JP2004283086A - Marker for type ii diabetes and arteriosclerosis, and probe and primer for detecting the marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a marker as an indicator of type II diabetes onset. <P>SOLUTION: The marker is as follows: (a) an HNF(human hepatocyte nuclear factor)-4α gene fragment comprising a specific base sequence where the C at the 31-site is mutated to T in the exon 4 of the HNF-4α gene, or (b) the HNF-4α gene including the gene fragment(a). Possibility of the onset of type II diabetes or arteriosclerosis is judged based on the presence or absence of the above mutation. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００２８】
前記表１に示すように、Ｔ１３０Ｉ−ＨＮＦ−４α変異の糖尿病患者が糖尿病と診断された平均年齢は４７．１±８．８歳（ｐ＝１．０×１０^−６）であり、表中「＃」に示す他のＨＮＦ−４α変異の糖尿病患者（ＭＯＤＹ１患者）の平均年齢２８．２±１５．２歳（ｎ＝４０、ｐ＝１．０×１０^−６）よりも、非常に高いことがわかった。この結果から、Ｔ１３０Ｉ−ＨＮＦ−４α変異の患者は、若年発症型糖尿病（例えば、ＭＯＤＹ）ではなく、通常の２型糖尿病を発症する確率が高いと言える。
【００２９】
Ｔ１３０Ｉ−ＨＮＦ−４α変異の糖尿病患者と、Ｔ１３０Ｉ−ＨＮＦ−４α変異を有さない糖尿病患者の間では、発症年齢、ＢＭＩ（ボディマス指数）、最大ＢＭＩ、空腹時血糖（ＦＰＧ）、ＨｂＡ１ｃ、インシュリン耐性のｈｏｍｅｏｓｔａｓｉｓ ｍｏｄｅｌ ａｓｓｅｓｓｍｅｎｔ （ＨＯＭＡ−ＩＲ）、全コレステロールおよびトリグリセリドのレベルに関して大きな有意差は無かった。しかしながら、血漿 ＨＤＬ−コレステロールレベルは、Ｔ１３０Ｉ変異糖尿病患者の方がより低い値を示した（ｐ＝０．００６）。この結果は測定レベルの違いは、有意差と判断できるため、Ｔ１３０Ｉ変異のＨＮＦ−４α遺伝子は、動脈硬化の発症可能性の判断における指標（マーカー）であることが確認された。
【００３０】
また、有意なデータが得られたＴ１３０Ｉ遺伝子型の糖尿病患者（４例）において、グルカゴンで刺激したＣ−ペプチド応答に、０．９ ｎｇ／ｍｌ、１．３ ｎｇ／ｍｌ、４．０ ｎｇ／ｍｌ、４．７ ｎｇ／ｍｌという変動が見られた（正常 ＞２．０ｎｇ／ｍｌ）。また、患者（６例）の尿中Ｃ−ｐｅｐｔｉｄｅ（ｕＣＰＲ）濃度は、それぞれ１４．７μｇ／２４ｈ、４７．８μｇ／２４ｈ、６７．２μｇ／２４ｈ、９３．７μｇ／２４ｈ、１０１μｇ／２４ｈおよび１４９μｇ／２４ｈ（ｎｏｒｍａｌ： ５０〜１２０μｇ／２４ｈ）であった。これらの結果は、インシュリン分泌が、Ｔ１３０Ｉ−ＨＮＦ−４α変異を持つ患者間において変動していることを示唆している。これは、Ｔ１３０Ｉ−ＨＮＦ−４α変異の糖尿病患者は、インシュリン分泌の特性欠陥を誘導するＭＯＤＹ１変異とは対照的であるといえる（前記非特許文献４）。
【００３１】
３．トランスアクティベーション活性の確認
（プラスミド）
Ｔ１３０Ｉ変異ＨＮＦ−４α遺伝子を、Ｃｈａｍｅｌｅｏｎ Ｄｏｕｂｌｅ−Ｓｔｒａｎｄｅｄ Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ Ｍｕｔａｇｅｎｅｓｉｓ ｋｉｔ （Ｓｔｒａｔａｇｅｎｅ， Ｌａ Ｊｏｌｌａ， ＣＡ）を用いてｈｕｍａｎ ＨＮＦ−４α２ ｃＤＮＡ から調製し、ｐｃＤＮＡ３．１ 発現ベクター（Ｉｎｖｉｔｒｏｇｅｎ， Ｓａｎ Ｄｉｅｇｏ， ＣＡ）によりクローニングした。なお、その構造は、ＤＮＡシークエンスにより確認した。異種ＴＫプロモーターとエンハンサーであるＨＮＦ−４α結合配列とのレポーター構築物（ｐＨＮＦ４−ｔｋ−Ｌｕｃ）、ＨＮＦ−１αレポーター、およびＰＫＬレポーター構築物は、文献（Ｙａｎｇ Ｑ， Ｙａｍａｇａｔａ Ｋ， Ｙａｍａｍｏｔｏ Ｋ ｅｔ ａｌ． （２０００） Ｒ１２７Ｗ−ＨＮＦ−４ａ ｉｓ ａ ｌｏｓｓ ｏｆ ｆｕｎｃｔｉｏｎ ｍｕｔａｔｉｏｎ ｂｕｔ ｎｏｔ ａ ｒａｒｅ ｐｏｌｙｍｏｒｐｈｉｓｍ ａｎｄ ｃａｕｓｅｓ Ｔｙｐｅ ＩＩ ｄｉａｂｅｔｅｓ ｉｎ ａ Ｊａｐａｎｅｓｅ ｆａｍｉｌｙ ｗｉｔｈ ＭＯＤＹ１． Ｄｉａｂｅｔｏｌｏｇｉａ ４３： ５２０−５２４）に従った。
【００３２】
（細胞培養）
マウスの初代肝細胞（Ｍｏｕｓｅ ｐｒｉｍａｒｙ ｈｅｐａｔｏｃｙｔｅｓ）を、文献（Ｓｈｉｍｏｍｕｒａ Ｉ， Ｍａｔｓｕｄａ Ｍ， Ｈａｍｍｅｒ ＲＥ， Ｂａｓｈｍａｋｏｖ Ｙ， Ｂｒｏｗｎ ＭＳ， Ｇｏｌｄｓｔｅｉｎ ＪＬ （２０００） Ｄｅｃｒｅａｓｅｄ ＩＲＳ−２ ａｎｄ ｉｎｃｒｅａｓｅｄ ＳＲＥＢＰ−１ｃ ｌｅａｄ ｔｏ ｍｉｘｅｄ ｉｎｓｕｌｉｎ ｒｅｓｉｓｔａｎｃｅ ａｎｄ ｓｅｎｓｉｔｉｖｉｔｙ ｉｎ ｌｉｖｅｒｓ ｏｆ ｌｉｐｏｄｙｓｔｒｏｐｈｉｃ ａｎｄ ｏｂ／ｏｂ ｍｉｃｅ． Ｍｏｌ Ｃｅｌｌ ６： ７７−８６）に従って、コラゲナーゼ潅流法（ｃｏｌｌａｇｅｎａｓｅ ｐｅｒｆｕｓｉｏｎ ｍｅｔｈｏｄ）により調製し、６−ｗｅｌｌ ｔｉｓｓｕｅ ｃｕｌｔｕｒｅ ｐｌａｔｅに３×１０^５ ｃｅｌｌｓ／ｗｅｌｌの密度で平板培養した。そして、ＨｅＬａ細胞、ＭＩＮ６細胞、ＨｅｐＧ２細胞および初代肝細胞に、ＬＩＰＯＦＥＣＴＡＭＩＮ ＰＬＵＳ ｒｅａｇｅｎｔを用いて、前述の発現ベクターおよびレポーターを、内部コントロールとなるｐＲＬ−ＴＫ（Ｐｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＷＩ）１０ｎｇと共に感染させた。
【００３３】
（ルシフェラーゼアッセイ）
トランスアクティベーション活性は、Ｄｕａｌ Ｌｕｃｉｆｅｒａｓｅ Ｒｅｐｏｒｔｅｒ Ａｓｓａｙ ｓｙｓｔｅｍ および Ｌｕｍａｔ ＬＢ９５０１ Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅを用いて、細胞に前記発現ベクターを感染させてから４８時間後に測定した（Ｙａｍａｇａｔａ Ｋ， Ｙａｎｇ Ｑ， Ｙａｍａｍｏｔｏ Ｋ ｅｔ ａｌ． （１９９８） Ｍｕｔａｔｉｏｎ Ｐ２９１ｆｓｉｎｓＣ ｉｎ ｔｈｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ ｈｅｐａｔｏｃｙｔｅ ｎｕｃｌｅａｒ ｆａｃｔｏｒ−１ａ ｉｓ ｄｏｍｉｎａｎｔ ｎｅｇａｔｉｖｅ． Ｄｉａｂｅｔｅｓ ４７： １２３１−１２３５）。
【００３４】
（ウエスタンブロット分析および電気泳動移動度シフトアッセイ（ＥＭＳＡ）
ウエスタンブロット分析は、前記Ｙａｍａｇａｔａらの文献（１９９８）に報告されている抗−ＨＮＦ−４α抗体（Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， Ｓａｎｔａ Ｃｒｕｚ， ＣＡ）を用いて行った。野生型ＨＮＦ−４αタンパク質およびＴ１３０Ｉ−ＨＮＦ−４αタンパク質は、ＴＮＴ Ｔ７ Ｑｕｉｃｋ Ｃｏｕｐｌｅｄ Ｔｒａｎｓｃｒｉｐｔｉｏｎ／Ｔｒａｎｓｌａｔｉｏｎ Ｓｙｓｔｅｍ （Ｐｒｏｍｅｇａ）を用いて合成した。そして、 ｉｎ ｖｉｔｒｏで翻訳されたタンパク質を、ＨＮＦ−１α遺伝子のＨＮＦ−４α結合部位（５’−ＧＧＣＴＧＡＡＧＴＣＣＡＡＡＧＴＴＣＡＧＴＣＣＣＴＴＣＧＣ−３’）を含む ^３２Ｐ−ラベルオリゴヌクレオチドのプローブと共に、２０μＬの反応混合液においてインキュベーションし、ＤＮＡ−タンパク質複合体を形成した（Ｙａｎｇ Ｑ， Ｙａｍａｇａｔａ Ｋ， Ｙａｍａｍｏｔｏ Ｋ ｅｔ ａｌ． （１９９９） Ｓｔｒｕｃｔｕｒｅ／ｆｕｎｃｔｉｏｎ ｓｔｕｄｉｅｓ ｏｆ ｈｅｐａｔｏｃｙｔｅ ｎｕｃｌｅａｒ ｆａｃｔｏｒ−１ａ， ａ ｄｉａｂｅｔｅｓ−ａｓｓｏｃｉａｔｅｄ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ． Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ ２６６： １９６−２０２）。また、競合結合分析（ｃｏｍｐｅｔｉｔｉｏｎ ｂｉｎｄｉｎｇ ａｓｓａｙ）においては、５０倍の非ラベル化プローブ（前記ラベル化プローブと同じ配列）をｃｏｍｐｅｔｉｔｏｒとして使用した。前記インキュベーション後のタンパク質（ＤＮＡ−タンパク質複合体）を、０．５×ＴＢＥ 緩衝液を用いた５％ポリアクリルアミドゲルで電気泳動し、オートラジオグラフィーによってＥＭＳＡ分析を行った。また、ポリクローナル抗−ＨＮＦ−４α抗血清（α４４５）を、移動度分析（ｓｕｐｅｒｓｈｉｆｔ ａｎａｌｙｓｉｓ）に使用した（前記非特許文献１参照）。さらに、Ｔ１３０Ｉ−ＨＮＦ−４αの相対的な結合親和性を確認するために、非平衡状態となるように、非ラベル化ｃｏｍｐｅｔｉｔｏｒの量を増加して（１０、２５および５０倍）添加し、５分間インキュベートした（前記Ｙａｎｇ Ｑらの文献（１９９９）参照）。
【００３５】
まず、Ｈｅｌａ細胞に、野生型（ＷＴ）−ＨＮＦ−４αの発現ベクターおよびＴ１３０Ｉ−ＨＮＦ−４αの発現ベクターをそれぞれ感染させ、ＷＴ−ＨＮＦ−４αとＴ１３０Ｉ−ＨＮＦ−４αの発現を確認した。この結果を図１Ａに示す。図１Ａは、前記各発現ベクター（８μｇ）をＨｅｌａ細胞に感染させ、４８時間後に行ったウエスタンブロットの結果を示す写真であり、レーン１はＷＴ−ＨＮＦ−４α、レーン２はＴ１３０Ｉ−ＨＮＦ−４α、レーン３はコントロール（発現ベクター無し）の結果を示す。図示のように、ＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ−４αの発現レベルは、ほぼ同様であった。このことから、Ｔ１３０ＩのＳＮＰの有無によっては、ＨＮＦ−４αの発現自体には影響が無いと言える。
【００３６】
つぎに、ＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーション活性をルシフェラーゼレポーター遺伝子アッセイによって算出した。これらの結果を図２のＢ、Ｃおよび図３のＤに示す。これらの図は、ｐＨＮＦ４−ｔｋ−Ｌｕｃ １００ｎｇ およびｐＲＬ−ＴＫ １０ｎｇ と共に、前述の発現ベクター５００ｎｇを各細胞に感染させ、４８時間後の各細胞におけるＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ４αのトランスアクティベーション活性を示すグラフである。なお、図２ＢはＨｅｌａ細胞、図２Ｃはマウスインスリノーマセルライン ＭＩＮ６細胞、図３Ｄはヒトヘパトーマセルライン ＨｅｐＧ２細胞についての結果である。各図において、縦軸は内部標準で補正したトランスアクティベーション活性を示し、「ｖｅｃｔｏｒ」とは、ＨＮＦ−４α遺伝子を含まないベクターを感染させた結果である（以下同様）。図２Ｂおよび２Ｃに示すように、Ｈｅｌａ細胞とＭＩＮ６細胞において、Ｗｔ−ＨＮＦ−４αとＴ１３０Ｉ−ＨＮＦ−４αとの間には、トランスアクティベーション活性に有意差は見られなかった（Ｎ．Ｓ．）。しかしながら、図３Ｄに示すように、同じ量の発現ベクターをヒトヘパトーマセルライン ＨｅｐＧ２細胞に感染した場合には、Ｔ１３０Ｉ−ＨＮＦ−４αの転写活性は、ＷＴ−ＨＮＦ−４αと比較して、４６．２％までに減少した（ｐ＜０．００１）。
【００３７】
そこで、発現ベクターの用量を増加させ、ＨｅｐＧ２細胞におけるＴ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーション活性を確認した。この結果を図４Ｅに示す。同図は、前記発現ベクターの用量を増加し（５０ｎｇ、１００ｎｇ、２００ｎｇ）、ｐＨＮＦ−４−ｔｋ−Ｌｕｃと共にＨｅｐＧ２細胞に感染させた、４８時間後におけるＴ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーション活性の結果を示すグラフである。同図において、それぞれの結果は、６回の試験の「ｍｅａｎ±ＳＤ」値を示す。ＭＩＮ６細胞の場合、前記発現ベクターの用量を変化させても、Ｔ１３０Ｉ−ＨＮＦ−４αはＷＴ−ＨＮＦ−４αと同様のトランスアクティベーションに達したが（データ表示せず）、図示のように、ＨｅｐＧ２においては、発現ベクターの量を変化させても、全ての用量において、Ｔ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーション活性の減少が見られた。
【００３８】
使用したＨｅｐＧ２細胞は、ＨＮＦ−４α遺伝子においてＤ６９Ａ変異を有している（Ｌａｕｓｅｎ Ｊ， Ｔｈｏｍａｓ Ｈ， Ｌｅｍｍ Ｉ ｅｔ ａｌ． （２０００） Ｎａｔｕｒａｌｌｙ ｏｃｃｕｒｒｉｎｇ ｍｕｔａｔｉｏｎｓ ｉｎ ｔｈｅ ｈｕｍａｎ ＨＮＦ４ａ ｇｅｎｅ ｉｍｐａｉｒ ｔｈｅ ｆｕｎｃｔｉｏｎ ｏｆ ｔｈｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ ｔｏ ａ ｖａｒｙｉｎｇ ｄｅｇｒｅｅ． Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ ２８： ４３０−４３７）。そこで、このＤ６９Ａ変異が、ＨｅｐＧ２細胞における、前述のようなＴ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーション減少の原因となるか否かを、マウスの初代培養肝細胞を用いて、前記ルシフェラーゼレポーター遺伝子アッセイにより確認した。具体的には、３×１０^５ｃｅｌｌｓ／ｗｅｌｌの密度のマウス初代肝細胞に、各レポーター遺伝子（ｐＨＮＦ−４−ｔｋ−Ｌｕｃ、ＨＮＦ−１αプロモーターまたはＰＫＬプロモーター）１００ｎｇと共に前記発現ベクター０．５μｇを感染させ、４８時間後の初代肝細胞におけるＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ４αのトランスアクティベーション活性を測定した。この結果を図５Ｆ〜Ｈに示す。各図は、初代肝細胞におけるトランスアクティベーション活性を示すグラフであり、図５Ｆはレポーター遺伝子としてｐＨＮＦ−４−ｔｋ−Ｌｕｃ、図５Ｇはレポーター遺伝子としてＨＮＦ−１αプロモーター、図５Ｈはレポーター遺伝子としてＰＫＬプロモーターを使用した結果を示すグラフである。各図において、それぞれの結果は、６回の試験の「ｍｅａｎ±ＳＤ」値を示す。図５Ｆ（レポーター遺伝子：ｐＨＮＦ−４−ｔｋ−Ｌｕｃ）に示すように、初代肝細胞においてもＴＴ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーションの減少が見られた（ＷＴ−ＨＮＦ−４αの活性の２７．９％、ｐ＝９．７×１０^−５）。また、ＨＮＦ−１αおよびＬ型ピルベートキナーゼ（ＰＫＬ）は、ＨＮＦ−４αにとってのターゲット遺伝子であり（Ｙａｍａｇａｔａ Ｋ， Ｏｄａ Ｎ， Ｋａｉｓａｋｉ ＰＪ ｅｔ ａｌ． （１９９６） Ｍｕｔａｔｉｏｎｓ ｉｎ ｔｈｅ ｈｅｐａｔｏｃｙｔｅ ｎｕｃｌｅａｒ ｆａｃｔｏｒ−１ａ ｇｅｎｅ ｉｎ ｍａｔｕｒｉｔｙ−ｏｎｓｅｔ ｄｉａｂｅｔｅｓ ｏｆ ｔｈｅ ｙｏｕｎｇ （ＭＯＤＹ３）． Ｎａｔｕｒｅ ３８４： ４５５−４５８）、図５Ｇおよび図５Ｈに示すように、Ｔ１３０Ｉ−ＨＮＦ−４αによって、ＨＮＦ−１α遺伝子のトランスアクティベーション活性はＷＴの７８．２％（ｐ＝０．０２４）に、ＰＫＬ遺伝子のトランスアクティベーション活性はＷＴの７７．１％（ｐ＝０．００２）に減少した。これらの結果は、Ｔ１３０Ｉ−ＨＮＦ−４αが、Ｈｅｌａ細胞やマウスのＭＩＮ６細胞ではなく、肝細胞の環境において機能欠損の変異（ａ ｌｏｓｓ−ｏｆ−ｆｕｎｃｔｉｏｎ ｍｕｔａｔｉｏｎ）として作用していることを強く示唆するといえる。
【００３９】
つぎに、ＨＮＦ−４αのＡ−ｂｏｘ ｒｅｇｉｏｎは前述のようにＤＮＡ結合に重要であると考えられているため、さらにＴ１３０Ｉ−ＨＮＦ−４αのＤＮＡ結合活性を調べた。まず、ＷＴ−ＨＮＦ−４αタンパク質およびＴ１３０Ｉ−ＨＮＦ−４αタンパク質を、ｐｃＤＮＡ３．１発現ベクター（１μｇ）から前記ＴＮＴ Ｔ７ Ｑｕｉｃｋ Ｃｏｕｐｌｅｄ Ｔｒａｎｓｃｒｉｐｔｉｏｎ／Ｔｒａｎｓｌａｔｉｏｎ Ｓｙｓｔｅｍを用いてｉｎ ｖｉｔｒｏで翻訳し、これをＥＭＳＡに供した。ｉｎ ｖｉｔｒｏにおいて転写されたタンパク質の発現レベルは、ウェスタンブロット分析によって確認した（データ表示せず）。なお、ｃｏｍｐｅｔｉｔｏｒとしては、前述のように過剰（５０倍）の非ラベル化オリゴヌクレオチドを使用した。
【００４０】
これらの結果を図６Ａに示す。同図は、ｉｎ ｖｉｔｒｏで発現させたＷＴ−ＨＮＦ−４αとＤＮＡ、Ｔ１３０Ｉ−ＨＮＦ−４αとＤＮＡのＥＭＳＡ結果を示す写真である。同図において、レーン１はＷＴ−ＨＮＦ−４α、レーン２はＷＴ−ＨＮＦ−４αおよびｃｏｍｐｅｔｉｔｏｒ、レーン３はＴ１３０Ｉ−ＨＮＦ−４α、レーン３はＴ１３０Ｉ−ＨＮＦ−４α、レーン４がＴ１３０Ｉ−ＨＮＦ−４αおよびｃｏｍｐｅｔｉｔｏｒ、レーン５がコントロール（ベクター無し）、レーン６がコントロール（ベクター無し＋ｃｏｍｐｅｔｉｔｏｒ）、レーン７がＷＴ−ＨＮＦ−４および抗−ＨＮＦ−４α抗体、レーン８がＴ１３０Ｉ−ＨＮＦ−４αおよび抗−ＨＮＦ−４α抗体の結果であり、矢印はＨＮＦ−４α／ＤＮＡ複合体の位置を示す。同図に示すように、レーン１および３において矢印の位置にバンドが確認できたことから、ＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ−４αは、前記オリゴヌクレオチドと特異的に結合したことがわかる。また、レーン７および８では、レーン１および２に比べて、バンドのｓｕｐｅｒｓｈｉｆｔが確認されたことから、ＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ−４αがさらに抗−ＨＮＦ−４α抗体とも結合したことがわかる。
【００４１】
また、添加する非ラベル化ｃｏｍｐｅｔｉｔｏｒの量を増加して（１０、２５、５０モル）、非平衡状態においてＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ−４αと前記ラベル化オリゴヌクレオチドとを結合させ、ＥＭＳＡを行った。この結果を図６Ｂに示す。図示のように、Ｔ１３０Ｉ−ＨＮＦ−４αのＤＮＡ結合は、前記図６Ａの平衡状態においても、図６Ｂに示す非平衡状態においても同様の結果を示した。この結果から、Ｔ１３０Ｉ−ＨＮＦ−４αのアミノ酸変化は、ｉｎ ｖｉｔｒｏにおけるＤＮＡとの結合に影響がないことがわかった。
【００４２】
【発明の効果】
以上のように、本発明の第１のマーカーによれば、優れた信頼性で２型糖尿病の発症可能性を判断し、本発明の第２のマーカーによれば、動脈硬化の発症の可能性を判断することができる。このため、例えば、臨床分野における２型糖尿病および動脈硬化の診断や治療に非常に有用であるといえる。
【００４３】
【配列表】

【図面の簡単な説明】
【図１】Ａは、ＷＴ−ＨＮＦ−４αおよびＴ１５０Ｉ−ＨＮＦ−４αの発現ベクターをＨｅｌａ細胞に感染させた後のウエスタンブロットの結果を示す写真である。
【図２】ＢおよびＣは、ＷＴ−ＨＮＦ−４αおよびＴ１５０Ｉ−ＨＮＦ−４αの発現ベクターを細胞に感染させた後のＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ４αのトランスアクティベーション活性を示すグラフであり、ＢはＨｅｌａ細胞、Ｃはマウスインスリノーマセルライン ＭＩＮ６細胞についての結果である。
【図３】Ｄは、ＷＴ−ＨＮＦ−４αおよびＴ１５０Ｉ−ＨＮＦ−４αの発現ベクターを細胞に感染させた後のＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ４αのトランスアクティベーション活性を示すグラフであり、ヒトヘパトーマセルライン ＨｅｐＧ２細胞についての結果である
【図４】ＨｅｐＧ２細胞におけるＴ１３０Ｉ−ＨＮＦ−４αのトランスアクティベーション活性を示すグラフである。
【図５】マウス初代肝細胞にレポーター遺伝子と共にＷＴ−ＨＮＦ−４αおよびＴ１５０Ｉ−ＨＮＦ−４αの発現ベクターを感染させた後のＷＴ−ＨＮＦ−４αおよびＴ１３０Ｉ−ＨＮＦ４αのトランスアクティベーション活性を示すグラフであり、ＦはｐＨＮＦ−４−ｔｋ−Ｌｕｃ、ＧはＨＮＦ−１αプロモーター、ＨはＰＫＬプロモーターをレポーター遺伝子として使用した結果である。
【図６】ＡおよびＢは、ＷＴ−ＨＮＦ−４αとＤＮＡ、Ｔ１３０Ｉ−ＨＮＦ−４αとＤＮＡのＥＭＳＡ結果を示す写真であって、Ａは平衡状態、Ｂは非平衡の結果を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a marker that is an indicator of the onset of type 2 diabetes and arteriosclerosis, a probe and a primer for detecting the marker, and a DNA chip.
[0002]
[Prior art]
Diabetes is broadly classified into two types, namely, type 1 diabetes and type 2 diabetes. Among them, type 2 diabetes is known to involve a genetic predisposition to its onset. With respect to type 2 diabetes, for example, the insulin gene and the nuclear receptor-type hepatocyte nuclear factor (HNF) -4α gene have been identified as causes of diabetes due to a single gene abnormality.
[0003]
HNF-4α is a member of a superfamily of steroid hormone receptors expressed in liver, islets of Langerhans, kidney, small intestine and the like (Non-patent Document 1), and has several functional domains, that is, an N-terminal transcriptionally active region (AF). -1), a DNA-binding domain (A-box region), a dimer-forming domain / ligand-binding domain / transcriptionally active domain (AF-2) complex. This HFN-4α forms a homodimer and binds to DNA, thereby activating transcription of a target gene. For example, it is known that HFN-4α is involved in the transcription control of molecules related to sugar metabolism and lipid metabolism. Have been. The HNF-4α gene abnormality occurs in a relatively young age (usually 25 years or younger), and is a young-onset type of monogenic type 2 diabetes identified by autosomal dominant inheritance. It has been clarified to be a genetic predisposition to diabetes maturity-onset diabetes of the young (MODY; MODY1) (Non-Patent Document 2).
[0004]
However, such HNF-4α gene and insulin gene are genes that can directly cause diabetes due to abnormality in the gene alone, but diabetes that clearly causes such a genetic abnormality directly causes diabetes. Is estimated to be a few percent of the total. For this reason, normal type 2 diabetes (late-onset type), which is considered to be a multifactorial disease caused by both genetic factors and environmental factors, instead of type 2 diabetes such as MODY1 caused by a single genetic abnormality, is used. There is a need for analysis of risk factors for II diabetes mellitus.
[0005]
Thus, in recent years, P12A mutant SNPs of the PPARγ gene (Non-Patent Document 3) and SNPs of the calpain 10 gene (Non-Patent Document 4) have been reported as genetic factors associated with the onset of type 2 diabetes. However, the former shows a correlation with type 2 diabetes as a risk factor for type 2 diabetes, but questions remain regarding its effectiveness in diagnosis. There is a problem that the relevance is weak. On the other hand, with respect to arteriosclerosis, it is also required to analyze risk factors involved in the onset thereof.
[0006]
[Non-patent document 1]
Sladek FM, Zong W., Darnell J. Jr. (Sradek FM, Zhong WM, Lai E, Darnell JE Jr.) Hepatic hypertranscription factor HNF-4α-French-rich (Hover-enriched), a novel element of the steroid hormone receptor superfamily. 4 is a novel member of the stereoid horne receptor superfamily. "Genes Dev" (1990) Vol. 4, p. 2353-2365
[Non-patent document 2]
Yamagata K, Furuta H, Oda Net et al. Mutations in the hepatocyte nuclear factor-4α gene in juvenile-onset diabetes mellitus. diabetes of the young (MODY1)) "Nature" (1996) vol. 384, p. 458-460
[Non-Patent Document 3]
Hiroyuki Mori, Hiroshi Ikegami, Yoshihiko Kawaguchi et al., PPAR-γ involved in the resistance to the development of diabetes in a general population of Hiroyuki Mori, Hiroshi Ikegami, Yoshihiko Kawaguchi et al.¹²→ Ala substitution (The Pro¹²→ Ala Substitution in PPAR-γ is associated with resistance to development of diabetes in the general population. ) "Diabetes" April 2001, Volume 50, p. 891-894
[Non-patent document 4]
(Yukio Horikawa et al.) Genetic variation in the gene encoding calpain-10 associated with type 2 diabetes (Genetic variation in the gene encoding calendar. "(2000) 26 volumes p. 163-175
[0007]
[Problems to be solved by the invention]
Therefore, a first object of the present invention is to provide a new marker for determining the possibility of onset of type 2 diabetes, and a second object of the present invention is to determine the possibility of onset of arteriosclerosis. To provide new markers for
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the first marker of the present invention is a marker for determining the possibility of the onset of type 2 diabetes, and is the following marker (a) or (b).
(A) SEQ ID NO: 1 (ccgtcccagaa tgagcgggac cggatcagca wherein C at position 31 is mutated to T in exon 4 of the human hepatocyte nuclear factor (HNF) -4α genetHNF-4α gene fragment comprising the nucleotide sequence of tcgaaggtc aagctatgag gacagcagcc tgccctccat caatgcgctc ctgcaggcgg aggtcctgtc ccgacag)
(B) HNF-4α gene containing the gene fragment of (a)
[0009]
The present inventors have conducted intensive studies on SNPs that can be a risk factor for type 2 diabetes, and as a result, obtained the following findings and reached the present invention. That is, as described above, the HNF-4α gene is known to cause MODY1 as a direct cause when a gene abnormality occurs, but the HNF-4α gene does not have the gene abnormality and exon 4 It has been found that when cytosine (C) at position 31 has an SNP mutated to thymine (T), type 2 diabetes develops with a high probability. Specifically, as a result of performing gene analysis on a total of 777 Japanese (patient 423, normal control 354), the SNP was found in only 0.8% of healthy subjects, whereas 2 It was present in 3.5% of type 2 diabetic patients, showing a very significant difference with p = 0.015 and odds ratio of 4.3, confirming a very strong association between the SNP and type 2 diabetes. It was done. Therefore, it can be said that the mutation in the first marker of the present invention is clearly a risk factor for susceptibility to type 2 diabetes. In addition, conventional SNPs alone have low reliability, and thus, for example, it was necessary to determine the susceptibility of type 2 diabetes from a combination of multiple SNPs. According to the first marker of the present invention, As described above, since it shows a high significant difference by itself, it can be said that it is much more reliable and more effective than the conventional SNP. Conventionally, the HNF-4α gene has been implicated in diabetes mellitus, such as the HNF-4α gene mutation causing MODY1, as described above, and the cytosine (C) at position 31 of exon 4 has been mutated to thymine (T). Although the existence of the SNP itself was known, it is a fact discovered for the first time by the present inventors that the SNP can be an indicator of the onset of type 2 diabetes. In addition, this SNP is also detected in a general population, and is a missense mutation in the DNA binding region (A-box region), and the A-box region is homodimerized with HNF-4α, and Although it is known that it is important in terms of high affinity in binding, it has already been confirmed that this single nucleotide mutation alone does not cause MODY1 (Non-Patent Document 4). Therefore, the present inventors first proved that the SNP, whose association with type 2 diabetes had not been expected at all, was associated with type 2 diabetes. Thus, the first marker of the present invention was detected, that is, By confirming the presence or absence of the mutation at position 31 of exon 4 (c → t), it was possible to effectively determine the possibility of the onset of type 2 diabetes. For this reason, the first marker of the present invention can be said to be a very effective index in the field of clinical medicine.
[0010]
Further, the second marker of the present invention is a marker for determining the possibility of the onset of arteriosclerosis, and is the following marker (a) or (b).
(A) HNF-4α gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 in which C at position 31 in exon 4 of human hepatocyte nuclear factor (HNF) -4α gene is mutated to T
(B) HNF-4α gene containing the gene fragment of (a)
[0011]
The present inventors further conducted research on the first marker of the present invention involved in the type 2 diabetes. As a result, patients having the SNPs had higher plasma HDL-cholesterol levels than patients having no SNPs. Was found to be low, and reached the second marker of the present invention. Specifically, as a result of measuring HDL cholesterol in the patient having the SNP, the marker showed a significantly lower value (p = 0.006) as compared with the patient not having the SNP. It also found that it also served as a risk factor. Therefore, by detecting the second marker of the present invention, that is, by confirming the presence or absence of a mutation at position 31 (C → T) of exon 4, it is possible to effectively determine the possibility of onset of arteriosclerosis. In other words, this is a new index that is very effective in the field of clinical medicine. Note that the relationship between the SNP and arteriosclerosis is the first evidenced by the present inventors, as is the relationship with the type 2 diabetes.
Useful as
[0012]
In the first and second markers of the present invention, as shown in the nucleotide sequence of SEQ ID NO: 1, position 31 of exon 4 of the HNF-4α gene is acfrom t to atSince the HNF-4α protein is expressed, the amino acid at position 130 is accompanied by an amino acid substitution from Thr to Ile. Therefore, in the present invention, hereinafter, the HNA-4α gene having an SNP in which exon 4 position 31 is mutated from C to T is referred to as “T130I mutant HNF-4α gene”, and the exon 4 having the SNP is referred to as “T130I mutation. Exon 4 ”.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
As described above, the marker of the present invention is a marker for determining the possibility of the onset of type 2 diabetes or arteriosclerosis, and is the marker of (a) or (b), respectively.
[0014]
The genomic DNA sequence of the gene encoding HNF-4α is disclosed in the database of GenBank (HYPERLINK “http://www.ncbi.nlm.nih.gov/Web/Genbank/”) (AH005099, U72959). , U72960).
[0015]
Methods for determining the possibility of onset of type 2 diabetes or arteriosclerosis using the marker of the present invention include the following methods. For example, a method of amplifying a target DNA by a PCR method, determining the base sequence of the obtained PCR product, and confirming the presence of the marker of the present invention (that is, the presence of a T130I mutation SNP); And a method of confirming the presence of the marker by hybridizing the probe of the present invention described below. Further, when the position 31 of exon 4 of the HNF-4α gene is mutated (c → t), the sequence containing the mutatedBsm  The PCR product is used as a cleavage site forBsm  After the treatment with I, the presence of the SNP can be confirmed by restriction fragment length polymorphism (RFLP). The type of the restriction enzyme is not particularly limited as long as it recognizes a sequence containing a SNP.Bsm  I is not restricted. According to such a method, when the presence of the SNP, that is, the marker of the present invention is confirmed, it is possible to determine that susceptibility to type 2 diabetes and arteriosclerosis is high. The conditions for the PCR, hybridization, and RFLP are not particularly limited, and can be appropriately determined based on conventionally known methods and conditions. Further, the method for confirming the marker of the present invention is not limited to these methods.
[0016]
The primer of the present invention used for detecting the marker associated with type 2 diabetes and arteriosclerosis is not particularly limited as long as it can amplify a partial fragment containing the base at position 31 of exon 4 of the HNF-4α gene. Not done. The length of the partial fragment containing the base at position 31 is not particularly limited. For example, when the RFLP method or the like is applied, a length of about 100 to 500 bp is easy to handle. Thus, it is preferable to set primers. Those skilled in the art can appropriately design variations of the primer based on, for example, the nucleotide sequence of the HNF-4α gene, the nucleotide sequence of exon 4, and the nucleotide sequence of SEQ ID NO: 1. The length of the primer can be set in the same manner as a conventionally known primer, and is not particularly limited, but is usually 15 to 30 mer.
[0017]
Specific examples of the primer of the present invention include, for example, (e) an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 (ccaccccctactccatccctgt), and (f) an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 (cccccccgtcagctgctccca). The former oligonucleotide is a forward primer for exon 4 in the HNP-4α gene, and the latter oligonucleotide is a reverse primer for exon 4, and exon 4 and flanking intron are amplified by PCR using these primers. A DNA (271 bp) having the nucleotide sequence of SEQ ID NO: 4 is obtained.
[0018]
In determining the possibility of the onset of type 2 diabetes or arteriosclerosis, the probe of the present invention for detecting the marker of the present invention is complementary to a partial fragment containing the base at position 31 of exon 4 in the HNF-4α gene. The polynucleotide is not particularly limited as long as it can specifically bind to the marker of the present invention. Therefore, those skilled in the art can design the probe of the present invention based on the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of the HNF-4α gene, and the nucleotide sequence of exon 4. Further, it may be a probe for the complementary strand of the marker of the present invention. Specifically, for example, the following probes (c) to (e) are mentioned.
(C) a polynucleotide that is complementary to the HNF-4α gene fragment of (a) and contains a base A complementary to T at position 31 in the base sequence of SEQ ID NO: 1
(D) a polynucleotide that is complementary to the HNF-4α gene of (b) and contains a base A that is complementary to T at position 31 in the base sequence of SEQ ID NO: 1
(E) a polynucleotide complementary to the polynucleotide of the above (c) or (d)
[0019]
If the probe of the present invention is used, the marker of the present invention is detected by the above-described hybridization method or the like, whereby the base at position 31 in exon 4 of the HNF-4α gene in the diagnostic subject is cytosine (c). Since it can be determined whether it is thymine (t), it is possible to diagnose the possibility of developing type 2 diabetes and arteriosclerosis.
[0020]
In the probe of the present invention, the type of the polynucleotide is not particularly limited, for example, polydeoxyribonucleotides composed of deoxyribonucleotides, polyribonucleotides composed of at least one of ribonucleotides and modified ribonucleotides, or deoxyribonucleotides and ribonucleotides And chimeric polynucleotides consisting of both. The length of the polynucleotide is not particularly limited as described above, but the number of bases is, for example, in the range of 5 to 50, preferably 10 to 30, and more preferably 15 to 30. The target to be hybridized with the probe of the present invention may be any of DNA, cDNA, and mRNA. The combination of the type of the target and the type of the polynucleotide may be a known combination. Can be
[0021]
Further, the probe of the present invention can be used for a DNA chip. The DNA chip of the present invention is characterized in that the probe of the present invention is immobilized on a substrate, and has the same configuration as a conventionally known DNA chip except that the probe of the present invention is used. It can be used based on the method of. If such a DNA chip is used, the first marker and the second marker of the present invention can be detected by hybridizing the probe with a prepared sample such as mRNA and detecting the intensity of hybridization. Therefore, by detecting the first and second markers of the present invention with this DNA chip, the possibility of the onset of type 2 diabetes or arteriosclerosis can be easily and quickly diagnosed.
[0022]
【Example】
(Example 1)
In the following examples of the present invention, allele frequency was tested by a chi-square test and a Fishers exact test as statistical significance. All clinical data were expressed as mean ± SD. Comparison of variables between different genotype groups was performed by at least one of the two-tailed Student's t test and the Mann-Whitney non-parametric test. Statistical analysis was performed using publicly known software "StatView 5.0 software (SAS institute, Cary, NC)." Unless otherwise indicated, a p-value of less than 0.05 was considered significant.
[0023]
1. HNF-4 in the α gene T130I Mutation screening
(patient)
423 Japanese type 2 diabetic patients (male 253, female 170; age 63.9 ± 8.9 years; BMI 23.0 ± 3.2 kg / m)²HbA1c 7.6 ± 1.6%, mean ± SD, 354 Japanese non-diabetic patients (control) (male 158, female 196; age 63.8 ± 18.9 years; BMI 22.4 ± 2) .8 kg / m², HbA1c 4.9 ± 0.4%). Type 2 diabetes was diagnosed based on WHO criteria and all type 2 diabetic patients were diagnosed as having type 2 diabetes after age 35. Also excluded were patients with type 1 diabetes and other diabetes (eg, MODY).
[0024]
(Method)
Using the genomic DNA of a patient extracted according to a conventional method as a template, exon 4 and flanking intron in the HNF-4α gene were converted into specific forward primers (SEQ ID NO: 2: 5′-CCACCCCCCACTCCATCCCTGT-3 ′) and reverse primers (SEQ ID NO: 3). : 5'-CCCTCCCGTCAGCTGCTCCA-3 ') and amplified by PCR (see Non-Patent Document 4). The T130I mutation siteBsmThe resulting amplification product is generated by restriction enzymeBsm  I-treated and detected by the PCR-RFLP method. As a result, SEQ ID NO: 4 (ccacccccta ctccatccct gttctcccctc ctcacctctc tgtgcctcct cacagccgtc cadagaatgag gggaccgcatgagcattcga aggtcaagct atgaggacag cagcctgccc tccatcaatg cgctcctgca ggcggaggtc ctgtcccgac aggtaccggg gtgatcctgc cacccaccca gggatccccc acactacaga ggagctcacc tcctccacct ccattctccc cagccaggcc ctggagcagc tgacgggagg g) fragments of 81bp and 190bp in the amplification of T30I mutation consisting of the nucleotide sequence of, in the amplification of unmutated (T130T) is A 271 bp fragment was identified.
[0025]
(result)
As a result of the SNP analysis, the T130I mutation was confirmed in 15 of 423 cases of type 2 diabetes (3.5%) and in 3 of 354 non-diabetic patients (0.8%). This result indicates that the mutation frequency in type 2 diabetic patients is significantly different from that in non-diabetic patients and is very high (p = 0.015, odds ratio 4.3, 95% CI 1.24-14.98). In addition, previous literature (Sakurai K, Seki N, Fujii R et al (2000) Mutations in the hepatocyte nuclear factor-4a gene in Japanese with non-insulin-dependent diabetes:. A nucleotide substitution in the polypyrimidine tract of intron 1b. Home Metab Res 32: 316-322) shows that the frequency of the same mutation is 2.0% (2/100) and 0% (0/100) in Japanese type 2 diabetics and control patients, respectively. From the disclosure, these two results can be summed up to 2 Diabetes mutation frequency 17/523 (3.3%), control mutation frequency 3/454 (0.7%), p = 0.0053, odds ratio 5.05, 95% CI 1.47-17.35. Is the result. From this, it can be said that the strong association between the T130I mutation and type 2 diabetes was fully supported. In the literature, no significant difference in mutation frequency was observed, nor was the connection between the SNP and type 2 diabetes established. It is clear that the inventors have proved heading for the first time.
[0026]
2. T130I Clinical characteristics of diabetic patients with mutation
Table 1 below shows the clinical characteristics of diabetic patients having the Ile codon at position 130 (having the T130I-HNF-4α mutation) and diabetic patients having the Thr codon (having no T130I-HNF-4α mutation). In Table 1 below, “Thr / Thr” is a diabetic patient without a T130I-HNF-4α mutation “Thr / Ile” is a diabetic patient with a T130I-HNF-4α mutation, “n” is the number of patients, “M / “F” indicates the “number of men / female” of the patient, “age” indicates the age of the patient, and “age at diagnosis of diabetes” indicates the age at which diabetes was diagnosed. In the table, the measured value of each test item such as BMI is represented by “Means ± SD”, and when p> 0.05, it was determined that there was no significant difference (NS).
[0027]
[Table 1]

[0028]
As shown in Table 1, the average age at which a diabetic patient with the T130I-HNF-4α mutation was diagnosed with diabetes was 47.1 ± 8.8 years (p = 1.0 × 10^-6), And the average age of the other HNF-4α mutant diabetic patients (MODY1 patients) indicated by “#” in the table is 28.2 ± 15.2 years (n = 40, p = 1.0 × 10^-6Turned out to be very high. From these results, it can be said that patients with the T130I-HNF-4α mutation have a high probability of developing normal type 2 diabetes, not juvenile-onset diabetes (eg, MODY).
[0029]
Age of onset, BMI (body mass index), maximum BMI, fasting blood glucose (FPG), HbA1c, insulin resistance between diabetic patients with T130I-HNF-4α mutation and diabetic patients without T130I-HNF-4α mutation Homeostasis model assessment (HOMA-IR), total cholesterol and triglyceride levels were not significantly different. However, plasma HDL-cholesterol levels were lower in T130I mutant diabetic patients (p = 0.006). This result indicates that the difference in the measurement level can be determined to be a significant difference, and thus it was confirmed that the HNF-4α gene of the T130I mutation was an index (marker) in determining the possibility of developing arteriosclerosis.
[0030]
In addition, in diabetic patients of T130I genotype (4 cases) for which significant data was obtained, the response of C-peptide stimulated with glucagon was 0.9 ng / ml, 1.3 ng / ml, and 4.0 ng / ml. ml, 4.7 ng / ml (normal> 2.0 ng / ml). The urinary C-peptide (uCPR) concentrations of the patients (6 cases) were 14.7 μg / 24 h, 47.8 μg / 24 h, 67.2 μg / 24 h, 93.7 μg / 24 h, 101 μg / 24 h, and 149 μg / 24 h (normal: 50 to 120 μg / 24 h). These results suggest that insulin secretion is fluctuating among patients with the T130I-HNF-4α mutation. This can be said to be in contrast to the MODY1 mutation that induces a characteristic defect in insulin secretion in diabetic patients with the T130I-HNF-4α mutation (Non-Patent Document 4).
[0031]
3. Confirmation of transactivation activity
(Plasmid)
The T130I mutant HNF-4.alpha. Gene, prepared from human HNF-4α2 cDNA using Chameleon Double-Stranded Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and, pcDNA3.1 expression vector (Invitrogen, San Diego, CA) Was cloned. In addition, the structure was confirmed by DNA sequence. A reporter construct (pHNF4-tk-Luc) comprising a heterologous TK promoter and an HNF-4α binding sequence as an enhancer, an HNF-1α reporter, and a PKL reporter construct are described in the literature (Yang Q, Yamagata K, Yamamoto K et al. (2000). ) R127W-HNF-4a is a loss of function mutation but not a rare polymorphism and causes Type II diabetes in a Japanese family with MODY1 Diabetologia 43:. 520-524 according to).
[0032]
(Cell culture)
Mouse primary hepatocytes (Mouse primary hepatocytes), literature (Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL (2000) Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity Mol Cell 6: 77-86), prepared by a collagenase perfusion method according to in rivers of lipodystrophy and ob / obmice, 6-well tissue culture. 3 × 10⁵  Cells were plated at a density of cells / well. Then, HeLa cells, MIN6 cells, HepG2 cells and primary hepatocytes were infected with 10 ng of the above-described expression vector and reporter together with pRL-TK (Promega, Madison, WI) as an internal control using LIPOFECTAMIN PLUS reagent. .
[0033]
(Luciferase assay)
The transactivation activity was measured 48 hours after the cells were infected with the expression vector using a Dual Luciferase Reporter Assay system and Lumat LB9501 Chemiluminescence (Yamagata K, Yamagata K., Yamagata K. P291fsinsC in the transcription factor hepatocyte nuclear factor-1 a is dominant negative. Diabetes 47: 1231-1235).
[0034]
(Western blot analysis and electrophoretic mobility shift assay (EMSA)
Western blot analysis was performed using an anti-HNF-4α antibody (Santa Cruz Biotechnology, Santa Cruz, CA) reported in the above-mentioned Yamagata et al. (1998). Wild-type HNF-4α protein and T130I-HNF-4α protein were synthesized using TNT T7 Quick Coupled Transcription / Translation System (Promega). Then, the protein translated in vitro contains the HNF-4α binding site (5′-GGCTGAAGTCCAAAGTTCAGTCCCTCTCGC-3 ′) of the HNF-1α gene.³²Incubation with a probe of a P-labeled oligonucleotide in a 20 μL reaction mixture resulted in the formation of a DNA-protein complex (Yang Q, Yamagata K, Yamamoto K et al. (1999) Structure / function studies of hepatic phenomena) 1a, a diabetes-associated transscription factor. Biochem Biophys Res Commun 266: 196-202). In the competition binding assay, a 50-fold unlabeled probe (the same sequence as the labeled probe) was used as a competator. The protein (DNA-protein complex) after the incubation was electrophoresed on a 5% polyacrylamide gel using 0.5 × TBE buffer, and EMSA analysis was performed by autoradiography. In addition, polyclonal anti-HNF-4α antiserum (α445) was used for mobility analysis (see non-patent document 1). Furthermore, in order to confirm the relative binding affinity of T130I-HNF-4α, the amount of unlabeled comparator was increased (10, 25 and 50 times) and added to reach a non-equilibrium state. (See Yang Q et al., 1999).
[0035]
First, Hela cells were infected with a wild-type (WT) -HNF-4α expression vector and a T130I-HNF-4α expression vector, respectively, and the expression of WT-HNF-4α and T130I-HNF-4α was confirmed. The result is shown in FIG. 1A. FIG. 1A is a photograph showing the results of western blotting performed 48 hours after infecting Hela cells with each of the above expression vectors (8 μg). Lane 1 is WT-HNF-4α, and lane 2 is T130I-HNF-4α. And lane 3 shows the results of the control (without the expression vector). As shown, the expression levels of WT-HNF-4α and T130I-HNF-4α were almost similar. From this, it can be said that the expression itself of HNF-4α is not affected by the presence or absence of the T130I SNP.
[0036]
Next, the transactivation activity of WT-HNF-4α and T130I-HNF-4α was calculated by a luciferase reporter gene assay. These results are shown in FIGS. 2B and C and FIG. 3D. These figures show that each cell was infected with 500 ng of the aforementioned expression vector together with 100 ng of pHNF4-tk-Luc and 10 ng of pRL-TK, and transactivation of WT-HNF-4α and T130I-HNF4α in each cell 48 hours later. It is a graph which shows activity. 2B shows the results for Hela cells, FIG. 2C shows the results for mouse insulinoma cell line MIN6 cells, and FIG. 3D shows the results for human hepatoma cell line HepG2 cells. In each figure, the vertical axis indicates transactivation activity corrected by the internal standard, and “vector” indicates the result of infection with a vector not containing the HNF-4α gene (the same applies hereinafter). As shown in FIGS. 2B and 2C, there was no significant difference in transactivation activity between Wt-HNF-4α and T130I-HNF-4α in Hela cells and MIN6 cells (N.S. ). However, as shown in FIG. 3D, when the same amount of the expression vector was used to infect human hepatoma cell line HepG2 cells, the transcriptional activity of T130I-HNF-4α was lower than that of WT-HNF-4α by 46%. 0.2% (p <0.001).
[0037]
Therefore, the dose of the expression vector was increased, and the transactivation activity of T130I-HNF-4α in HepG2 cells was confirmed. The result is shown in FIG. 4E. The figure shows that the transactivation activity of T130I-HNF-4α at 48 hours after increasing the dose of the expression vector (50 ng, 100 ng, 200 ng) and infecting HepG2 cells with pHNF-4-tk-Luc. It is a graph which shows a result. In the figure, each result shows the “mean ± SD” value of six tests. In the case of MIN6 cells, T130I-HNF-4α achieved the same transactivation as WT-HNF-4α even when the dose of the expression vector was changed (data not shown). In all cases, the transactivation activity of T130I-HNF-4α was reduced at all doses even when the amount of the expression vector was changed.
[0038]
The used HepG2 cells have a D69A mutation in the HNF-4α gene (Lausen J, Thomas H, Lemm I et al. (2000) Naturally occurring mutations in the country HNF4 gene exchange empirical action empirical action empirical action empirical action empirical action empirical action empirical action empirical action empirical action empirical mechanism). varying degree. Nucleic Acids Res 28: 430-437). Therefore, it was determined whether the D69A mutation causes a decrease in the transactivation of T130I-HNF-4α in HepG2 cells as described above by using the luciferase reporter gene assay using primary mouse hepatocytes. confirmed. Specifically, 3 × 10⁵Cells / well primary mouse hepatocytes were infected with 0.5 μg of the expression vector together with 100 ng of each reporter gene (pHNF-4-tk-Luc, HNF-1α promoter or PKL promoter), and the primary liver cells 48 hours later were infected. The transactivation activity of WT-HNF-4α and T130I-HNF4α in the cells was measured. The results are shown in FIGS. Each figure is a graph showing the transactivation activity in primary hepatocytes. FIG. 5F shows pHNF-4-tk-Luc as a reporter gene, FIG. 5G shows HNF-1α promoter as a reporter gene, and FIG. 5H shows PKL as a reporter gene. It is a graph which shows the result of using a promoter. In each figure, each result indicates the “mean ± SD” value of 6 tests. As shown in FIG. 5F (reporter gene: pHNF-4-tk-Luc), a decrease in TT130I-HNF-4α transactivation was also observed in primary hepatocytes (27. 9%, p = 9.7 × 10^-5). Also, HNF-1α and L-type pyruvate kinase (PKL) are target genes for HNF-4α (Yamagata K, Oda N, Kaisaki PJ et al. (1996) Mutations in the hepatocyte nuclear enzyme). As shown in FIG. 5G and FIG. 5H, the transactivation activity of the HNF-1α gene is 78.% of WT by the T130I-HNF-4α, as shown in FIG. 5G and FIG. 5H, where the nature-onset diabetes of the young (MODY3). Nature 384: 455-458). By 2% (p = 0.024), the transactivation activity of the PKL gene was reduced to 77.1% of WT (p = 0.002). These results strongly suggest that T130I-HNF-4α acts as a loss-of-function mutation in a hepatocyte environment, but not in a Hela cell or mouse MIN6 cell. I can say.
[0039]
Next, since the A-box region of HNF-4α is considered to be important for DNA binding as described above, the DNA binding activity of T130I-HNF-4α was further examined. First, the WT-HNF-4α protein and the T130I-HNF-4α protein were translated in vitro from the pcDNA3.1 expression vector (1 μg) using the TNT T7 Quick Coupled Transcription / Translation System, and provided for EMSA. . Expression levels of the transcribed protein in vitro were confirmed by Western blot analysis (data not shown). As described above, the excess (50-fold) unlabeled oligonucleotide was used as the comparator.
[0040]
These results are shown in FIG. 6A. The figure is a photograph showing the EMSA results of WT-HNF-4α and DNA, and T130I-HNF-4α and DNA expressed in vitro. In the figure, lane 1 is WT-HNF-4α, lane 2 is WT-HNF-4α and a competor, lane 3 is T130I-HNF-4α, lane 3 is T130I-HNF-4α, and lane 4 is T130I-HNF-4α. Lane 5 is control (no vector), lane 6 is control (no vector + competrator), lane 7 is WT-HNF-4 and anti-HNF-4α antibody, lane 8 is T130I-HNF-4α and anti-HNF This is the result of the -4α antibody, and the arrow indicates the position of the HNF-4α / DNA complex. As shown in the figure, bands were confirmed at the positions of the arrows in lanes 1 and 3, indicating that WT-HNF-4α and T130I-HNF-4α specifically bound to the oligonucleotide. In lanes 7 and 8, compared to lanes 1 and 2, supershift of the band was confirmed, indicating that WT-HNF-4α and T130I-HNF-4α further bound to the anti-HNF-4α antibody. Understand.
[0041]
In addition, the amount of the non-labeled comparator added was increased (10, 25, 50 mol), and WT-HNF-4α and T130I-HNF-4α were bound to the labeled oligonucleotide in a non-equilibrium state, to give EMSA Was done. The result is shown in FIG. 6B. As shown, the DNA binding of T130I-HNF-4α showed the same results both in the equilibrium state shown in FIG. 6A and in the non-equilibrium state shown in FIG. 6B. From these results, it was found that the amino acid change of T130I-HNF-4α did not affect the binding to DNA in vitro.
[0042]
【The invention's effect】
As described above, according to the first marker of the present invention, the onset possibility of type 2 diabetes is judged with excellent reliability. According to the second marker of the present invention, the possibility of onset of arteriosclerosis is determined. Can be determined. Therefore, it can be said that, for example, it is very useful for diagnosis and treatment of type 2 diabetes and arteriosclerosis in the clinical field.
[0043]
[Sequence list]

[Brief description of the drawings]
FIG. 1A is a photograph showing the result of Western blot after infecting Hela cells with expression vectors of WT-HNF-4α and T150I-HNF-4α.
FIGS. 2B and 2C are graphs showing the transactivation activity of WT-HNF-4α and T130I-HNF4α after infecting cells with expression vectors of WT-HNF-4α and T150I-HNF-4α. , B shows the results for Hela cells, and C shows the results for mouse insulinoma cell line MIN6 cells.
FIG. 3D is a graph showing the transactivation activity of WT-HNF-4α and T130I-HNF4α after infecting cells with the expression vectors of WT-HNF-4α and T150I-HNF-4α. It is a result about a hepatoma cell line HepG2 cell.
FIG. 4 is a graph showing the transactivation activity of T130I-HNF-4α in HepG2 cells.
FIG. 5 is a graph showing the transactivation activity of WT-HNF-4α and T130I-HNF4α after infecting mouse primary hepatocytes with a WT-HNF-4α and T150I-HNF-4α expression vector together with a reporter gene. Yes, F is the result of using pHNF-4-tk-Luc, G is the result of using the HNF-1α promoter, and H is the result of using the PKL promoter as the reporter gene.
FIGS. 6A and 6B are photographs showing EMSA results of WT-HNF-4α and DNA, and T130I-HNF-4α and DNA, wherein A shows an equilibrium state and B shows a non-equilibrium result.

Claims (12)

A marker for determining the possibility of onset of type 2 diabetes, which is the following marker (a) or (b).
(A) a human hepatocyte nuclear factor (HNF) -4α gene exon 4 wherein the C at position 31 is mutated to T, and a HNF-4α gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 (b) the gene of (a) above HNF-4α gene containing fragment A probe for detecting the marker according to claim 1, wherein at least one probe selected from the group consisting of the following (c) to (e).
(C) a polynucleotide (d) which is complementary to the HNF-4α gene fragment of (a) according to claim 1 and comprises a base A complementary to T at position 31 in the base sequence of SEQ ID NO: 1 (d A polynucleotide (e) complementary to the HNF-4α gene of (b) according to claim 1 and comprising a base A complementary to T at position 31 in the base sequence of SEQ ID NO: 1. a polynucleotide complementary to the polynucleotide of c) or (d) The probe according to claim 2, wherein the number of bases of the polynucleotide ranges from 5 to 50. The probe according to claim 2, wherein the polynucleotide is a polydeoxynucleotide or a polyribonucleotide. A DNA chip for determining the possibility of onset of type 2 diabetes, wherein the probe according to any one of claims 2 to 4 is immobilized on a substrate. A primer used for detecting the marker according to claim 1, wherein at least one of the following primers (e) and (f):
(E) oligonucleotide consisting of the base sequence of SEQ ID NO: 2 (f) oligonucleotide consisting of the base sequence of SEQ ID NO: 3 A marker for determining the possibility of developing arteriosclerosis, wherein the marker comprises the following (a) or (b):
(A) a human hepatocyte nuclear factor (HNF) -4α gene exon 4 wherein the C at position 31 is mutated to T, and a HNF-4α gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 (b) the gene of (a) above HNF-4α gene containing fragment A probe for detecting the marker according to claim 7, wherein at least one probe selected from the group consisting of the following (c) to (e).
(C) a polynucleotide (d) that is complementary to the HNF-4α gene fragment of (a) according to claim 7 and that contains a base A complementary to T at position 31 in the base sequence of SEQ ID NO: 1 (d A) a polynucleotide complementary to the HNF-4α gene of (b) according to claim 7 and comprising a base A complementary to T at position 31 in the base sequence of SEQ ID NO: 1; a polynucleotide complementary to the polynucleotide of c) or (d) The probe according to claim 8, wherein the number of bases of the polynucleotide ranges from 5 to 50. The probe according to claim 8 or 9, wherein the polynucleotide is a polydeoxynucleotide or a polyribonucleotide. A DNA chip for judging the possibility of arteriosclerosis, wherein the probe according to any one of claims 8 to 10 is immobilized on a substrate. A primer used for detecting the marker according to claim 7, wherein at least one of the following primers (e) and (f):
(E) oligonucleotide consisting of the base sequence of SEQ ID NO: 2 (f) oligonucleotide consisting of the base sequence of SEQ ID NO: 3

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