor protein. These methods of treatment include the step of administering to the patient a modulator of protein activity in a pharmaceutical composition as disclosed herein, as needed for treatment.
[0097]
The invention further belongs to the novel reagents identified by the screening assays described above. Therefore, it is within the scope of the present invention to further use a reagent identified as disclosed herein in a suitable animal model. For example, a reagent identified as disclosed herein (e.g., an estrogen receptor modulating reagent, an antisense estrogen receptor nucleic acid molecule, an estrogen receptor specific antibody, or an estrogen receptor binding partner) can be treated with such a reagent. Can be used in animal models to determine the efficacy, toxicity, and side effects of the drug. Alternatively, reagents identified as disclosed herein can be used in animal models to determine the mechanism of action of such reagents. Further, the present invention relates to the use of the novel reagents identified by the above-described screening assays for treatment as disclosed herein.
[0098]
The estrogen receptor proteins of the present invention are also useful for providing a subject for diagnosing a disease or susceptibility to a disease mediated by estrogen receptors. Accordingly, the invention provides a method for detecting the presence or level of an estrogen receptor variant (or encoding mRNA) of the invention in a cell, tissue, or organism. The method includes contacting a biological sample with a compound capable of interacting with a receptor protein (or a gene encoding a receptor, mRNA) such that the interaction is detected.
[0099]
One reagent for detecting a protein in a sample is an antibody that can selectively bind to a mutant form of the estrogen receptor protein. Such samples include tissues, cells, and biological fluids present in the patient as well as tissues, cells, and biological fluids separated from the patient.
[0100]
The estrogen receptor protein of the present invention also provides a subject for diagnosing ongoing illness, susceptibility to illness in a patient having a mutant estrogen receptor. The diseases herein include those via estrogen, such as bone growth and cell differentiation. For example, the receptor can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in an aberrant form of receptor activity. This includes amino acid substitutions, deletions, insertions, rearrangements (as a result of aberrant splicing phenomena), and the appropriate post-translational corrections given in FIG. Analytical methods include altered electrical conductivity, altered trypsin digestion, altered receptor activity in cell-based or cell-free assays, altered ligand or antibody binding patterns, altered isoelectric points, direct amino acid sequencing, and mutations in proteins. And other known assay techniques useful for detecting Particularly useful are the variations given in FIG.
[0101]
In vitro techniques for detecting peptides include enzyme linked immunosorbent assays (ELISAs), western blots, immunoprecipitations, and immunofluorescence. Alternatively, the peptide can be detected in the patient in vivo by introducing a labeled anti-peptide antibody into the patient. For example, an antibody can be labeled with a radioactive marker whose presence and location in a patient can be detected by standard imaging techniques. Particularly useful are methods for detecting specific allelic variants of the estrogen receptors disclosed herein, as well as methods for detecting fragments of a peptide in a sample, which are expressed in a patient.
[0102]
The peptides are also useful for pharmacogenomic analysis. Pharmacogenomics deals with genetic variants that are clinically significant for response to drugs due to alterations in the drug processing process and abnormal effects in patients. See, for example, Eichelbaum, M .; (Clin. Exp. Pharmacol. Physiol. 23 (10-11): 983-985 (1996)) and Linder, M .; W. (Clin. Chem. 43 (2): 254-266 (1997)). The clinical consequences of these mutations are that in some individuals the therapeutic agent becomes severely toxic as a result of mutations in metabolic individuals, and in others the drug treatment fails. Thus, an individual's genotype may determine the way in which the therapeutic compound acts on the body, or the way in which the body metabolizes the compound. In addition, the activity of drug metabolizing enzymes affects both the strength and length of drug action. Thus, the pharmacogenomics of an individual can select an effective compound and an effective dosage of the compound for prophylactic or therapeutic treatment based on the genotype of the individual. The discovery of genetic polymorphisms in some drug metabolizing enzymes indicates that standard drug administration may not achieve the expected drug effect, show excessive drug effect, or experience severe toxicity in some patients I will explain. Polymorphisms can be expressed in broad metabolic and poor metabolic gene phenotypes. Thus, genetic polymorphisms lead to allelic variation of a receptor protein, where the function of one or more receptors in one population differs from that of other populations. Thus, the genetic predisposition that can affect the subject's therapy with the peptide can be ascertained. For example, in ligand-based therapies, polymorphisms cause amino-terminal extracellular domains and / or other ligand binding regions where ligand binding is somewhat active, and result in receptor activation. Thus, the dosage of the ligand will, of course, be adjusted to maximize the therapeutic effect in a given population containing the polymorphism / haplotype. Instead of genotype, specific polymorphic peptides can be identified.
[0103]
antibody
The present invention also provides an antibody that selectively binds to the estrogen receptor protein of the present invention, and a fragment thereof. Here, the antibody selectively binds to a target protein if it does not significantly bind to an unrelated protein. Even if the antibody binds to another protein that is not substantially homologous to the target protein, it still binds to the protein selectively as long as the protein has homology to a fragment or domain of the target protein of the antibody. It is regarded. In this case, it is understood that the antibody binding to the protein is still selective despite having some cross-reactivity.
[0104]
Here, antibody is defined by terms consistent with those recognized in the art. These are multi-subunit proteins produced by mammalian organisms in response to antigen challenge. The antibodies of the present invention include polyclonal and monoclonal antibodies, and Fab or F (ab ')₂And Fv fragments, including, but not limited to, antibody fragments.
[0105]
Many methods for generating and / or identifying antibodies to a given peptide of interest are known. Some of the methods are described in Harlow, Antibodies, Cold Spring Harbor Press, (1989). Generally, to produce antibodies, the isolated peptide is used as an immunogen and administered to a mammalian organism such as a rat, rabbit, or mouse. Full length proteins, antigenic peptide fragments, or fusion proteins can be used.
[0106]
Antibodies are preferably prepared from regions or discrete fragments of the estrogen receptor protein. Antibodies can be prepared in any region of the peptides disclosed herein. However, preferred regions include those involved with function / activity and / or receptor / binding partner interactions. Antigenic fragments usually contain at least 10 contiguous amino acid residues. However, the antigenic peptide can include at least 12, 14, 20, or more amino acid residues. Such fragments can be selected for their physical properties, for example, fragments corresponding to regions located on the surface of the protein, such as hydrophobic regions.
[0107]
Detection of an antibody of the invention can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include forcella dish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic base complexes include streptavidin / biotin, and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanase, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of luminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include¹²⁵I,¹³¹I,³⁵S, or³H is included.
[0108]
Use of antibodies
Antibodies can be used to isolate the estrogen receptor proteins of the invention by standard techniques such as affinity chromatography or immunoprecipitation. Antibodies can facilitate the purification of natural proteins from cells and from recombinantly produced proteins expressed in host cells. Furthermore, the antibodies are useful in detecting the presence of the estrogen receptor protein of the present invention in cells or tissues to determine the expression pattern of the protein in various tissues of the organism and during normal development. is there. In addition, the antibodies can be used in situ, in vitro, or for detection of proteins in cell lysates or supernatants to assess expression levels and patterns. This antibody can also be used to assess abnormal tissue distribution or abnormal expression during development. Antibody detection of circulating fragments of the full-length estrogen receptor protein can be used to identify turnover.
[0109]
In addition, the antibodies can be used to assess expression in disease states, such as the active stages of the disease, or in individuals prone to diseases associated with protein function (especially bone growth / formation / deterioration). Accompanying illness). If the modulation is caused by inappropriate tissue distribution, growth expression, protein expression levels, or expressed / engineered forms, the antibodies can be prepared against normal proteins. If the modulation is characterized by a specific mutation in the protein, antibodies specific for the mutant protein can be used in assays for the presence of the specific mutant protein.
[0110]
The antibodies can also be used to assess normal and aberrant gene localization of cells in various tissues of an organism. Diagnostic uses can be applied not only to genetic testing, but also to monitoring therapies. Thus, where treatment is intended to ultimately alter expression levels, or the presence and abnormal tissue distribution of aberrant sequences, or growth-directed expression, it may be directed to that protein or related fragments. Antibodies can be used to monitor therapeutic efficiency.
[0111]
In addition, antibodies are useful for pharmacogenomic analysis. For example, antibodies prepared against a polymorphic protein can be used to identify individuals in need of a modified therapy. This antibody is used as a diagnostic tool as an immunological marker for aberrant estrogen receptor proteins analyzed by electrical conductivity, isoelectric point, tryptic peptide digestion, and other physical assays known in the art. Is also useful.
[0112]
This antibody is also useful for inhibiting protein function, for example, inhibiting binding of estrogen receptor protein to a binding partner such as a ligand. These uses can also be applied to therapeutic situations where treatment involves inhibition of protein function. Antibodies can be used, for example, to inhibit binding, thereby modulating (acting or antagonizing) the activity of a protein. Antibodies can be prepared against specific fragments containing sites where function is required, or against intact proteins associated with cells or cell membranes.
[0113]
The invention also includes a kit for using the antibody to detect the presence of a protein in a biological sample. The kit comprises an antibody, such as a labeled or labelable antibody, and a compound or reagent for detecting an estrogen receptor protein in a biological sample; a means for determining the amount of protein in the sample; Means for comparing the amount of estrogen receptor protein of the present invention to a reference; and guidance for use.
[0114]
Nucleic acid molecule
The present invention further provides an isolated nucleic acid molecule encoding an estrogen receptor protein according to the present invention. The nucleic acid molecule consists of, consists essentially of, or comprises the nucleotide sequence encoding one of the estrogen receptor proteins of the present invention.
[0115]
As used herein, an "isolated" nucleic acid molecule is one that is distinguished from other nucleic acids that are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid was derived (ie, sequences located at the 5 'or 3' end of the nucleic acid). However, it may include some flanking nucleotide sequences, such as up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, especially sequences that encode the peptide continuously and the peptide within the same gene. A sequence that encodes but is separated from introns of the genomic sequence. The important point is that the nucleic acid was isolated from distant, unimportant flanking sequences, which are specialized for recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequence described herein. That is, it is a target of operation.
[0116]
In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, can substantially bind other cellular material, or media when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Not included. However, the nucleic acid molecule can be fused to other coding or regulatory sequences, which are also considered isolated.
[0117]
For example, a recombinant DNA molecule contained in a vector is considered isolated. Further examples of an isolated DNA molecule include a recombinant DNA molecule maintained in a heterologous host cell, or a purified (partially or substantially) DNA molecule in solution. An isolated RNA molecule comprises an RNA transcript of the isolated DNA molecule of the present invention in vivo or in vitro. Further, the isolated nucleic acid molecules according to the present invention include synthetically produced molecules.
[0118]
Accordingly, the present invention provides a nucleic acid molecule comprising one or more of the sequence polymorphisms provided in FIG. 2, consisting of the nucleotide sequence shown in FIG. A nucleic acid molecule consists of a nucleotide sequence because the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.
[0119]
The present invention further provides a nucleic acid molecule comprising one or more of the sequence polymorphisms provided in FIG. 2, consisting essentially of the nucleotide sequence shown in FIG. A nucleic acid molecule consists essentially of a nucleotide sequence in which some additional nucleic acid residues are present in the final nucleic acid molecule.
[0120]
The invention further provides a nucleic acid molecule comprising one or more of the sequence polymorphisms provided in FIG. 2, comprising the nucleotide sequence of FIG. A nucleic acid molecule includes a nucleotide sequence if the nucleotide sequence is a nucleic acid sequence that is part of the final nucleotide sequence of the nucleic acid molecule. Thus, a nucleic acid molecule can have only a nucleotide sequence or additional nucleic acid residues, where the nucleic acid residues are naturally related to the nucleotide sequence or heterologous nucleotide sequence. Such nucleic acid molecules have few additional nucleotides or contain hundreds or more additional nucleotides. A brief description of how these various types of nucleic acid molecules were made / isolated is provided below.
[0121]
An isolated nucleic acid molecule may encode a mature protein and additional amino acids or carboxyl terminal amino acids, or amino acids within a mature peptide (eg, where the mature form has one or more peptide chains). Such sequences may play a role in promoting protein transport, increasing or decreasing protein half-life, or increasing the efficiency of protein manipulation during assays or production, or other reasons, when the protein grows from a precursor to a mature form. . Generally, in situ, additional amino acids are cleaved from the mature protein by cellular enzymes.
[0122]
As noted above, the isolated nucleic acid molecule encodes the mature peptide and additional coding sequences, such as sequences encoding only the estrogen receptor protein, major or minor sequences (eg, pre-pro or pro-protein sequences). In addition to sequences that encode mature peptides, with or without additional coding sequences, and genomic regulatory sequences such as promoters, for example, introns and non-coding 5 'and 3' sequences are transcribed. Are not translated and include, but are not limited to, additional non-coding sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and mRNA stabilization. Further, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide useful for purification.
[0123]
An isolated nucleic acid molecule can be in the form of RNA, such as mRNA, or in the form of DNA, including genomic DNA obtained by cDNA and cloning or chemical synthesis techniques, or a combination thereof. Nucleic acids, especially DNA, are double-stranded or single-stranded. A single strand can be the coding strand (sense strand) or the non-coding strand (antisense strand).
[0124]
Fragments comprise a contiguous nucleotide sequence of 12 or more nucleotides. Further, fragments can be at least 30, 40, 50, 100, 250, or 500 nucleotides in length. The length of the fragment depends on the purpose of use. For example, fragments encode the epitope-behaving portions of the peptide or are useful as DNA probes or primers. Such fragments can be isolated using known nucleotide sequences for synthesizing oligonucleotide probes. Labeled probes can be used to isolate nucleic acids corresponding to the coding portion, to screen cDNA libraries, genomic DNA libraries or mRNA. In addition, primers can use PCR reactions to clone specific portions of the gene.
[0125]
The probe / primer typically comprises a substantially purified oligonucleotide or oligonucleotide pair. Oligonucleotides typically include a portion of a nucleotide sequence that hybridizes under stringent conditions to be at least about 12, 20, 25, 40, 50 or more contiguous nucleotides.
[0126]
As used herein, the term "hybridizes under stringent conditions" refers to a residue which typically hybridizes to each other and hybridizes until the nucleotide sequences encoding the peptides have at least 50-55% homology to each other. And cleaning. The conditions typically remain hybridized to each other and have at least about 65%, at least about 70%, or at least about 75% or more homology with each other. Such stringent conditions are known to those of skill in the art and are described in Current Protocols in Molecular Biology, John Wiley & Sons, N.W. Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions is to hybridize at 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C. and wash at least once with 0.2 × SSC, 0.1% SDS at 50-65 ° C. Condition.
[0127]
Use of nucleic acid molecules
The nucleic acid molecules of the invention are useful for probes, primers, chemical intermediates, and biological assays. Probes can span any sequence over the entire length of the nucleic acid molecule consisting of the nucleotide sequence of FIG. 1, including one or more of the sequence polymorphisms shown in FIG. Thus, it can be derived from a 5 'non-coding part, a coding part, and a 3' non-coding part. However, as mentioned above, fragments do not include fragments published prior to the present invention.
[0128]
Nucleic acid molecules are useful for amplifying any given portion of the nucleic acid molecule by PCR and for the synthesis of anti-sensitizing molecules of desired length and sequence.
[0129]
Nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express part or all of the peptide sequence. Vectors include insertion vectors and are used in fusions with other nucleic acid molecule sequences, for example, in the genome of a cell, to alter the expression status of a gene and / or gene product. For example, the endogenous coding sequence can be homologously recombined with one or more specifically mutated coding portions for all or a portion thereof.
[0130]
Nucleic acid molecules are also useful for indicating the antigenic portion of a protein.
Nucleic Acid Molecules Also useful from the nucleic acid molecules described herein to design ribozymes corresponding to full or partial mRNA.
The nucleic acid molecules are also useful for constructing host cells that express some or all of the nucleic acid molecules and peptides.
[0131]
The nucleic acid molecules are also useful for constructing transgenic animals that express all or a portion of the nucleic acid molecules and peptides.
Nucleic acid molecules are also useful for making vectors that express some or all of the peptide.
[0132]
Nucleic acid molecules are also useful as hybridizing probes to determine the presence, level, morphology and distribution of nucleic acid molecule expression. Accordingly, probes can be used to detect or determine the level of a particular nucleic acid molecule in cells, tissues and organisms. Nucleic acid levels are determined in DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to evaluate expression and / or gene copy number in a given cell, tissue, or organism. The use of these is equivalent to the diagnosis of abnormalities associated with increased or decreased estrogen receptor protein expression compared to normal.
[0133]
In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for DNA detection include Southern hybridizations and in situ hybridizations.
[0134]
The probe can be used as part of a diagnostic test kit to identify cells or tissues that express the estrogen receptor protein, which is a receptor-encoding nucleic acid, such as mRNA or genomic DNA, in a sample of cells from a subject. Or by detecting whether the estrogen receptor gene is mutated.
[0135]
Nucleic acid expression assays are useful as screening reagents to identify compounds that modulate estrogen receptor nucleic acid expression.
[0136]
Thus, the present invention provides methods for identifying compounds that can be used to treat abnormalities associated with estrogen receptor gene nucleic acid expression. The method typically assayes the ability of the compound to modulate estrogen receptor nucleic acid expression and determines if the agent can be used to treat an abnormality characterized by unwanted estrogen receptor nucleic acid expression. These assays can be performed in cell-based and cell-free systems. Cell-based assays include cells that naturally express estrogen receptor nucleic acid, or recombinant cells that are genetically designed to express a particular nucleic acid sequence.
[0137]
Assays of estrogen receptor nucleic acid expression are performed by direct assays at the nucleic acid level, such as at the mRNA level, or by secondary compounds involved in signal pathways. In addition, increased or decreased regulation of gene expression on the estrogen receptor protein signaling pathway is assayed. In this embodiment, the regulatory portion of these genes can be linked to a reporter gene such as luciferase.
[0138]
The modulator of estrogen receptor gene expression is identified by contacting the cell with a candidate compound and detecting mRNA expression. The level of estrogen receptor mRNA expression in the presence of the candidate compound is compared to the level of estrogen receptor mRNA expression in the absence of the candidate compound. Candidate compounds are determined as modulators of nucleic acid expression based on this comparison and are used, for example, in treating abnormalities characterized by abnormal nucleic acid expression. If the expression of the mRNA is statistically significantly greater in the presence of the candidate compound compared to its absence, the candidate compound is identified as a stimulator of nucleic acid expression. If the expression of the mRNA is statistically significantly lower in the presence of the candidate compound compared to its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
[0139]
The present invention provides a method for treating a nucleic acid using a compound specified through drug screening as a gene modulator that modulates estrogen receptor nucleic acid expression in cells or tissues that express the estrogen receptor. Modulation includes the modulation of both up-modulation (eg, activation or agonism) or down-regulation (suppression or antagonism), or nucleic acid expression.
[0140]
Alternatively, the modulator of estrogen receptor nucleic acid expression is a screening assay described herein, so long as the agent or small molecule inhibits estrogen receptor nucleic acid expression in cells or tissues in which the protein is expressed. Are small molecules or drugs identified using
[0141]
Nucleic acid molecules are useful in clinical trials or in treatment management to monitor the effectiveness of modulatory compounds on estrogen receptor gene expression or activity. The gene expression pattern is a barometer of the continuous effect of the treatment with the compound, and is particularly useful for compounds for which the patient is resistant. Gene expression patterns are also useful as markers that indicate the physiological response of cells affected by the compound. Thus, such a monitor would permit an increase in the dose of the compound or the administration of another compound to which the patient is not resistant. Similarly, if the level of nucleic acid expression falls below the desired level, the administration of the compound can be proportionally reduced.
[0142]
Nucleic acid molecules are also useful in diagnostic assays for qualitative changes in estrogen receptor nucleic acid expression, and particularly elicit qualitative changes in disease states. Nucleic acid molecules can be used to detect mutations in gene expression products such as estrogen receptor genes and mRNA. Nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the estrogen receptor gene, and to determine whether a subject with a mutation is at risk for an abnormality caused by the mutation. it can. Mutations can include deletions, additions, or substitutions of one or more nucleotides in a gene; chromosomal rearrangements such as transposition or transfer; changes in genomic DNA such as abnormal methylation patterns, changes in the number of gene copies such as amplification, and the like. Including. Detection of a mutant form of the estrogen receptor gene associated with inverse function can be used to assess activity, or to determine susceptibility to the disease if the disease is the result of overexpression, underexpression, or mutation of the estrogen receptor protein. It is a diagnostic method.
[0143]
Individuals having a mutation in the estrogen receptor gene can be detected at the genetic level by various techniques. Genomic DNA can be analyzed directly or amplified using PCR prior to analysis. RNA or cDNA can be used as well. In some applications, the detection of mutations involves the use of probes / primers in the polymerase chain reaction (PCR) (U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR. Or, differently, to the ligation chain reaction (LCR) (see Landegran et al., Science 241: 1077-1080 (1988); and Nakazawa et al., PNAS 91: 360-364 (1994)). However, the latter is particularly effective in detecting point mutations in genes (see Abravaya et al., Nucleic Acids Res. 23: 675-682 (1995)). The method comprises the steps of collecting a cell sample from a patient, isolating a nucleic acid (eg, genomic, mRNA, or both) from the cells of the sample, and subjecting the nucleic acid sample to hybridization (if present) and amplification of the gene (if present). Contacting the gene with one or more primers specifically hybridized to the gene, detecting the presence or absence of the amplification product, or detecting the size of the amplification product and comparing it to the length of a control sample. . Absences and insertions can be detected by changes in size by comparing the amplification product to a normal genotype. Point mutations can be identified by hybridizing the amplified DNA with normal RNA or unsensitized DNA.
[0144]
Alternatively, mutations in the estrogen receptor gene can be identified directly, for example, by determining the modified restricted enzyme digestion pattern by gel electrophoresis.
In addition, sequence-specific ribozymes (U.S. Patent No. 5,498,531) can be scored for the presence of specific mutations by growing or reducing ribozyme cleavage sites. Perfectly matched sequences can be distinguished from unmatched sequences by nuclease cleavage digestion assays or differences in melting points.
[0145]
Sequence changes at specific positions can be assessed by nuclease protection assays such as RNase and S1 protection or by chemical cleavage methods. In addition, sequence differences between the mutant estrogen receptor gene and the wild-type gene are determined by direct DNA sequencing. Various types of automatic sequencing means are effective when performing diagnostic assays (Naeve, CW, (1995) Biotechniques 19: 448) and include sequencing by mass spectrum (PCT International Publication No. WO 94/94). Cohen et al., Adv. Chromatogr. 36: 127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38; 147-159 (1993)).
[0146]
Other methods for detecting gene mutations include protecting against cleavage reagents to detect incompatible bases in RNA / RNA or RNA / DNA duplexes (Myers et al., Science 230; 1242 (1985)); Cotton et al. , PNAS 85: 4397 (1988); Saleeba et al. , Meth. Enzymol. 217: 286-295 (1992)), comparing the electrophoretic amounts of mutant and wild-type nucleic acids (Orita et al., PNAS 86; 2766 (1989); Cotton et al., Mutat. Res. 285: 125-). 144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9: 73-79 (1992)) and denaturing gradient gel electrophoresis, in polyacrylamide gels containing gradient concentrations of denaturing agents. Which assay for the migration of a mutant or wild type fragment (Myers et al., Nature 313: 495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.
[0147]
Nucleic acid molecules are also useful for testing individuals of genotype that do not unnecessarily cause disease and affect therapeutic modality. The nucleic acid molecule can then be used to study the correlation between an individual's genotype and the individual's response to the compound used for therapy (pharmacogenomic correlation). Accordingly, the nucleic acid molecules described herein can be used to evaluate the amount of estrogen receptor gene mutation in an individual to select preferred compounds and therapeutic dosages.
[0148]
And the nucleic acid molecule which shows the gene mutation which affects a therapy provides the diagnostic subject which can be used for the tailor therapy for an individual. Therefore, the production of recombinant cells and animals having these polymorphisms / haplotypes allows for effective clinical design of drugs and dosages.
[0149]
Nucleic acid molecules regulate estrogen receptor gene expression in cells, tissues and organisms, and are therefore useful for insensitivity formation. DNA-insensitized nucleic acid molecules are designed to be complementary to the portion of the gene involved in transcription to prevent transcription and subsequent production of the estrogen receptor protein. The insensitive RNA or DNA nucleic acid molecule hybridizes to the mRNA and blocks translation of the mRNA into estrogen receptor protein.
[0150]
Alternatively, one of the desensitizing molecules is used to inactivate mRNA to reduce expression of estrogen receptor nucleic acid. Thus, these molecules can treat abnormalities characterized by abnormal or unwanted estrogen receptor nucleic acid expression. This technique involves cleavage by ribozymes in which the nucleic acid sequence that reduces the ability of the mRNA to be translated is complementarily included in one or more portions of the mRNA. Possible moieties are coding moieties associated with the catalytic and other functional activities of estrogen receptor proteins, such as coding moieties and especially substrate binding moieties.
[0151]
The nucleic acid molecule further provides a vector for gene therapy in a patient comprising cells having abnormal estrogen receptor gene expression. The recombinant cells, including the patient's cells that have been modulated ex vivo and returned to the patient, are then introduced into a position where the patient's cells produce the desired estrogen receptor protein for treatment of the individual.
[0152]
The invention also includes a kit for detecting the presence of an estrogen receptor nucleic acid in a biological sample. For example, the kit comprises a labeled or labelable nucleic acid or an agent capable of detecting estrogen receptor nucleic acid in a biological sample; a means for determining the amount of estrogen receptor nucleic acid in the sample; Means for comparing the amount of estrogen receptor nucleic acid with a standard. The compound or agent is enclosed in a suitable container. The kit further includes instructions for using the kit to detect estrogen receptor protein mRNA or DNA.
[0153]
Design of method for detecting SNP-containing nucleic acid
The SNP nucleic acid molecules of the invention are useful in probes, primers, chemical intermediates, and biological assays for SNPs. Probes / primers are consistent with one or more of the SNPs shown in FIG. 2, or with 5 'and / or 3'. However, as noted above, fragments do not include fragments that are not associated with the SNPs of the present invention, or that are known in the art of SNP detection. The SNP-containing nucleic acid molecules and information provided herein are also useful for PCR to amplify the SNPs provided in the present invention and to design PCR primers to design formatted SNP detection reagents / kits.
[0154]
The probe / primer typically comprises a substantially purified oligonucleotide or oligonucleotide pair. Oligonucleotides typically include a portion of a nucleotide sequence that has been hybridized under stringent conditions to be at least about 12, 20, 25, 40, 50 or more contiguous nucleotides. Based on certain conditions, the contiguous nucleotides can include the SNP position of interest or become a specific portion sufficiently close to the nearby 5 'and / or 3' SNP that is the SNP position performing the desired assay. You can also.
[0155]
Preferred primer and probe sequences can be readily determined using the sequences provided in FIGS. 1, 2, and 9. It will be apparent to those skilled in the art that such primers and probes are useful as diagnostic or amplification primers for genotyping the SNPs of the present invention and can be incorporated into kit forms.
[0156]
To analyze SNPs, it is appropriate to use oligonucleotides specific to SNP alleles (designated as "allele-specific oligonucleotides", "allele-specific probes", or "allele-specific primers"). . The design and use of allele-specific probes for analyzing polymorphisms is described, for example, in Saiki et al. , Nature 324, 163-166 (1986); Datatagta, EP 235,726, Saiki, WO 89/11548.
[0157]
In a hybridization-based assay, an allele-specific probe is designed to hybridize to a segment of DNA of interest from one human, but not to another human-derived segment where different polymorphisms are present. . Hybridization conditions should be sufficiently stringent because there are significant differences in hybridization intensity between alleles, preferably significant binary responses, so that the probe hybridizes to only one of the alleles. Soy. Since some probes are designed to hybridize to a segment of the DNA of interest, the polymorphic portion is arranged at the central position of the probe (eg, 15-mer at position 7, 16-mer at position 8 or 9). . The design of the probe allows the hybridization between the different alleles to be well distinguished.
[0158]
Allele specific probes are often used in pairs, where "pairs" are identical except for a single nucleotide mismatch representing an allelic variation at the SNP position. One of the pair is completely identical to the subject sequence and the other is completely identical to the mutant. In a sequence, several pairs of probes can be immobilized on the same carrier for simultaneous analysis of multiple polymorphisms within the same sequence of interest.
[0159]
In one type of PCR-based assay, the allele-specific primer hybridizes to the portion of the DNA of interest that overlaps the SNP position, and only primers of the allele type, for which the primers exhibit perfect complementarity, are amplified. (See Gibbs, Nucleic Acid Res. 17 2427-2448 (1989)). This primer is used in conjunction with a second primer that hybridizes at the end. Amplification results from the two primers, resulting in a product that can indicate the presence of a particular allelic type. Controls are usually performed with two pairs of primers. One pair is mismatched at the single base of the polymorphic portion and the other pair is completely complementary to the peripheral portion. Single base mismatches prevent amplification and no product is formed. This method works best when the mismatch is included at the 3'-most position of the polymorphic oligonucleotide. This is because this position extends from the primer and is most unstable (see, for example, WO 93/22456). This PCR-based assay is utilized as part of the TaqMan assay and is described as follows.
[0160]
SNP detection kit, nucleic acid sequence, and integration system
The present invention further provides SNP detection kits, such as sequences or microsequences of nucleic acid molecules, probe / primer sets, based on the SNPs provided in FIGS. 1, 2, 4, 8, and 9.
[0161]
In one embodiment of the invention, a kit is provided that includes the necessary reagents to perform one or more assays that detect one or more SNPs disclosed herein. The present invention also provides a multi-component incorporation system for analyzing SNPs in the present invention.
[0162]
A SNP detection kit can include one or more oligonucleotide probes, or pairs of probes, that hybridize at or near an individual SNP location. To test multiple SNPs, including at least one of the SNPs of the present invention, multiple pairs of allele specific oligonucleotides can be included in the kit simultaneously. In some kits, such as sequences, allele specific oligonucleotides are provided and immobilized on a substrate. For example, the same substrate may be at least 1; 10; 100; 1000; 10000; 100,000; 300,000, or alleles to detect nearly all of the polymorphisms shown in FIGS. 1, 2, 4, 8, and 9. Gene specific oligonucleotide probes can be included.
[0163]
In particular, the present invention relates to a method comprising the steps of: (a) providing an allele-specific oligonucleotide capable of binding one of the nucleic acid probes, for example, a fragment of the SNP-containing human genome described herein; And (b) a compartmentalized kit comprising one or more containers that can include one or more of a detergent or a reagent capable of detecting the presence of a bound probe.
In particular, compartmentalized kits each have an isolated container in which a plurality of reagents are placed. Such containers include small glass containers, plastic containers, plastic stripes, glass or paper, or ordered materials such as silica. Such containers efficiently transfer reagents from one compartment to another, do not cause cross-contamination of samples and reagents, and quantitatively add reagents or solutions from each container from one container to another. Is done. Such containers include a container for holding a test sample, a container for holding a nucleic acid probe, a container for holding a washing solution (phosphate buffer, Tris buffer, etc.), and a container for holding a reagent for detecting a bound probe. Further, the kit can include reagents for PCR or other enzymatic reactions, and instructions for using the kit. A person skilled in the art can identify the unspecified SNP of the present invention, identify it routinely using the sequences described herein, and use it by folding it into a well-known kit format.
[0164]
Further, the invention provides a sequence or microsequence of a nucleic acid molecule based on the sequence information of FIG. 1, comprising one or more of the variants of FIG.
[0165]
As used herein, `` sequence '' or `` microarray '' refers to a sequence of discrete polynucleotides or oligonucleotides synthesized on a substrate, wherein the substrate is paper, nylon or other types of membranes, filters, chips, glass slides or Other suitable solid carriers. In one embodiment, the microarray is a U.S.A. S. Patent 5,837,832, Chee et al. , PCT Application WO 95/11995 (Cee et al.), Lockhart, D .; J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Shena, M .; et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), which are incorporated herein by reference. In other embodiments, such sequences are described in Brown et al. , U.S. S. Patent No. 5,807,522. Sequences or microarrays are generally referred to as "DNA chips."
[0166]
In individual probes or pairs of probes corresponding to different SNP positions, multiple oligonucleotide probes, such as allele specific oligonucleotides, are sequenced. The oligonucleotide is synthesized in a specific region of the substrate by light irradiation chemical treatment. The substrate is a paper, nylon or other type of membrane, filter, chip, glass slide, or other suitable solid carrier.
[0167]
Hybridization assays based on oligonucleotide sequences rely on differences in perfectly matched and mismatched sequence variations of interest in the hybridization stability of short oligonucleotide probes. Polymorphism information is efficiently obtained by a basic structure that includes a high density sequence of oligonucleotide probes attached to a solid support (eg, chip) at selected locations. Individual DNA chips are arranged in a grid, miniaturized to dime size, and correspond to individual specific SNP positions or allelic variations, and contain thousands to millions of DNA probes. Preferably, the probes are bound to a solid support in an addressable sequence.
[0168]
Array / chip technology has already been successfully applied in many cases. For example, the BRCA1 gene, s. Mutation screening was performed on cerevisiae mutant strains and the HIV-1 virus protease gene (Hasia et al., 1996; Shomaker et al., 1996; Kozal et al., 1996). Mutant chips used for SNP detection can be manufactured on customized substrates.
[0169]
Useful sequence-based tiling schemes for detecting SNPs are described in EP 785280. That is, the sequence is generally “tiled” to many specific polymorphisms. "Tiling" refers to a defined synthesis of an oligonucleotide probe consisting of a sequence complementary to the sequence of interest, as well as a sequence variant selected previously, eg, one of one or more monomer bases. The above substitution at the given position, that is, nucleotide. The tiling plan is described in PCT Application No. This is further described in WO 95/11995. In certain aspects, sequences are tiled to many specific SNPs. In particular, the sequences are tiled such that each detection block contains many detection blocks specific to a particular SNP or set of SNPs. For example, the detection block is tiled to include a number of probes that measure a sequence segment that includes a particular SNP. Probes are synthesized in different pairs at SNP positions to obtain probes that exhibit complementarity to individual alleles. Mono-substituted probes, as well as probes that differ at SNP positions, are also generally tiled within the detection block. This method is immediately applicable to the SNP information described here.
[0170]
These monosubstituted probes have a fixed number of bases in either direction from the polymorphism replaced by nucleotide residues (selected from A, T, G, C, and U). Generally, the tiling detection block probe contains a substitution at a sequence position 5 bases away from the SNP. Monosubstituted probes provide an internal control to the tiling sequence to distinguish actual hybridization from artificial hybrid hybridization. In completing a hybrid with a subject sequence and a wash of the sequence, the sequence is considered to determine its position in the sequence to which the subject sequence hybridizes. Hybridization data from the scanned sequence is analyzed to identify the presence of the allele or SNP allele in the sample. Hybridization and scanning are described in PCT Application No. WO 92/10092, WO 95/11995 and U.S. Pat. S. Patent No. Performed as described in 5424186.
[0171]
Thus, in some embodiments, the chip comprises a fragment nucleic acid sequence of about 15 nucleotides in length. In other embodiments, the chip comprises at least one sequence selected from the group described in FIGS. 1, 2, 8, and 9, a complementary sequence, a fragment, at least eight or more, preferably 10, 15, Fragments containing 20, more preferably 25, 30, 40, 47 or 50 contiguous nucleotides and polymorphic bases. In some embodiments, the polymorphic base is within 5, 4, 3, 2, or 1 nucleotides from the center of the polynucleotide, more preferably at the center of the polynucleotide. In another embodiment, the chip comprises a sequence having some of the polynucleotides of the invention.
[0172]
Oligonucleotides are synthesized on the substrate by chemical bonding and inkjet processing equipment, which is described in PCT application WO 95/251116 (Baldeshweiler et al.), All of which are incorporated herein by reference. In another example, a "lattice" sequence is one in which dots (or slots) place and bind cDNA fragments or oligonucleotides to the surface of the substrate by a vacuum system, heating, UV, mechanical or chemical bonding processes. Arrays such as those described above may be manual or suitable machines (slot blot or dot blot equipment), materials (either suitable solid carriers) and machines (robotized equipment), and 8,24,96,384,1536. , 6144 or more, or oligonucleotides that make efficient use of commercially used equipment.
[0173]
Using such a sequence, the present invention provides a method for identifying a SNP according to the present invention. In particular, such methods include incubating a test sample with one or more nucleic acid molecules and assaying the binding of the nucleic acid molecules to components in the test sample. This assay comprises a sequence comprising a number of genes, at least one SNP according to the invention and / or one allele of a SNP according to the invention.
[0174]
Incubation conditions for the test sample and the nucleic acid molecule vary. Incubation conditions will depend on the form used in the assay, the detection method employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art can prepare the commonly applied hybridization, amplification, or sequence assay formats as appropriate for using the novel SNPs of the human genome described herein. Examples of such assays are described in Chard, T, An Introduction to Radioimmunoassay and Related Technologies, Elsevier Science Publishers, Amsterdam, The Netherlands, The Netherlands, The Netherlands, The Netherlands; R. et al. , Technologies in Immunochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P .; , Practice and Theory of Enzyme Immunoassay: described in Laboratory Technologies in Biochemistry and Molecular Biology, Physiological Sciences, 85, 85, 85, 85, 85.
[0175]
The test sample of the present invention can be obtained from proteins, nucleic acid extracts, cell membrane extracts obtained from body fluids (blood, urine, saliva, sputum, gastric juice, etc.), cultured cells, biopsy materials, or other tissue preparations. It is possible, but not limited to these. The test sample used by the above method will vary based on the assay format, the nature of the detection method, and the tissues, cells, and extracts used as the sample. Methods for preparing nucleic acids, proteins, or cell extracts are well known to those skilled in the art and can be readily adapted to obtain samples that are compatible with the system utilized.
[0176]
Multi-component integration systems can also be used for SNP analysis. Such systems miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. Examples of such techniques are described in US patent 5,589,136 and describe PCR amplification and capillary electrophoresis of chips.
[0177]
Embedded systems can be considered mainly when microfluidic systems are used. These systems include microchannel types contained in microchips designed on glass, silicon, quartz, or plastic wafers. The movement of the sample is controlled by electrical, electroosmotic, or hydrostatic forces applied to various areas of the microchip to create a functional microscopic valve and pump without any moving parts. Changing the voltage controls the liquid flow rate at the intersection between the micromachined channels and changes the liquid flow rate of the pump across the various cross sections of the microchip.
[0178]
To genotype SNPs, microfluidic systems synthesize detection methods such as, for example, nucleic acid amplification, miniature sequence primer extension, capillary electrophoresis, and laser-induced fluorescence detection.
[0179]
In a first step, the DNA sample is amplified, preferably by PCR. The amplification products are then hybridized immediately upstream of the targeted polymorphic base (ddNTPs (specific fluorescence for individual ddNTPs)) and automated mini-sequences using appropriate oligonucleotide mini-sequence primers. Follow the reaction. Once the 3 'end extension is completed, the primers are isolated by capillary electrophoresis from the unencapsulated fluorescent ddNTPs. The isolation medium used in capillary electrophoresis can be, for example, polyacrylamide, polyethylene glycol, or dextran. The ddNTPs contained in a single nucleotide primer extension product are identified by laser-induced fluorescence detection. This microchip can be used to process at least 96 to 384 or more samples in parallel.
[0180]
Vector / host cell
The invention also provides a vector comprising a nucleic acid molecule described herein. "Vector" means a vehicle, preferably a nucleic acid molecule, for transporting a nucleic acid molecule. When the vector is a nucleic acid molecule, the nucleic acid molecule is covalently linked to the vector nucleic acid. In this aspect of the invention, vectors include plasmids, single- or double-stranded phage, single- or double-stranded RNA or DNA viral vectors, or artificial chromosomes, for example, BAC, PAC, YAC, MAC And the like.
The vector can be maintained in the host cell as an extrachromosomal component, where it replicates and produces another copy of the nucleic acid molecule. Alternatively, the vector may integrate into the host cell genome and produce another copy of the nucleic acid molecule as the host cell replicates.
[0181]
The present invention provides a vector for maintenance (cloning vector) or a vector for expression of a nucleic acid molecule (expression vector). Vectors can function in prokaryotic or eukaryotic cells, or both (shuttle vectors).
The expression vector contains a cis-acting regulatory region, which is operably linked to the nucleic acid molecule in the vector such that transcription of the nucleic acid molecule can occur in a host cell. A nucleic acid molecule can be introduced into a host cell along with another nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor that interacts with the cis-regulatory control region to allow transcription of the nucleic acid molecule from the vector. Alternatively, the trans-acting factor may be supplied by a host cell. Also, trans-acting factors can be produced from the vector itself. However, it is understood that in some experiments, transcription and / or translation of the nucleic acid molecule can occur in a cell-free system.
[0182]
The regulatory sequences to which the nucleic acid molecules described herein are operably linked include a promoter that directs mRNA transcription. This includes, but is not limited to, the remaining promoters from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the immediate CMV promoter, the adenovirus early and late promoters, and the retrovirus. Contains the viral long terminal repeat.
In addition to a control site that promotes transcription, the expression vector may also include a transcription regulatory site such as a repressor binding site or an enhancer. Examples include the SV40 enhancer, cytomegalovirus immediate enhancer, polyoma enhancer, adenovirus enhancer, retrovirus LTR enhancer.
[0183]
In addition to including sites for initiation and control of transcription, the expression vector can include sequences necessary for termination of transcription, and, in the site to be transcribed, may include a ribosome binding site for translation. It is possible. Other regulatory control elements for expression also include start and stop codons, as well as a polyadenylation signal. One of skill in the art would know many regulatory sequences useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. , Molecular Cloning: Laboratory Manual. 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
[0184]
A variety of expression vectors can be used for expressing the nucleic acid molecule. Such expression vectors include chromosomal vectors, episomal vectors, and virus-derived vectors, for example, bacterial plasmid derived, bacteriophage derived, yeast episomal derived, yeast chromosome elements including yeast artificial chromosomes, baculovirus, papovavirus such as SV40 , Vaccinia virus, adenovirus, poxvirus, pseudorabies virus, retrovirus and the like. Vectors may be derived from combinations of these sources, such as from plasmids and bacteriophage genetic elements, eg, cosmids, phagemids, and the like. Suitable cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al. , Molecular Cloning: Laboratory Manual. 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
[0185]
The regulatory sequence may provide for constitutive expression in one or more host cells (ie, tissue-specific), or may be one such as by temperature, nutrient additives, or an exogenous factor such as a hormone or another ligand. Inducible expression in one or more cell types may be provided. A variety of vectors that provide for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of skill in the art.
[0186]
The nucleic acid molecule can be inserted into the vector nucleic acid by well-known methodologies. Generally, the final expressed DNA sequence is ligated to the expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes, and then linking the fragments together. Procedures for digestion and ligation of restriction enzymes are well known to those skilled in the art.
Vectors containing appropriate nucleic acid molecules can be introduced into host cells suitable for growth or expression using well known techniques. Bacterial cells include, but are not limited to, E. coli, actinomycetes, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
[0187]
As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the present invention provides a fusion vector capable of producing a peptide. Fusion vectors can enhance recombinant protein expression, increase the solubility of the recombinant protein, and aid in protein purification, for example, by acting as a ligand for affinity purification. A cleavage site for protein hydrolysis may be introduced at the junction of the fusion moiety, so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Representative fusion expression vectors include pGEX (Smith et al., Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), And glutathione S- against recombinant proteins of interest. PRIT5 (Pharmacia, Piscataway, NJ) fused to transferase (GST), maltose E-binding protein, or protein A, respectively.
Examples of suitable inducible non-fused Escherichia coli expression vectors include pTrc (Amann et al., Gene 69: 301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods 18th org. -89 (1990)).
[0188]
Recombinant protein expression can be maximized in host bacteria by providing a genetic background with a reduced ability of the host cell to proteolytically cleave the recombinant protein (Gottesman, S. et al. , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be modified to provide preferential codon usage for a particular host cell, such as E. coli (Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992)).
[0189]
The nucleic acid molecule can be expressed by an expression vector that acts in yeast. Yeasts such as S. Examples of vectors for expression in S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6: 229-234 (1987)) and pMFa (Kurjan et al., Cell 30: 933-943 (1982)). ), PJRY88 (Schultz et al., Gene 54: 113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, CA).
[0190]
Nucleic acid molecules can also be expressed in insect cells, for example, using a baculovirus expression vector. Baculovirus vectors useful for protein expression in cultured insect cells (eg, Sf 9 cells) include the pAc system (Smith et al., Mol. Cell Biol. 3: 2156-2165 (1983)) and the pVL system (Lucklow). et al., Virology 170: 31-39 (1989)).
In one embodiment of the invention, the nucleic acid molecules described herein are used in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329: 840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6: 187-195 (1987)).
[0191]
The expression vectors listed here are provided by way of example only among well-known vectors useful for expressing nucleic acid molecules and available to those of skill in the art. One of skill in the art would know of other vectors suitable for maintaining, propagating or expressing the nucleic acid molecules described herein. These are described, for example, in Sambrook, J .; Fritsh, E .; F. , And Maniatis, T .; Molecular Cloning: A Laboratory Manual. 2nd, ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
[0192]
The invention also includes vectors where the nucleic acid sequences described herein have been cloned in the reverse orientation into the vector, but are operably linked to regulatory sequences that permit transcription of the antisense RNA. Thus, antisense transcripts can be produced for all or a portion of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. The expression of the antisense RNA is in accordance with the above-mentioned parameters relating to the expression of the sense RNA (regulatory sequence, constitutive or inducible expression, tissue-specific expression).
[0193]
The present invention also relates to a recombinant host cell comprising the vector described herein. Host cells thus include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
Recombinant host cells are produced by introducing the vector constructs described herein into cells by techniques readily available to those of skill in the art. These include calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, lipofection, and Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;
[0194]
A host cell can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, a nucleic acid molecule can be introduced alone or with other nucleic acid molecules unrelated to the nucleic acid molecule that provides the trans-acting factor for the expression vector. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced, or ligated to a nucleic acid molecule vector.
[0195]
In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated viruses by standard methods of infection and transduction. Viral vectors can be replication competent or replication defective. When viral replication is defective, replication occurs in a host cell that functions to complement the defect.
[0196]
Vectors generally contain a selectable marker from which a subpopulation of cells containing the recombinant vector construct can be selected. The marker may be included on the same vector containing the nucleic acid molecules described herein, or may be on a separate vector. Markers include the tetracycline or ampicillin resistance gene for prokaryotic host cells and the dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that selects a phenotypic trait may be effective.
[0197]
While mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of appropriate regulatory sequences, cell-free transcription and translation systems can be performed using RNA derived from the DNA constructs described herein. They can also be used to produce these proteins.
If secretion of the peptide is required, it is difficult to perform on a multi-transmembrane domain containing proteins such as the estrogen receptor, but an appropriate secretion signal is added into the vector. The signal sequence may be endogenous to the peptides or may be atypical to these peptides.
[0198]
If the peptide is not secreted into the medium, which is a typical case of the estrogen receptor, the protein can be isolated from host cells by standard disruption methods, including freeze-thaw, sonication, mechanical disruption, use of lytic agents, and the like. Can be isolated from The peptide is then precipitated by ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography, etc. And can be recovered and purified by the well-known purification method described above.
[0199]
Depending on the host cell in the recombinant production of the peptides described herein, the peptide may not be glycosylated as in cells produced in bacteria, but may have different glycosylation patterns. It is also understood that there is. Further, in some cases, such as the result of a host-mediated process, the peptide may include an initial modified methionine.
[0200]
Use of vectors and host cells
Recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing estrogen receptor proteins or for producing peptides that can be further purified to produce desired amounts of estrogen receptor proteins and fragments. Therefore, host cells containing the expression vector are useful for peptide production.
[0201]
Host cells are also useful in cell-based assays involving estrogen receptor proteins or estrogen receptor protein fragments as described above, as well as in formats known in the art. Therefore, recombinant host cells that express native estrogen receptor protein are useful for assaying substances that stimulate or suppress estrogen receptor protein function.
Host cells are also useful for identifying estrogen receptor protein mutants that affect their function. If the mutant occurs spontaneously and causes pathology, the host cell containing the mutation may have an effect on the native estrogen receptor protein that is It is useful for analyzing a compound having a desired effect (for example, a stimulating function or a suppressing function) on the mutant.
[0202]
Genetically engineered host cells can be further used to produce non-human transgenic animals. The transgenic animal is preferably a mammal, for example a rodent such as a rat or mouse, wherein one or more of the animal's cells contains the transgene. A transgene is exogenous DNA that is integrated into the genome of the cell in which the transgenic animal has developed and remains in the genome of the adult animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of estrogen receptor protein, and for identifying and evaluating modulators of estrogen receptor protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0203]
Transgenic animals can be produced by introducing a nucleic acid into the male pronucleus of a fertilized oocyte. For example, it can be produced by microinjection, retrovirus infection, development of oocytes in pseudopregnant female foster animals, and the like. Any of the estrogen receptor protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.
Any regulatory sequences or other sequences useful in expression vectors can form part of the transforming sequence. This includes the intron sequence and polyadenylation signal if not already included. Tissue-specific regulatory sequences are operably linked to the transgene and direct specific cells to express estrogen receptor protein.
[0204]
Methods for creating transgenic animals by embryo manipulation and microinjection are routine in the art, especially for animals such as mice, and are described, for example, in US Pat. Nos. 4,736,866 and No. 4,870,009 (both Leder et al.), U.S. Pat. No. 4,873,191 (Wagner et al.), And Hogan, B.A. , Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Similar methods are used for the production of other transgenic animals. The basal animal for transformation can be identified based on the transgene in its genome and / or the expression of the transformed mRNA in tissues or cells of the animal. The transgenic animal can then be used to generate another animal that carries the transgene. In addition, a transgenic animal carrying a transgene can be improved to another transgenic animal carrying another transgene. Transgenic animals also include animals that produce whole animals or their tissues using the homologous recombination host cells described herein.
[0205]
In another embodiment, transgenic non-human animals can be produced comprising a selection system capable of regulating transgene expression. One example of such a system is the cre / oxP recombinase system of bacteriophage P1. Regarding the cre / oxP recombinase system, see, for example, Lakso et al. PNAS 89: 6232-6236 (1992). Another example of a recombinase system is the S. recombinase system. cerevisiae FLP recombinase system (O'Gorman et al. Science 251: 1351-1355 (1991)). If the cre / oxP recombinase system is used to regulate transgene expression, an animal containing the transgene encoding both the Cre recombinase and the selection protein is required. Such animals can be constructed, for example, through the construction of a "double" transgenic animal, for example, comprising two transgenic animals, one containing a transgene encoding a selection protein and the other containing a transgene encoding a recombinase. By breeding two transformed animals.
[0206]
Clones of the non-human transgenic animals described herein are also described in Wilmut, I .; et al. Nature 385: 810-813 (1997) and the methods described in PCT International Publication Nos. WO 97/07668 and WO 97/07669. Briefly, cells, eg, somatic cells, escape from the growth cycle and₀Can be isolated and derived from the transgenic animal to enter the phase.
Resting cells can then be fused, for example, by using an electrical pulse on annucleated oocytes from an animal of the same species as which the resting cells were isolated. This reconstructed oocyte is then cultured to develop into a morula or tail vesicle and transferred to a pseudopregnant female foster animal. The offspring of the female foster animal will be a clone from the animal from which the cell, eg, a somatic cell, was isolated.
[0207]
Transgenic animals containing recombinant cells expressing the peptides described herein are useful for performing the assays described herein in an in vivo situation. The various physiological factors present in vivo and capable of effecting ligand binding, estrogen receptor protein activation, and signal transduction may not be apparent from in vitro cell-free or cell-based assays. Thus, the provision of non-human transgenic animals may provide for in vivo estrogen receptor protein function, such as ligand interaction, the effects of specific mutant estrogen receptor proteins on estrogen receptor protein function, and the effect of chimeric estrogen receptor protein. Useful for analyzing functions including effects. The effect of a null mutation, ie, a mutation that substantially or completely eliminates one or more estrogen receptor protein functions, can also be evaluated.
[0208]
【Example】
1. Identification and evaluation of SNP
Each exon of the estrogen receptor alpha (ESR1) was PCR amplified using primers flanking each exon flanking sequence (exon / intron boundary) and the sequence of the amplified fragment was analyzed. Genomic DNA (see FIG. 2 (b)) from the Colliere Diversity Panel (10 each of 5 ethnic groups) and / or Liverpool clinical breast tumors and corresponding blood samples from 48 patients (Liverpool, UK) as PCR templates (See FIG. 2 (a)). PolyPhred version 2.0 (DA Nickerson, S. Taylor, N. Kolker, Univ. Of Washington, 1998) was run on the array (with default settings) to visualize potential heterozygotes. The properties of the labeling site were tested to confirm the polymorphism.
[0209]
Thirty-six SNPs with a frequency of ≧ 2 and a trait score of ≧ 20 were identified with a clinical sample specific 13 Fifteen SNPs showed at least one change in heterozygosity of the clinical sample, and four SNPs showed at least one defect in heterozygosity. For analysis, PCR primers were used for SNP identification and detection (see FIG. 2 (e)).
Furthermore, the primer set and M13 primer of FIG. 2 (d) were used for overlapping PCR and clone sequencing.
[0210]
[Table 1]
Summary of SNPs found in clinical samples: All SNPs had a frequency of 2 or more and a characteristic score of 20 or more (see FIG. 8).

[0211]
[Table 2]
Summary of heterozygous changes in clinical samples: SNPs had a frequency of ≧ 2 and a characteristic score of ≧ 20 (see FIG. 8).

[0212]
FIG. 2 (c) included the analyzed SNPs in the Liverpool control group versus the blood or tumor group.
FIG. 5 shows the domain structure of the ESR1 protein and many of the SNP positions disclosed herein. FIG. 6 is a graph showing the SNP and the frequency of occurrence in the test sample.
FIG. 8 (a) (1) shows SNPs and incidence in collier samples collected from ethnic groups of Northern Europeans, Chinese, Indo-Pakistani, African American, and Southwestern Native Americans. . In 3 'flanking exon 8, the location of the SNP is based on the sequence of AL078582 in the gene bank. The results can be used for detection between specific ethnic groups. FIG. 8 (a) (2) shows genotyping of the SNP at site 167989 (Intron 1D, # 9) in the Collier panel. The panel includes 100 Caucasians, 51 male and 49 female. The results show that the frequency of the minor allele is 9%.
[0213]
FIG. 8 (b) (1) shows the SNP and occurrence frequency in Liverpool samples selected from the group of blood samples from Nordic people and breast cancer samples.
FIG. 8 (b) (2) shows the results of tack human genotyping of SNP sites 167,989 (intron 1D, # 9) in 60 additional Liverpool patients. This result indicates that the frequency of the minor allele (G) was 14% in blood and 10% in tumor. This is similar to the SNP genotyping of the collier sample shown in FIG. 8 (b) (1). The frequency of the minor allele was 16% in blood and 17% in tumor. The Liverpool control group (95 cases) shows that the frequency at the minor allele was 10.5% (FIG. 2 (c)).
[0214]
2. Haplotype analysis
The method developed for SNP discovery was designed to obtain haplotype data. SNPs could be associated with specific haplotypes. Sample cDNA was obtained from a random group present at an unknown ratio. SNPs from specific clones were clustered into haplotypes and made.
[0215]
The data consists of two sample types (FIGS. 4 (a) and (b)). Liverpool samples were from 48 patients, each patient having a tumor and a corresponding blood sample. The Collier samples were controls, but they were not the corresponding controls. Rather, they were included in a mix of Europeans, Chinese, Indo-Pakistani, and African Americans. Forty-six SNPs in ESR1 were recorded in the Liverpool sample. The same 46 SNPs and an additional 6 SNPs 3 'of the RSR1 gene were recorded in the Collier sample.
[0216]
These data were subjected to analysis to infer the most appropriate haplotype phase of the individual. The results are shown in FIG. 4 (a), where each haplotype has a number and the number with a dash is the count (or frequency) of that haplotype.
FIG. 4 (b) shows non-single haplotype data adapted to a close-junction tree diagram. If the tree diagram is cut at the arrow, the branch including 3L, 10-4,... 73L is defined as "branch X" from the rest of the tree diagram. The differences in tumor incidence in the haplotypes in Branch X versus the rest of the dendrogram are shown below. The incidence of each haplotype was counted by first adding the numbers with dashes. L indicates a neoplastic Liverpool sample, and L indicates a Collier control.
[0217]

X²Was calculated to be 21.29 with a degree of freedom having a probability of less than 0.0001 based on a 2 × 2 table. Thus, branch X of the ER1 gene has more opportunities to associate with tumors. This full fork is very rare elsewhere in the world. Even among Europeans, there was only one out of 20 haplotypes.
[0218]
The sites where branching was identified with a large frequency difference between the Colliere control, Liverpool control and Liverpool tumor samples are as follows.

[0219]
3 . Estrogen receptor 1 promoter region and SNP
The CAAT and TATA boxes are the first 200 b. Of the sequence. p. It is thought that these distances may be functional as basal promoters.
The distance between the CAAT-TATA sites and the actual start of transcription specified is about 300 b. p. (Usually about 20-40 bp), and this region includes multiple TF regions such as NFAT, NFKB, SP1-family, CEBP and EBOX. Most of these have been implicated in the regulation of specific (tissue, cell type) expression.
[0220]
The region of T (SNP) has interesting properties. It not only has a maintenance (TC) 6-repeat, but also has a 5-prime (CA) 6. The maintenance motif is CACAYTCTC in the same region. None of the known TF sites fit this region significantly, and it appears to be a novel TF site. Very close to T (SNP) is the TF site called AHRARNT_02 for the aryl hydrocarbon / Arnt heterodimer. The CACAYTCTC site aids in the correct placement of the cofactor attachment point or Arnt complex. A single mutation in the TF binding site may reduce the affinity of protein binding by several folds and lead to disease pathways.
[0221]
In the present invention, the SNP occurs exactly 13 bp upstream of exon 1C in a short GA repeat (GAGAGAGA). Of the related SNPs in the branch (FIG. 4), three are silent mutations, one is in the 3'UTR and the rest are in the intron region.
An important T to G SNP (# 9) is located at site 167989 (intron 1D) in the promoter region (FIGS. 2 (a), (b) and (c)). Loss of gene copy is associated with cancer risk. Promoter mutations that reduce gene expression have a similar effect.
[0222]
G to T transversions are in alternating promoter sites. There are certain effects on estrogen physiology, perhaps the overall level of signaling in the nucleus. The "effect" of the associated branch (and the haplotype of the branch) (FIG. 4) is to increase overall estrogen receptor sensitivity and reactivity, or that the cycle is in the direction of unregulated (non-physiological) growth signals. It is expected that it can be guided towards some other growth process. Estrogen exposure plays a crucial role in the cause of breast cancer in virtually all epidemiological studies.
[0223]
Furthermore, the TF binding site and SNP were detected in the ESR1 gene in which 5 SNPs occurred in the transcription binding site, as described below.

[0224]
4 . ESR1 Genome Sequencing-Complete Genome Structure of Estrogen Receptor Alpha
The estrogen receptor (ER) is a member of the nuclear hormone receptor gene superfamily. This family of genes is characterized by a modular structure of three distinct domains: a mutated (N) -terminal domain, a highly maintained DNA binding domain, and a maintained (C) -terminal domain (see references 1, 2). . Functionally, the (N) -terminal domain is for transactivation, the DNA binding domain is for dimerization and DNA binding, and the (C) -terminal domain is for transactivation, dimerization, ligand binding, nuclear translocation. , Silencing, and heat shock protein binding, respectively. The function of each domain of the nuclear hormone gene superfamily is independent of the receptor in which they are found, and retains its function even when the domains are located in different atypical proteins (see references 3, 4, and 5). ). Because the major subfamilies of these genes evolved early in evolution through simple gene replication, there is a domain modularity of the nuclear hormone receptor gene superfamily (Ref. 6). The nuclear hormone receptor gene family can be separated according to two different classification schemes. One is based on hormone binding and the other is based on dimerization and the method of binding the receptor to the DNA response element (see reference 2).
[0225]
The ERα cDNA was first cloned and sequenced from the MCF-7 breast cancer cell line and was found to have 27% identity and 41% conservation with respect to the v-erb-A gene (Ref. 7). ). ERa was mapped to chromosome 6q25.1 using fluorescence in situ hybridization (FISH) and chromosome binding (8). In 1996, a novel estrogen receptor (ERβ) was identified by degenerate PCR (ref. 9) and mapped to 14q22-24 by FISH (ref. 10). ERα and ERβ have 96% sequence identity in the DNA binding domain and 58% identity in the ligand binding domain, and are identical at the 5 ′ and 3 ′ ends as in the hinge (domain D). Was shown to be low. A variety of ERα and ERβ variants have been described previously, including transcripts containing single and multiple exon deletions, truncated transcripts, and insertions (refs. 11, 12, 13). These variants have been isolated from a variety of sources including normal tissues, tumor tissues and cell lines. The tumor ER status of breast cancer patients has been used as an indicator of response to endocrine therapy (Refs. 14, 15) and many studies have suggested a role for ERs in breast cancer tumor progression, ER-negative status, and hormone antagonist resistance. Has been studied (see reference 16).
[0226]
Due to the importance of the ER gene, we cloned it whole and sought to determine its complete structure. Initially, standard bacterial artificial chromosome (BAC) sequencing was used to generate sequence information for the coding region of the gene. As the human genome sequencing of Celera progressed, the remaining regions of the ER were filled in using Celera Legion assembly. Regions smaller than 25 kb were filled in on ERa using known BACs (Al353611.6, positions 1,497-25,941).
[0227]
Materials and methods
1) BAC screening
Appropriate markers were designed for the ERa and ERβ exons and used to obtain commercially available BAC clones from Research Genetics (Huntsville, AL). Several positive BACs were selected and each clone was rescreened for confirmation.
[0228]
2) Isolation of DNA and preparation of library
BAC DNA was isolated from the confirmed clones using a QIAGEN column (QIAGEN, Inc., Valencia, CA) according to the manufacturer's specifications. Shotgun libraries were made according to standard protocols (Ref. 17). Briefly, isolated BAC DNA was sonicated, polished, and size fractionated. The size selected DNA fragments were then subcloned into pUC19 using standard ligation techniques. The ligated DNA was transformed into electrocompetent cells (Life Technologies, Rockville, MD) and grown overnight.
[0229]
3) DNA sequencing and annotation
Sequencing reactions were performed using Big Dye Terminator chemistry, Applied Biosystems, Foster City, CA, and performed on an ABI PRISM 3700 DNA Analyzer (Applied Bios). Phred (ref. 18), Prap and Consed (ref. 19) were used for base calling, assembly and finishing, respectively. Exon positions were determined using cross-matching and the published gene sequence was compared to the genomic contig.
[0230]
result
1. Estrogen receptor alpha
Alignment of the ERα genomic sequence and the published mRNA sequence of ERα shows that the gene consists of 14 exons and covers a 446,296 bp genomic sequence (FIG. 7, Table 3).
2. Estrogen receptor beta
Alignment of the ERβ genomic sequence and the published mRNA sequence of ERβ shows that the gene consists of 17 exons and covers a 253,748 bp genomic sequence (Figure 2, Table 1). The gene, human synaptic nucleus expression gene 2 (syn-2, accession number NM_015180.1) was identified by analysis with the Serera genome browser. It is completely contained within intron 9 of ERβ on the opposite strand. Further analysis of the syne-2 gene showed that it consisted of 21 exons and covered a genomic sequence of 51,471 bp.
[0231]
Consideration
Alignment of the complete ERα genomic sequence with the various ERβ transcripts indicates that the gene covers a 446,296 bp genomic sequence and consists of 14 exons. Alignment of the published sequence of exon 1E (AJ002561) (ref. 20) with the ERα genomic sequence revealed that exon 1E actually consists of two separate exons. The newly indicated exon follows the nomenclature established so far and is referred to herein as exon 1G. Exon 1G is located approximately 45 kb upstream of exon 1E and corresponds to the GT / AG splice site consensus sequence (Figure 1, Table 1).
[0232]
Alignment of the various ERβ transcripts to the complete ERβ genomic sequence shows a more complex organization than heretofore accepted (13). The 5′UTR of the ERβcx mutant (AB006589) is actually composed of seven untranslated exons (herein referred to as exons-1 to -7), all of which correspond to the GT / AG splice site consensus sequence (FIG. 2). , Table 1). Sequence alignment of the ERβ mutants AF061055 and AF061054 (ref. 12) indicated that these transcripts both contained intron sequences and were probably partially mature transcripts. Both of these partially mature transcripts contain exon 7 and part of exon 9, but do not match the splice site consensus sequence where the intron sequence is present.
[0233]
By examining the ER genomic sequence using the Cellera Genome Browser, the isolated gene completely contained within intron 9 of ERβ could be identified. This gene is a human synaptic nucleus expression gene 2 (syn-2), which has been shown to cover a 50 Kb genomic sequence and consist of 21 exons. These all correspond to the GT / AG splice consensus sequence (Table 4). The syne-2 gene is located on the antisense strand of ERβ.
Completion of the sequence and structure of ERα and ERβ contributes to further understanding and characterization of these key receptors.
[0234]
[Table 3]
Exon-intron boundaries and locations at the human estrogen receptor:
Exon sequences are in upper case, intron sequences are in lower case, and splice sites are in bold.

[0235]
[Table 4]
Exon-Intron Boundary and Location in Human Synaptic Nuclear Expression Gene 2:
Exon sequences are in upper case, intron sequences are in lower case, and splice sites are in bold.

[0236]
References
1. Ribeiro RC, Kushner PJ, Baxter JD, Tenbaum S, Baniahmad A. The nuclear hormone receptor gene superfamily. Annu Rev Med 1995; 46: 443-53.
2. Tenbaum S, Baniahmad A. Nuclear receptors: structure, function and evolution in disease. Int J Biochem Cell Biol. 1997 Dec; 29 (12): 1325-41.
3. Baniahmad C, Baniahmad A, O'Malley BW. A rapid method combining a functional test of fusion proteins in vivo and the air purification. Biotechniques. 1994 Feb; 16 (2): 194-6.
4. Parker MG, White R. Nuclear receptors spring into action. Nat Struct Biol. 1996 Feb; 3 (2): 113-5.
5. Schwabe JW. Transcriptional control: how nuclear receptors get turned on. Curr Biol. 1996 Apr 1; 6 (4): 372-4.
[0237]
6. Laudet V, Hanni C, Coll J, Catzeflis F, Stehelin D. Evolution of the nuclear receptor gene superfamily. EMBO J.S. 1992 Mar; 11 (3): 1003-13.
7. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986 Mar 13-19; 320 (6058): 134-9.
8. Menasce LP, White GR, Harrison CJ, Boyle JM. Localization of the estrogen receptor locus (ESR) to chromasome 6q25.1 by FISH and a simple post-FISH banding technique. Genomics 1993 Jul; 17 (1): 263-5.
9. Mosselman S, Polman J, Dijkema R. ER beta: identification and characterisation of a novel human estrogen receptor. FEBS Lett. 1996 Aug 19; 392 (1): 49-53.
10. Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G, Nordenskjold M, Gustafsson JA. Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab. 1997 Dec; 82 (12): 4258-65.
[0238]
11. Murphy LC, Dotzlaw H, League E, Douglas D, Coutts A, Watson PH. Estrogen receptor variants and mutations. J Steroid Biochem Mol Biol. 1997 Aug; 62 (5-6): 363-72.
12. Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, Su JL, Kliewer SA, Lehmann JM, Willson TM. Cloning and characteristics of human estrogen receptor beta isoforms. Biochem Biophys Res Commun. 1998 Jun 9; 247 (1): 75-8.
13. Ogawa S, Inoue S, Watanabe T, Orimo A, Hosoi T, Ouchi Y, Muramatsu M. Molecular cloning and characterisation of human estrogen receptor betax: a potential inhibitor of estrogen action in human. Nucleic Acids Res. 1998 August 1: 26 (15): 3505-12.
14． Osborne CK, Yochmowitz MG, Knight WA 3d, McGuire WL. The value of estrogen and progesterone receptors in the treatment of breast cancer. Cancer 1980 Dec 15; 46 (12 Suppl): 2884-8.
15. DeSombre ER, Carbone PP, Jensen EV, McGuire WL, Wells SA Jr, Wittliff JL, Lipsett M Special report. Stereoceptors in breast cancer. N Engl J Med 1979 Nov 1; 301 (18): 1011-2.
[0239]
16. Parl, Fritz F.A. , Estrogens, Estrogen receptor, and Breast Cancer. IOS Press, Amsterdam, Netherlands, 2000.
17． Birren B, Green ED, Klapolz S, Myers, R, Riethman H, Roskams J, (eds.) (1997), Genome Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
18. Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 1998 Mar; 8 (3): 175-85.
19. Gordon D, Abajan C, Green P. Consed: a graphical tool for sequence finishing. Genome Res 1998 Mar; 8 (3): 195-202.
20. Flouriot G, Griffin C, Knealy M, Sonntag-Buck V, Gannon F. Differentially expressed messenger RNA isoforms of the human estrogen receptor-alpha gene are generated by the alternating splicing and promoter. Mol Endocrinol 1998 Dec; 12 (12): 1939-54.
[0240]
All publications and patents mentioned in the above specification are herein incorporated. Various modifications and variations of the described method and system will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology and related fields are within the scope of the claims.
[Sequence list]

[Brief description of the drawings]
FIG. 1. Complete genomic sequence of the estrogen receptor alpha gene.
FIG. 2. Sequence polymorphisms found in estrogen receptor alpha genomic DNA (nucleotide positions are based on the sequence given in FIG. 1).
(A) SNP in Liverpool clinical tissue sample
(B) SNPs in the Collier Diversity Panel
(C) SNPs in Liverpool control population
(D) PCR primer
(E) Sequencing primer
FIG. 3. Amino acid sequence of estrogen receptor alpha protein.
FIG. 4: Estrogen receptor haplotype (see haplotype).
(A) Liverpool samples from 48 patients, each patient has a tumor and a corresponding blood sample. The Collier sample is a control.
(B) Non-single haplotype data suitable for neighboring trees (L is Liverpool sample).
FIG. 5. Domain structure of ESR1 protein and SNP positions disclosed herein.
FIG. 6: Distribution and frequency of many SNPs according to the invention.
FIG. 7 is a graphical representation of the human ESR1 locus.
(A) Complete structure of human estrogen receptor alpha (ERα). Exons are filled squares, and introns are horizontal lines.
(B) The order and name of the contigs used to complete the genome sequence. The GA number represents a cellera contig number. Research Genetics BAC clones are designated by reference plate and detail number.
FIG. 8: ESR alpha SNPs: a) in Collier sample, b) in Liverpool sample (T = tumor sample, B = blood sample, LC = Liverpool control), c) in Liverpool control sample.
FIG. 9: ESR alpha exon with SNP (see FIG. 2 for “N”, “C”, “I”, “A”, “S”). The underlined sequence represents the primer sequence.

Claims (17)

An isolated peptide comprising an amino acid sequence selected from the group consisting of the following (a) and (b):
(A) Amino acid sequence of mutant estrogen receptor protein given in FIG.
(B) A fragment of the amino acid sequence of the mutant estrogen receptor protein provided in FIG. 2, comprising at least 10 contiguous amino acids. An isolated peptide comprising an amino acid sequence selected from the group consisting of the following (a) and (b).
(A) Amino acid sequence of mutant estrogen receptor protein given in FIG.
(B) A fragment of the amino acid sequence of the mutant estrogen receptor protein provided in FIG. 2, comprising at least 10 contiguous amino acids. An isolated antibody that selectively binds to the peptide of claim 1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of the following (a), (b) and (c):
(A) Nucleotide sequence encoding the amino acid sequence of the mutant estrogen receptor protein provided in FIG.
(B) Nucleotide sequence encoding a fragment of the amino acid sequence of the mutant estrogen receptor protein provided in FIG.
(C) a nucleic acid molecule that is the complement of the nucleic acid molecule of (a)-(b). An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of (a), (b) and (c) below.
(A) Nucleotide sequence encoding the amino acid sequence of the mutant estrogen receptor protein provided in FIG.
(B) Nucleotide sequence encoding a fragment of the amino acid sequence of the mutant estrogen receptor protein provided in FIG.
(C) a nucleic acid molecule that is the complement of the nucleic acid molecule of (a)-(b). A nucleic acid vector comprising the nucleic acid sequence according to claim 4. A nucleic acid vector comprising the nucleic acid sequence according to claim 5. A host cell comprising the vector according to claim 6. A host cell comprising the vector according to claim 7. 2. The method for producing a peptide according to claim 1, wherein a nucleotide sequence encoding any of the peptide sequences (a)-(b) is introduced into a host cell, and a protein is expressed from the nucleic acid sequence. A method for producing a peptide, comprising culturing the host cell under conditions. 3. The method for producing a peptide according to claim 2, wherein a nucleotide sequence encoding any of the peptide sequences (a) and (b) is introduced into a host cell, and a protein is expressed from the nucleic acid sequence. A method for producing a peptide, comprising culturing the host cell under conditions. 2. A method for detecting the presence of any of the peptides according to claim 1 in a sample, comprising contacting the sample with an agent capable of specifically detecting the presence of the peptide in the sample, and then detecting the presence of the peptide. A detection method comprising: 13. A kit comprising a reagent for use in the method of claim 12, wherein said reagent comprises an agent that specifically binds to said peptide. 5. A method for detecting the presence of a nucleic acid sequence according to claim 4 in a sample, wherein the sample is contacted with an oligonucleotide that hybridizes to the nucleic acid sequence under stringent conditions, and A method of detecting, comprising determining whether or not binds. 15. A kit comprising a reagent for use in the method of claim 14, wherein the reagent comprises a compound that hybridizes under stringent conditions to any of the nucleic acid molecules. The method for identifying an agent that binds to any peptide according to claim 1, wherein the peptide is contacted with a substance, and the contact mixture is analyzed to determine whether the substance binds to the peptide to form a complex. A specific method that includes determining A method for identifying a human having or at risk of developing a disease mediated by a mutant estrogen receptor, comprising analyzing nucleic acid molecules isolated from the human for alterations in the estrogen receptor gene sequence, A method of identifying, comprising identifying an alteration in said estrogen receptor gene selected from the group consisting of the variants provided in FIG. 4 as having or at risk of developing a bone disease.

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