JP2004141013A - Method for judging onset risk to diabetes - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for judging the relative onset risk to diabetes, especially type 2 diabetes. <P>SOLUTION: The method for judging the onset risk to the diabetes comprises detecting any one or two or more genetic polymorphisms present in the -34642nd nucleotide in the 5' upstream region of an HIRIP5 gene, the -26376th nucleotide in the 5' upstream region of the HIRIP5 gene, the -350th nucleotide of an intron 3 of the HIRIP5 gene, the 2721st nucleotide of an intron 4 of the HIRIP5 gene, the -28256th nucleotide in the 5' upstream region of the HIRIP5 gene, the -27915th nucleotide in the 5' upstream region of the HIRIP5 gene, the 3270th nucleotide of the intron 3 of the HIRIP5 gene, the 3832nd nucleotide of the intron 3 of the HIRIP5 gene, the -168th nucleotide of the intron 3 of the HIRIP5 gene and the 36th nucleotide of the first intron of a GFAT1 gene and/or a haplotype composed of a combination thereof. <P>COPYRIGHT: (C)2004,JPO

Description

ＰＣＲ条件：９４℃３０秒、５８℃３０秒、７２℃１分を３７サイクル
【０１０２】
２）ＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）がｇ（グアニン）であるか、ｃ（シトシン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１０ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．３：　５’−ＣＴＴＣＴＧＧＧＡＡＡＧＡＡＴＴＣＡＴＣＣＴ−３’（配列番号１３）、
プライマーＮｏ．４：　５’−ＡＣＡＧＴＣＴＧＡＧＴＡＴＣＴＧＧＧＣＴ−３’（配列番号１４）
（Ｎｏ．３、Ｎｏ．４ともに、ＨＥＸで５’末端を標識）
【０１０３】
【表２】

ＰＣＲ条件：９４℃３０秒、５６℃３０秒、７２℃１分を３７サイクル
【０１０４】
３）ＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）ｔ（チミン）の繰り返しが１回であるか２回であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１４ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．５：　５’−ＡＣＣＡＧＴＡＡＡＴＴＧＡＧＡＡＡＣＴＧＡ−３’（配列番号１５）
プライマーＮｏ．６：　５’−ＡＡＧＣＣＡＧＴＡＡＡＴＡＴＧＣＡＧＡＣ−３’（配列番号１６）
（Ｎｏ．５、Ｎｏ．６ともに、ＮＥＤで５’末端を標識）
【０１０５】
【表３】

ＰＣＲ条件：９４℃３０秒、５６℃３０秒、７２℃１分を３７サイクル
【０１０６】
４）ＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）がｃ（シトシン）であるか、ｔ（チミン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１５ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．７：　５’−ＡＣＡＣＣＡＴＴＧＣＡＣＴＣＴＧＧＴＣＡ−３’（配列番号１７）、
プライマーＮｏ．８：　５’−ＴＴＡＣＣＡＴＴＴＡＣＴＧＴＴＴＣＣＴＡＴＴＴＴ−３’（配列番号１８）
（Ｎｏ．７、Ｎｏ．８ともに、ＨＥＸで５’末端を標識）
【０１０７】
【表４】

ＰＣＲ条件：９４℃３０秒、５３℃３０秒、７２℃１分を３７サイクル
【０１０８】
５）ＨＩＲＩＰ５遺伝子の５’上流域の−２８２５６番目のヌクレオチド（配列番号５のヌクレオチド番号２５８）がｇ（グアニン）であるか、ｃ（シトシン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１７ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．９：　５’−ＧＴＡＧＣＣＴＧＡＡＴＣＡＧＧＴＴＣＣＡ−３’（配列番号１９）
プライマーＮｏ．１０：　５’−ＡＣＣＣＴＧＴＡＧＡＡＧＡＣＡＣＧＡＧＴ−３’（配列番号２０）
（Ｎｏ．９、Ｎｏ．１０ともに、ＮＥＤで５’末端を標識）
【０１０９】
【表５】

ＰＣＲ条件：９４℃３０秒、５９℃３０秒、７２℃１分を３７サイクル
【０１１０】
６）ＨＩＲＩＰ５遺伝子の５’上流域の−２７９１５番目のヌクレオチド（配列番号６のヌクレオチド番号２９９）がｔ（チミン）であるか、ｃ（シトシン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１６ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．１１：　５’−ＧＣＡＴＴＡＴＧＣＧＴＴＡＧＣＣＡＧＡＣ−３’（配列番号２１）
プライマーＮｏ．１２：　５’−ＧＧＡＧＣＧＡＧＡＴＡＣＡＣＴＧＧＡＡＡ−３’（配列番号２２）
（Ｎｏ．１１はＦＡＭで５’末端を標識、Ｎｏ．１２はＨＥＸで５’末端を標識）
【０１１１】
【表６】

ＰＣＲ条件：９４℃３０秒、５６℃３０秒、７２℃１分を３７サイクル
【０１１２】
７）ＨＩＲＩＰ５遺伝子のイントロン３の３２７０番目のヌクレオチド（配列番号７のヌクレオチド番号２８８）がｇ（グアニン）であるか、ａ（アデニン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１９ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．１３：　５’−ＧＧＴＣＴＴＣＡＡＧＴＧＡＴＣＣＴＣＣＴ−３’（配列番号２３）
プライマーＮｏ．１４：　５’−ＧＣＴＣＡＣＡＣＡＡＴＣＴＧＣＴＧＣＣＴ−３’（配列番号２４）
（Ｎｏ．１３はＮＥＤで５’末端を標識、Ｎｏ．１４はＮＥＤで５’末端を標識）
【０１１３】
【表７】

ＰＣＲ条件：９４℃３０秒、６１℃３０秒、７２℃１分を３７サイクル
【０１１４】
８）ＨＩＲＩＰ５遺伝子のイントロン３の３８３２番目のヌクレオチド（配列番号８のヌクレオチド番号３１０）がｃ（シトシン）であるか、ｔ（チミン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．２０ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．１５：　５’−ＧＴＧＧＴＴＡＣＡＴＴＡＧＴＴＣＡＴＣＴＴ−３’（配列番号２５）
プライマーＮｏ．１６：　５’−ＣＡＴＡＣＡＣＡＴＡＴＧＡＣＡＧＴＡＴＣＴ−３’（配列番号２６）
（Ｎｏ．１５はＦＡＭで５’末端を標識、Ｎｏ．１６はＦＡＭで５’末端を標識）
【０１１５】
【表８】

ＰＣＲ条件：９４℃３０秒、５６℃３０秒、７２℃１分を３７サイクル
【０１１６】
９）ＨＩＲＩＰ５遺伝子のイントロン３の−１６８番目のヌクレオチド（配列番号９のヌクレオチド番号３１３）がｔ（チミン）であるか、ａ（アデニン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．２２ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．１７：　５’−ＣＣＴＡＡＣＡＴＣＡＴＴＡＧＣＡＴＡＴＣＡ−３’（配列番号２７）
プライマーＮｏ．１８：　５’−ＧＣＡＡＧＴＡＣＧＡＧＴＡＴＴＡＡＡＡＣＡ−３’（配列番号２８）
（Ｎｏ．１７はＦＡＭで５’末端を標識、Ｎｏ．１８はＦＡＭで５’末端を標識）
【０１１７】
【表９】

ＰＣＲ条件：９４℃３０秒、５６℃３０秒、７２℃１分を３７サイクル
【０１１８】
１０）ＧＦＡＴ１遺伝子の第１イントロンの３６番目（配列番号１０のヌクレオチド番号６３２）のヌクレオチドがｃ（シトシン）であるかｔ（チミン）であるかを検出するためのＰＣＲ：
以下のプライマーおよび反応条件でＰＣＲを行い、多型部位を含む０．１６ｋｂｐのＤＮＡ断片を増幅した。
ＰＣＲプライマー：
プライマーＮｏ．１９：　５’−ＣＡＣＣＧＡＡＧＣＴＣＧＴＧＴＧＴＧＣＡ−３’（配列番号２９）
プライマーＮｏ．２０：　５’−ＣＧＣＣＴＴＧＧＧＣＣＴＴＡＧＣＧＧＣＡ−３’（配列番号３０）
（Ｎｏ．１９はＨＥＸで５’末端を標識、Ｎｏ．２０はＨＥＸで５’末端を標識）
【０１１９】
【表１０】

ＰＣＲ条件：９４℃１分、５５℃１分、７２℃２分３０秒を４０サイクル
【０１２０】
実施例３：　ＨＩＲＩＰ５遺伝子、およびＧＦＡＴ１遺伝子の多型の検出
１）ＤＮＡシークエンサーによる解析
ＨＩＲＩＰ５遺伝子、およびＧＦＡＴ１遺伝子の各多型部位を同定するためのヌクレオチド配列の決定は、ＤＮＡシークエンサー（ＡＢＩ　ＰＲＩＳＭ　３７７：アプライドバイオシステムズ社製）を用いて常法により行った。
【０１２１】
その結果、以下の位置に各多型が存在することが確認された。
ａ）ＨＩＲＩＰ５遺伝子の５’上流域の−３４６４２番目のヌクレオチド（配列番号１のヌクレオチド番号３５２）；
ｂ）ＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）；
ｃ）ＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）；
ｄ）ＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）；
ｅ）ＨＩＲＩＰ５遺伝子の５’上流域の−２８２５６番目のヌクレオチド（配列番号５のヌクレオチド番号２５８）；
ｆ）ＨＩＲＩＰ５遺伝子の５’上流域の−２７９１５番目のヌクレオチド（配列番号６のヌクレオチド番号２９９）；
ｇ）ＨＩＲＩＰ５遺伝子のイントロン３の３２７０番目のヌクレオチド（配列番号７のヌクレオチド番号２８８）；
ｈ）ＨＩＲＩＰ５遺伝子のイントロン３の３８３２番目のヌクレオチド（配列番号８のヌクレオチド番号３１０）；
ｉ）ＨＩＲＩＰ５遺伝子のイントロン３の−１６８番目のヌクレオチド（配列番号９のヌクレオチド番号３１３）；
ｊ）ＧＦＡＴ１遺伝子の第１イントロンの３６番目（配列番号１０のヌクレオチド番号６３２）
【０１２２】
２）ＳＳＣＰ法
実施例２で得られた各ＰＣＲ産物を、ホルムアミド溶液：ローディングバッファー（０．０３ｇ／ｍｌのブルーデキストランを含む０．０５ＭのＥＤＴＡ水溶液）＝５：１の混合液に１／５量加え混合し、９５℃で２分間の熱変性後、氷冷して電気泳動用試料とした。この試料を非変性ポリアクリルアミドゲルで電気泳動し、アレルの相違に基づくＤＮＡ断片の移動度の違いにより多型を検出した。
【０１２３】
電気泳動のゲルは、１２％ポリアクリルアミドゲルにグリセロ―ルを５％添加したもの（ゲル厚さ０．２ｍｍ）を用いた。なお、測定条件の違いにより多型検出に変化がないことを確認するために、１０％ポリアクリルアミドにグルセロ―ルを５％添加したもの、および１２％ポリアクリルアミドゲルにグルセロ―ルを１０％添加したものの２条件で行った。
【０１２４】
電気泳動は、ＤＮＡシークエンサー（ＡＢＩ　ＰＲＩＳＭ　３７７：アプライドバイオシステムズ社製）を用いて、３０ワット定電力でＡフィルターを使用し、３〜１２時間通電することにより実施した。蛍光バンドはＤＮＡシークエンサーの検出機能を用いて検出した。電気泳動中は、ゲル板温度を保つため、冷却装置（ＲＴＥ−ＩＩＩ、ＮＥＳＬＡＢ社製）で２０℃に調節した水をゲル板外部に循環させるようにした。なお、各ＤＮＡ断片の遺伝子型は、前項１）において多型部位のヌクレオチド配列を決定したＤＮＡ断片をポジティブコントロールとして判定した。
【０１２５】
実施例４：　ＨＩＲＩＰ５遺伝子およびＧＦＡＴ１遺伝子の多型と糖尿病との関連
ＨＩＲＩＰ５遺伝子と２型糖尿病との関連を関連解析法（アソシエーション法）により解析した。有意差検定にはχ２乗法を用いた。（フィッシャーら、バイオスタティスティクス（Ｂｉｏｓｔａｔｉｓｔｉｃｓ）、Ｊｏｈｎ　Ｗｉｌｌｅｙ　＆　Ｓｏｎｓ社、１９９３年）。ハプロタイプ解析は、Ａｒｌｅｑｕｉｎプログラムを用いて行なった（Ｌａｕｒｅｎｔ　Ｅｘｃｏｆｆｉｅｒ　社製　統計解析ソフトウェア、ＵＲＬ：ｈｔｔｐ：／／ｌｇｂ．ｕｎｉｇｅ．ｃｈ／ａｒｌｅｑｕｉｎ／）。有意差検定には、Ｆｉｓｈｅｒの直接確率計算法を拡張したｅｘａｃｔ　ｔｅｓｔ　ｏｆ　ｐｏｐｕｌａｔｉｏｎ　ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ（ＰＤ検定）を用いた（「基礎医学統計学　改定第４版」、加納克己、高橋秀人共著、南江堂、１９９５年）。有意水準は危険率５％とし、危険率５％未満のものを有意差ありと判定した。
【０１２６】
１）２型糖尿病とＨＩＲＩＰ５遺伝子５’上流域の−３４６４２番目のヌクレオチド（配列番号１のヌクレオチド番号３５２）との関係
日本人の健常人１９０名、日本人の２型糖尿病患者３９３名について、ＨＩＲＩＰ５遺伝子５’上流域の−３４６４２番目のヌクレオチド（配列番号１のヌクレオチド番号３５２）におけるアレル頻度を算出し、アソシエーション法により２型糖尿病との関連を解析した。結果を表１１に示す。
【０１２７】
【表１１】

【０１２８】
表１１において、ＨＩＲＩＰ５遺伝子５’上流域の−３４６４２番目のヌクレオチド（配列番号１のヌクレオチド番号３５２）部位がチミンの場合は「Ｔ」、あるいは、シトシンの場合は「Ｃ」として表されている。表１１の結果より、健常人と２型糖尿病患者との間において、ＨＩＲＩＰ５遺伝子５’上流域の−３４６４２番目のヌクレオチド（配列番号１のヌクレオチド番号３５２）多型のアレル頻度が統計的に有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子５’上流域の−３４６４２番目のヌクレオチド（配列番号１のヌクレオチド番号３５２）の多型を調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１２９】
２）２型糖尿病とＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）との関係
日本人の健常人１９１名、日本人の２型糖尿病患者３９０名について、２型糖尿病とＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）多型のアレル頻度を算出し、アソシエーション法により２型糖尿病との関連を解析した。結果を表１２に示す。
【０１３０】
【表１２】

【０１３１】
表１２において、ＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）部位がグアニンの場合は「Ｇ」、あるいは、シトシンの場合は「Ｃ」として表されている。表１２の結果より、健常人と２型糖尿病患者との間において、ＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）多型のアレル頻度および遺伝子型頻度が統計的に有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子の５’上流域の−２６３７６番目のヌクレオチド（配列番号２のヌクレオチド番号２７８）の多型を調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１３２】
３）２型糖尿病とＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）との関係
日本人の健常人１９０名、日本人の２型糖尿病患者３９４名について、ＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）におけるアレル頻度を算出し、アソシエーション法により２型糖尿病との関連を解析した。結果を表１３に示す。
【０１３３】
【表１３】

【０１３４】
表１３において、ＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）におけるＴの繰り返しが１回の場合は、「Ｔ」、あるいは、２回の場合は「ＴＴ」として表されている。表１３の結果より、健常人と２型糖尿病患者との間において、ＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）多型のアレル頻度が統計的に有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子のイントロン３の−３５０番目のヌクレオチド（配列番号３のヌクレオチド番号３１１）の多型を調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１３５】
４）２型糖尿病とＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）の多型との関係
日本人の健常人１９１名、日本人の２型糖尿病患者３８８名について、２型糖尿病とＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）多型のアレル頻度を算出し、アソシエーション法により２型糖尿病との関連を解析した。結果を表１４に示す。
【０１３６】
【表１４】

【０１３７】
表１４において、ＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）部位がシトシンの場合は「Ｃ」、あるいは、チミンの場合は「Ｔ」として表されている。表１４の結果より、健常人と２型糖尿病患者との間において、ＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）多型のアレル頻度が統計的に有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子のイントロン４の２７２１番目のヌクレオチド（配列番号４のヌクレオチド番号２６２）の多型を調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１３８】
５）２型糖尿病とＨＩＲＩＰ５遺伝子の５’上流域の−２８２５６番目のヌクレオチド（配列番号５のヌクレオチド番号２５８）多型とＨＩＲＩＰ５遺伝子の５’上流域の−２７９１５番目のヌクレオチド（配列番号６のヌクレオチド番号２９９）多型を組み合わせたハプロタイプとの関係
日本人の健常人１９１名、日本人の２型糖尿病患者３８６名について、ＨＩＲＩＰ５遺伝子の５’上流域の−２８２５６番目のヌクレオチド（配列番号５のヌクレオチド番号２５８）多型とＨＩＲＩＰ５遺伝子の５’上流域の−２７９１５番目のヌクレオチド（配列番号６のヌクレオチド番号２９９）多型を組み合わせたハプロタイプ頻度を算出し、ＰＤ検定により２型糖尿病との関連を解析した。結果を表１５に示す。
【０１３９】
【表１５】

【０１４０】
表１５において、ＨＩＲＩＰ５遺伝子の５’上流域の−２８２５６番目のヌクレオチド（配列番号５のヌクレオチド番号２５８）がＧである遺伝子をホモで有する被験者は「Ｇ／Ｇ」、当該部位がＣ（シトシン）である遺伝子をホモで有する被験者は「Ｃ／Ｃ」、両遺伝子をヘテロで有する被験者は「Ｇ／Ｃ」として表されている。また、表１５において、ＨＩＲＩＰ５遺伝子の５’上流域の−２７９１５番目のヌクレオチド（配列番号６のヌクレオチド番号２９９）がＴである遺伝子をホモで有する被験者は「Ｔ／Ｔ」、当該部位がＣ（シトシン）である遺伝子をホモで有する被験者は「Ｃ／Ｃ」、両遺伝子をヘテロで有する被験者は「Ｔ／Ｃ」として表されている。表１５の結果より、健常人と２型糖尿病患者との比較において、それぞれのハプロタイプにおける頻度分布が有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子の５’上流域の−２８２５６番目のヌクレオチド（配列番号５のヌクレオチド番号２５８）多型とＨＩＲＩＰ５遺伝子の５’上流域の−２７９１５番目のヌクレオチド（配列番号６のヌクレオチド番号２９９）多型を組み合わせたハプロタイプを調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１４１】
６）２型糖尿病とＨＩＲＩＰ５遺伝子のイントロン３の３２７０番目のヌクレオチド（配列番号７のヌクレオチド番号２８８）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプとの関係
日本人の健常人１８５名、日本人の２型糖尿病患者３５７名について、ＨＩＲＩＰ５遺伝子のイントロン３の３２７０番目のヌクレオチド（配列番号７のヌクレオチド番号２８８）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプ頻度を算出し、ＰＤ検定により２型糖尿病との関連を解析した。結果を表１６に示す。
【０１４２】
【表１６】

【０１４３】
表１６においてＨＩＲＩＰ５遺伝子のイントロン３の３２７０番目のヌクレオチド（配列番号７のヌクレオチド番号２８８）がＧである遺伝子をホモで有する被験者は「Ｇ／Ｇ」、当該部位がＡ（アデニン）である遺伝子をホモで有する被験者は「Ａ／Ａ」、両遺伝子をヘテロで有する被験者は「Ｇ／Ａ」として表されている。また、表１６において、ＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）がＴである遺伝子をホモで有する被験者は「Ｔ／Ｔ」、当該部位がＣ（シトシン）である遺伝子をホモで有する被験者は「Ｃ／Ｃ」、両遺伝子をヘテロで有する被験者は「Ｔ／Ｃ」として表されている。表１６の結果より、健常人と２型糖尿病患者との比較において、それぞれのハプロタイプにおける頻度分布が有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子のイントロン３の３２７０番目のヌクレオチド（配列番号７のヌクレオチド番号２８８）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプを調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１４４】
７）２型糖尿病とＨＩＲＩＰ５遺伝子のイントロン３の３８３２番目のヌクレオチド（配列番号８のヌクレオチド番号３１０）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプとの関係
日本人の健常人１８５名、日本人の２型糖尿病患者３８２名について、ＨＩＲＩＰ５遺伝子のイントロン３の３８３２番目のヌクレオチド（配列番号８のヌクレオチド番号３１０）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプ頻度を算出し、ＰＤ検定により２型糖尿病との関連を解析した。結果を表１７に示す。
【０１４５】
【表１７】

【０１４６】
表１７においてＨＩＲＩＰ５遺伝子のイントロン３の３８３２番目のヌクレオチド（配列番号８のヌクレオチド番号３１０）がＣ（シトシン）である遺伝子をホモで有する被験者は「Ｃ／Ｃ」、当該部位がＴ（チミン）である遺伝子をホモで有する被験者は「Ｔ／Ｔ」、両遺伝子をヘテロで有する被験者は「Ｃ／Ｔ」として表されている。また、表１７において、ＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）がＴである遺伝子をホモで有する被験者は「Ｔ／Ｔ」、当該部位がＣ（シトシン）である遺伝子をホモで有する被験者は「Ｃ／Ｃ」、両遺伝子をヘテロで有する被験者は「Ｔ／Ｃ」として表されている。表１７の結果より、健常人と２型糖尿病患者との比較において、それぞれのハプロタイプにおける頻度分布が有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子のイントロン３の３８３２番目のヌクレオチド（配列番号８のヌクレオチド番号３１０）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプを調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１４７】
８）２型糖尿病とＨＩＲＩＰ５遺伝子のイントロン３の−１６８番目のヌクレオチド（配列番号９のヌクレオチド番号３１３）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプとの関係
日本人の健常人１８６名、日本人の２型糖尿病患者３８５名について、ＨＩＲＩＰ５遺伝子のイントロン３の−１６８番目のヌクレオチド（配列番号９のヌクレオチド番号３１３）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプ頻度を算出し、アソシエーション法により２型糖尿病との関連を解析した。結果を表１８に示す。
【０１４８】
【表１８】

【０１４９】
表１８においてＨＩＲＩＰ５遺伝子のイントロン３の−１６８番目のヌクレオチド（配列番号９のヌクレオチド番号３１３）がＴ（チミン）である遺伝子をホモで有する被験者は「Ｔ／Ｔ」、当該部位がＡ（アデニン）である遺伝子をホモで有する被験者は「Ａ／Ａ」、両遺伝子をヘテロで有する被験者は「Ｔ／Ａ」として表されている。また、表１８において、ＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）がＴである遺伝子をホモで有する被験者は「Ｔ／Ｔ」、当該部位がＣ（シトシン）である遺伝子をホモで有する被験者は「Ｃ／Ｃ」、両遺伝子をヘテロで有する被験者は「Ｔ／Ｃ」として表されている。表１８の結果より、健常人と２型糖尿病患者との比較において、それぞれのハプロタイプにおける頻度分布が有意に異なることが示された。このことは、ＨＩＲＩＰ５遺伝子のイントロン３の−１６８番目のヌクレオチド（配列番号９のヌクレオチド番号３１３）多型とＧＦＡＴ１遺伝子の第１イントロンの３６番目のヌクレオチド（配列番号１０のヌクレオチド番号６３２）多型を組み合わせたハプロタイプを調べることにより、糖尿病を発症する相対的危険度を判定できることを示すものである。
【０１５０】
【発明の効果】
本発明によれば、糖尿病、特に２型糖尿病の相対的危険度を簡便に判定することができる。すなわち、本発明は糖尿病の発症危険性判定方法と該方法のための材料を提供する。
【０１５１】
【配列表】

【０１５２】
【配列表フリーテキスト】
配列番号１−変異の説明：（Ｔ）と（Ｃ）の置換による一塩基多型
配列番号２−変異の説明：（Ｃ）と（Ｇ）の置換による一塩基多型
配列番号３−変異の説明：（Ｔ）の一塩基挿入による多型
配列番号４−変異の説明：（Ｔ）と（Ｃ）の置換による一塩基多型
配列番号５−変異の説明：（Ｃ）と（Ｇ）の置換による一塩基多型
配列番号６−変異の説明：（Ｔ）と（Ｃ）の置換による一塩基多型
配列番号７−変異の説明：（Ａ）と（Ｇ）の置換による一塩基多型
配列番号８−変異の説明：（Ｃ）と（Ｔ）の置換による一塩基多型
配列番号９−変異の説明：（Ｔ）と（Ａ）の置換による一塩基多型
配列番号１０−変異の説明：（Ｔ）と（Ｃ）の置換による一塩基多型
配列番号１１〜３０−人工配列の説明：プライマー[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for determining the risk of developing diabetes. More specifically, the present invention relates to a method for determining a potential risk of developing diabetes by detecting a novel gene polymorphism associated with the development of diabetes from a sample containing human genomic DNA.
[0002]
[Prior art]
Diabetes has been increasing in the number of patients in recent years and is attracting attention as one of adult diseases. Diabetes includes insulin-dependent type 1 diabetes and non-dependent type 2 diabetes. Among them, type 2 diabetes (non-insulin-dependent diabetes mellitus, NIDDM) is a type of diabetes that is common in Japanese and is detected early. Early treatment is important in terms of its prognosis.
[0003]
However, the causes of type 2 diabetes are diverse and little is known about the possible causes. Causes of insufficient insulin action in type 2 diabetes include abnormalities in the insulin sensitivity mechanism and decreased insulin secretion. In Europe and the United States, the former, that is, insulin resistance is the main cause of type 2 diabetes, but in Japan, insulin secretion deficiency is often the main cause.
[0004]
On the other hand, diabetes develops retinopathy, neuropathy, arteriosclerosis or nephropathy as its complications. In particular, when a diabetic suffers from nephropathy, the renal disorder causes hypertension and hyperlipidemia to worsen, further worsening the prognosis of the patient. Therefore, prevention and treatment of nephropathy are particularly important problems among diabetic complications.
[0005]
In recent years, with the rapid progress of molecular biology, knowledge on the mechanism of insulin action has been accumulated. Beginning to elucidate the structure of the insulin receptor, the signal transduction mechanism after the receptor has also been gradually elucidated. Under these circumstances, a therapeutic agent for diabetes [α-glycosidase inhibitor: Acarbose, Voglibose (for example, see Non-patent Documents 1 to 6), an insulin sensitizer: Troglitazone, Pioglitazone (for example, see Non-Patent Documents 7 to 11)] has also appeared and been released. On the other hand, in the United States, a biguanide agent was approved as a therapeutic agent for diabetes in 1996, and has been attracting attention because it can be used for daily medical treatment of diabetes (for example, see Non-Patent Documents 12 and 13). All of these drugs, unlike sulfonylurea (SU) drugs which have been widely used in daily medical practice since ancient times, have an action of lowering blood glucose without promoting insulin secretion from pancreatic β cells.
[0006]
At present, the following nine items are considered as an action mechanism of a therapeutic drug for diabetes having a function of improving insulin resistance. Activation of insulin receptor kinase, promotion of transfer of sugar transporter to cell membrane, action of rate-limiting enzyme of glucose metabolism and correction of abnormal glucose metabolism, suppression of hepatic gluconeogenesis, promotion of hepatic glucose uptake, enhancement of hepatic glycogen production, It is a decrease in blood lipid, a decrease in hepatic gluconeogenesis due to a decrease in blood lipid, and an increase in insulin sensitivity due to a decrease in blood lipid.
[0007]
Recent progress in pharmacogenomics research has made it possible to predict the effects and side effects of drugs on individual patients by genetic diagnosis from the relationship between genetic polymorphisms and drug effects or between genetic polymorphisms and side effects. In addition, by examining the relationship between a genetic polymorphism and a disease, it is becoming possible to make a preliminary diagnosis of some diseases and determine a prognosis. Examples of the former include gene polymorphisms of drug metabolizing enzymes. Known drug metabolizing enzymes whose activity increases or decreases due to polymorphism include cytochrome P4501A2, cytochrome P4502A6, cytochrome P4502C9, cytochrome P4502C19, cytochrome P4502D6, and cytochrome P4502E1. In addition, gene polymorphism also exists in a group of enzymes called conjugating enzymes such as thiopurine methyltransferase, N-acetyltransferase, UDP-glucuronosyltransferase, and glutathione S-transferase, and the activity decreases due to the polymorphism. (For example, see Non-Patent Document 14). As an example of the latter, a large number of disease-causing genes found from polymorphism analysis studies have been reported. For example, (1) HLA as a causative gene for ulcerative colitis, (2) TCRa as a causative gene for rheumatoid arthritis, (3) APOE4 as a causative gene for Alzheimer's disease, (4) a causative gene for schizophrenia Dopamine D3 receptor, 5) tryptophan hydroxylase as a causative gene for manic depression, 6) angiotensin precursor as a causative gene for albuminuria, 7) blood coagulation factor as a causative gene for myocardial infarction VII, (8) Leptin as a causal gene of obesity and the like (for example, see Non-Patent Document 15).
[0008]
TNF-α is considered to be an important substance involved in the pathogenesis of various inflammatory diseases. Recently, a polymorphism that enhances the expression of this gene is located in the 5′-terminal upstream region of the TNF-α gene. Was found to exist (for example, see Non-Patent Document 16). Since these polymorphisms enhance the expression level of the TNF-α gene, detection of the polymorphisms leads to advancement of TNF-α-related diseases such as juvenile rheumatoid arthritis, rheumatoid arthritis and diabetes. Diagnosis becomes possible (for example, see Patent Document 1).
[0009]
Regarding diabetes, the relationship between gene mutation and the onset of diabetes has been studied. Genes that have been identified so far to cause diabetes by a single abnormality include insulin, insulin receptor, insulin promoter factor, glucokinase, hepatocyte nucleus factor, amylin, and skeletal muscle glycogen synthesis. Enzymes and mitochondrial genes. In addition, genes considered to be involved in the development of type 2 diabetes include insulin receptor substrate, glucose transporter, mitochondrial glycerophosphate dehydrogenase, fatty acid binding protein, adrenergic receptor, glucagon receptor, potassium channel, CD38 and the like (for example, Non-Patent Document 17).
[0010]
Recently, it has been reported that the Arg140Trp polymorphism in CD38 increases the risk of diabetes (for example, see Non-Patent Document 18). Since this polymorphism is found in Japanese diabetic patients at a frequency of about 13%, it is considered to be useful for diagnosing risk for diabetes.
[0011]
By the way, glutamine: fructose-6-phosphate amide transferase (hereinafter referred to as “GFAT”) catalyzes the rate-limiting reaction of fructose-6-phosphate to glucosamine-6-phosphate in the hexosamine biosynthesis pathway. It is an important enzyme. It is thought that a drug that inhibits GFAT activity promotes the uptake of glucose into cells and can lower the blood glucose level, and is therefore expected to be developed as a therapeutic agent for diabetes. The mechanism of action of this GFAT inhibitor is explained as follows. The hexosamine biosynthesis pathway produces UDP-N-acetylglucosamine, CMP-N-acetylneuraminic acid, and the like in the metabolic process, and these flow as crude materials such as saccharification modification of proteins and proteoglycans or gangliosides. It is considered. On the other hand, when insulin binds to a receptor on the cell surface, an intracellular signal is activated, and a sugar transporter (GLUT4 or the like) pooled in the cell is transferred to the membrane surface, and glucose uptake is promoted. Incorporated glucose is metabolized by the glycolysis system and accumulates ATP as an energy source, but when incorporated in excess, fructose-6-phosphate, a glucose metabolite, will flow to the hexosamine biosynthesis pathway. . The flowing fructose-6-phosphate is converted to glucosamine-6-phosphate by GFAT. Although the detailed mechanism is unknown, various circumstantial evidences have shown that this glucosamine-6-phosphate metabolite suppresses the translocation of the sugar transporter to the membrane, thereby suppressing glucose uptake into cells. (For example, see Non-Patent Documents 19 to 23). From these facts, it is considered that the hexosamine biosynthetic pathway functions as a feedback mechanism for glucose uptake as one of its roles. And GFAT plays an important role as a rate-limiting enzyme in the pathway.
[0012]
In addition, recently, transgenic mice overexpressing antisense nucleotides of GFAT constitutively suppress the expression of GFAT, and administration of streptozotocin, a selective destroyer of pancreatic β cells, inhibits β cell apoptosis. Thus, it has been reported that the blood glucose level is improved (for example, see Non-Patent Document 24). In diabetics, this GFAT activity is generally known to be high, and is considered to be one of the causes of high blood sugar levels (for example, see Non-Patent Document 25). At present, two types of human-derived GFATs are known: GFAT1 (for example, see Non-Patent Document 26) and GFAT2 (for example, see Patent Document 2 and Non-Patent Document 27). It has been reported that GFAT1 is a 77 kDa protein consisting of 681 amino acids and GFAT2 is composed of 682 amino acids, but the full-length sequence of the GFAT1 gene has not yet been elucidated. The amino acid sequence homology between human GFAT1 and GFAT2 is 78%. In addition, mouse-derived (for example, see Non-Patent Document 28), yeast-derived GFAT (for example, see Non-patent Document 29), or Escherichia coli-derived GFAT (for example, see Non-Patent Document 30) have been reported. Shows high homology to human GFAT.
[0013]
On the other hand, the HIRA interacting protein 5 (hereinafter, referred to as “HIRP5”) gene located on the 5 ′ side of the GFAT1 gene on the genome is expected to bind to histones and participate in iron metabolism due to homology with known proteins. (See, for example, Non-Patent Document 31), but its detailed functions are completely unknown. Further, the relationship between the HIRIP5 gene and the aforementioned polymorphism present on the GFAT1 gene and diabetes has not been known at all.
[0014]
Diabetes is thought to be caused by multiple genetic polymorphisms or external factors such as eating habits rather than being caused by mutations or polymorphisms in a single gene, and the mechanism of its development remains unclear. There are many. Therefore, it is desired to further study the genes and polymorphisms involved in the onset of diabetes in the future.
[0015]
[Patent Document 1]
International Publication No. 98/54361 Pamphlet
[Patent Document 2]
JP-A-10-108683
[Non-patent document 1]
Diabetes Frontier, 3, 557-564 (1992)
[Non-patent document 2]
Drugs, 46, 1025-1054 (1994)
[Non-Patent Document 3]
History of Medicine, 149, 591-618 (1989)
[Non-patent document 4]
Clinical and research, 67, 219-233 (1990)
[Non-Patent Document 5]
Clinical and Research, 69, 919-932 (1992)
[Non-Patent Document 6]
Clinician 21 (extra number), 578-587 (1995)
[Non-Patent Document 7]
Diabetes, 37, 1549-1558 (1998)
[Non-Patent Document 8]
Life Science (Life Sci.), 67, 2405-2416 (2000)
[Non-Patent Document 9]
Metabolism, 44, 486-490 (1995)
[Non-Patent Document 10]
New England Journal of Medicine (New Engl. J. Med.), 331, 1188-1193 (1994).
[Non-Patent Document 11]
Edited by Yoshio Goto, "New Diabetes Drugs", Pharmaceutical Journal, Osaka, (1994)
[Non-Patent Document 12]
New England Journal of Medicine (New Engl. J. Med.), 333, 541-549 (1995)
[Non-patent document 13]
Diabetes Spectrum, 8, 194-197 (1995)
[Non-patent document 14]
Edited by Yusuke Nakamura, "Strategy for SNP gene polymorphism", Nakayama Shoten, Tokyo, (2000)
[Non-Patent Document 15]
Nature Genetics (Nat. Genet.), 22, 139-144 (1999)
[Non-Patent Document 16]
Tissue Antigens, 51, 605-612 (1998)
[Non-Patent Document 17]
Edited by Takashi Kadowaki, "The Forefront of Diabetes", Yodosha, Tokyo, (1997)
[Non-Patent Document 18]
Diabetologia, 41, 1024-1028 (1998)
[Non-Patent Document 19]
FASEB J., 5, 3031-3036 (1991)
[Non-Patent Document 20]
Diabetologia, 38, 518-524 (1995)
[Non-Patent Document 21]
Journal of Biological Chemistry (J. Biol. Chem.), 266, 10155-10161 (1991)
[Non-Patent Document 22]
Journal of Biological Chemistry (J. Biol. Chem.), 266, 4706-4712 (1991)
[Non-Patent Document 23]
Endocrinology, 136, 2809-2816 (1995)
[Non-Patent Document 24]
Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA), 97, 2820-2825 (2000)
[Non-Patent Document 25]
Diabetes, 45, 302-307 (1996)
[Non-Patent Document 26]
Journal of Biological Chemistry (J. Biol. Chem.), 267, 25208-25212 (1992)
[Non-Patent Document 27]
Genomics, 57, 227-234 (1999).
[Non-Patent Document 28]
Gene, 140, 289-290 (1994).
[Non-Patent Document 29]
Journal of Biological Chemistry (J. Biol. Chem.), 264, 8753-8758 (1989)
[Non-Patent Document 30]
Biochemical Journal (Biochem. J.), 224, 779-815 (1984).
[Non-Patent Document 31]
Biochemical. Biophys. Acta, 1517, 376-383 (2001).
[0016]
[Problems to be solved by the invention]
An object of the present invention is to provide a determination method for determining the risk of developing diabetes, particularly type 2 diabetes, and a reagent and a kit for the method.
[0017]
[Means for Solving the Problems]
The present inventors examined DNA samples isolated from diabetic patients and healthy subjects and examined the frequency of gene polymorphisms present in the HIRIP5 gene and the GFAT1 gene. We found that polymorphisms existed. Then, they have found that the potential risk of developing diabetes can be determined with a high probability by detecting the gene polymorphism, and completed the present invention.
[0018]
That is, the present invention provides the following (1) to (9).
(1) In a sample containing human genomic DNA, by detecting a gene polymorphism present at any one or two or more positions selected from the following a) to j), and / or a haplotype consisting of a combination thereof, A method for determining the risk of developing diabetes of the sample provider.
a) Nucleotide at −34642 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 352 of SEQ ID NO: 1)
b) Nucleotide 26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2)
c) Nucleotide at position −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
d) Nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4)
e) The nucleotide at position 28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 in SEQ ID NO: 5)
f) The nucleotide at position 27915 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6)
g) Nucleotide at position 3270 of intron 3 of HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7)
h) Nucleotide 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8)
i) The -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9)
j) The 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10)
[0019]
(2) The method according to the above (1), wherein the diabetes is type 2 diabetes.
[0020]
(3) The detection target is any one or two or more selected from gene polymorphisms present at the following positions a) to d) and haplotypes shown below w) to z). The method according to (1) or (2).
a) Nucleotide at −34642 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 352 of SEQ ID NO: 1)
b) Nucleotide 26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2)
c) Nucleotide at position −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
d) Nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4)
w) Present at the nucleotide -28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 of SEQ ID NO: 5) and at the nucleotide -27915 in the 5' upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6) Haplotype combining different genetic polymorphisms
x) A gene present at the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and the 3270th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7) Haplotype combining polymorphisms
y) The gene present at the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and the 3832th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8) Haplotype combining polymorphisms
z) It is present at the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and at the -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9). Haplotype combining genetic polymorphisms
[0021]
(4) Detection is performed by RFLP, PCR-SSCP, ASO hybridization, direct sequencing, ARMS, denaturing gradient gel electrophoresis, RNaseA cleavage, chemical cleavage, DOL, TaqMan PCR, Invader Method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele-specific hybridization method, padlock probe method, UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, and ECA method The method according to any one of the above (1) to (3), wherein the method is performed using one or more methods selected from the group consisting of:
[0022]
(5) The method according to the above (4), wherein the detection is performed using a PCR-SSCP method or a direct sequence method.
[0023]
(6) any one polynucleotide selected from the following a) to j), or a labeled product thereof;
a) On the nucleotide sequence represented by SEQ ID NO: 1, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide number 352
b) On the nucleotide sequence represented by SEQ ID NO: 2, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide number 278.
c) On the nucleotide sequence represented by SEQ ID NO: 3, a continuous polynucleotide having a length of 16 to 500 bases including the nucleotide of nucleotide No. 311
d) On the nucleotide sequence represented by SEQ ID NO: 4, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide number 262.
e) On the nucleotide sequence represented by SEQ ID NO: 5, a continuous polynucleotide having a length of 16 to 500 bases including nucleotide of nucleotide number 258
f) On the nucleotide sequence represented by SEQ ID NO: 6, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide number 299.
g) On the nucleotide sequence represented by SEQ ID NO: 7, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide number 288.
h) On the nucleotide sequence represented by SEQ ID NO: 8, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide No. 310
i) On the nucleotide sequence represented by SEQ ID NO: 9, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide No. 313
j) On the nucleotide sequence represented by SEQ ID NO: 10, a continuous polynucleotide having a length of 16 to 500 bases and including the nucleotide of nucleotide number 632
(However, when the polynucleotide is RNA, the base symbol “t” in the sequence listing should be read as “u”).
[0024]
(7) An oligonucleotide having a length of 15 to 30 bases or a label thereof, which hybridizes to a part of any one of the polynucleotides selected from the following a) to j) and specifically amplifies the polynucleotide. object;
a) On the nucleotide sequence represented by SEQ ID NO: 1, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide number 352
b) On the nucleotide sequence represented by SEQ ID NO: 2, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide number 278
c) On the nucleotide sequence represented by SEQ ID NO: 3, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide No. 311
d) On the nucleotide sequence represented by SEQ ID NO: 4, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide number 262
e) On the nucleotide sequence represented by SEQ ID NO: 5, a continuous polynucleotide having a length of 16 bases or more including nucleotide of nucleotide number 258
f) On the nucleotide sequence represented by SEQ ID NO: 6, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide number 299
g) On the nucleotide sequence represented by SEQ ID NO: 7, a continuous polynucleotide having a length of 16 bases or more including nucleotide of nucleotide number 288
h) On the nucleotide sequence represented by SEQ ID NO: 8, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide No. 310
i) On the nucleotide sequence represented by SEQ ID NO: 9, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide No. 313
j) On the nucleotide sequence represented by SEQ ID NO: 10, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide No. 632
(However, when the polynucleotide is RNA, the base symbol “t” in the sequence listing should be read as “u”).
[0025]
(8) An immobilized sample on which the polynucleotide according to (6) is immobilized.
[0026]
(9) A reagent or kit for determining the risk of developing diabetes, comprising at least one or more selected from the following 1) to 3).
1) The polynucleotide according to (6) or a labeled product thereof
2) The oligonucleotide according to (7) or a labeled product thereof
3) The solid-phased sample according to the above (8)
[0027]
BEST MODE FOR CARRYING OUT THE INVENTION
The method of the present invention is a method for determining the risk of developing diabetes of a sample provider by detecting a novel gene polymorphism present on the HIRIP5 gene and / or GFAT1 gene in a sample containing human genomic DNA. .
[0028]
In the present specification, “gene polymorphism” includes both so-called single nucleotide polymorphism (single nucleotide polymorphism: single nucleotide polymorphism: SNP) and polymorphisms over a plurality of consecutive nucleotides. That is, in a human population, a mutation such as substitution, deletion, insertion, transposition, or inversion of one or more nucleotides at a specific site in one or more other individual genomes with reference to the genome sequence of a certain individual. Is present, it is statistically certain that the mutation is not a mutation that occurred in the one or more individuals, or it is not a mutation within the individual and is present in the population at a frequency of 1% or more. If it is proven in a family, the mutation is called a “gene polymorphism”.
[0029]
Hereinafter, the method of the present invention will be described in detail.
1. Novel genetic polymorphisms associated with the development of diabetes
The gene polymorphism to be detected in the present invention is a novel gene polymorphism present on the HIRIP5 gene or the GFAT1 gene, which has been confirmed to appear frequently in diabetic patients.
[0030]
In this specification, the positions of the polymorphisms in the HIRIP5 gene are registered in GenBank as follows: (1) HIRIP5 cDNA (Accession number: NM_015700.1); (2) HIRIP5 genomic DNA (Accession number: AC114772). .3). That is, since the position of the open reading frame of the HIRIP5 gene in Accession Number: NM_015700.1 is the 259th to 849th bases, the position of the 259th position in NM_015700.1 (the translation start codon ATG of the HIRIP5 gene (Corresponding to A) as +1 of exon 1 of the HIRIP5 gene, and the position of each polymorphism is described. Incidentally, since there is no report on the N-terminal amino acid sequence of the HIRIP5 protein at this time, the exact position of the translation initiation codon of the HIRIP5 gene has not been determined (for example, the position of the above translation initiation codon ATG is determined by Laurent et al. (It differs from the position of the ATG in the report [Biochim. Biophys. Acta, 1517, 376-383 (2001)]). Therefore, in the present specification, the position of each polymorphism on the HIRIP5 gene is specified by performing the above definition “the 259th position in NM — 015700.1 is set to +1 of exon 1 of the HIRIP5 gene”. did.
[0031]
That is, the gene polymorphism according to the present invention exists at the following positions a) to j).
a) Nucleotide at −34642 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 352 of SEQ ID NO: 1)
b) Nucleotide 26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2)
c) Nucleotide at position −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
d) Nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4)
e) The nucleotide at position 28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 in SEQ ID NO: 5)
f) The nucleotide at position 27915 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6)
g) Nucleotide at position 3270 of intron 3 of HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7)
h) Nucleotide 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8)
i) The -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9)
j) The 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10)
[0032]
In the method of the present invention, a haplotype consisting of a genetic polymorphism and / or a combination thereof present at any one or more positions selected from the above a) to j) is detected, and the risk of developing diabetes is detected. Determine the degree. That is, the determination may be performed based on the detection result of one or more gene polymorphisms, or may be performed based on the detection result of a haplotype in which two or more gene polymorphisms are combined. Alternatively, the determination may be made by combining the polymorphism and the haplotype.
[0033]
2. Preparation of sample containing human genomic DNA
The “sample containing human genomic DNA” used in the method of the present invention is prepared using any cells (excluding germ cells), tissues, organs, and the like isolated from subjects and clinical samples. As the material, leukocytes or mononuclear cells separated from peripheral blood are preferable, and leukocytes are most preferable. These materials are isolated according to methods commonly used in clinical tests.
[0034]
For example, when using leukocytes as a material, first, leukocytes are separated from peripheral blood isolated from a subject according to a conventional method. Next, proteinase K and sodium dodecyl sulfate (SDS) are added to the obtained leukocytes to decompose and denature the protein, followed by phenol / chloroform extraction to obtain genomic DNA (including RNA). RNA can be removed by RNase if necessary. However, the extraction of genomic DNA is not limited to the above method, but is well known in the art (for example, Sambrook, J. et al. (1989): “Molecular Cloning: A Laboratory Manual (2nd Ed.)”). Cold Spring Harbor Laboratory, NY), or a commercially available DNA extraction kit or the like.
[0035]
3. Gene polymorphism detection
Next, from the sample containing the obtained human genomic DNA, a novel gene polymorphism which is elucidated by the present inventors and is highly related to diabetes is detected. Hereinafter, a typical gene polymorphism detection method will be described.
[0036]
(1) RFLP (restriction fragment length polymorphism) method
When a gene polymorphism site is included in a restriction enzyme recognition site, the gene polymorphism can be detected from a difference in the length of a DNA fragment generated by digestion of the restriction enzyme. In this case, (1) a method in which genomic DNA is digested with a restriction enzyme, followed by Southern blotting; and (2) a DNA fragment containing a polymorphic site is amplified by PCR, cut with a restriction enzyme, and the DNA cut by electrophoresis. A method of analyzing the size of a fragment is exemplified. The probe used in the method (1) may be a DNA containing a polymorphic site of interest and corresponding to a sequence ranging from about 0.05 to 2 kb on the 5 'end and 3' end from the polymorphic site, respectively. Fragments (labeled with an isotope, biotin, a fluorescent dye, or the like) are preferred. The PCR primer used in the method (2) is preferably a 15-mer to 30-mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 4 kb containing a polymorphic site.
[0037]
(2) PCR-SSCP method (single-stranded DNA higher-order structural polymorphism analysis)
The PCR-SSCP method is a method in which a DNA fragment containing a gene polymorphism site is amplified by PCR, heat-denatured, and single-stranded DNA having a different higher-order structure is separated by electrophoresis [Biotechniques, 16, 296- 297 (1994), Biotechniques, 21, 510-514 (1996)]. Since the migration distance of single-stranded DNA differs depending on the presence or absence of the gene polymorphism, the polymorphism can be typed by analyzing the pattern. In order to use it as a standard for typing, it is preferable to use a DNA sample derived from the human genome in which the nucleotide sequence of the polymorphic site has been confirmed in advance as a template DNA for PCR at the same time as the test sample. As a primer for PCR amplification, a 15-30 mer oligonucleotide for amplifying a DNA fragment of about 50-500 bp containing a polymorphic site, whose 5 ′ end is labeled with a fluorescent dye, is preferable.
[0038]
(3) ASO (Allele Specific Oligonucleotide) hybridization method
In the ASO hybridization method, a PCR product containing a gene polymorphism site is dot-blotted on a support such as a nylon filter, hybridized with a probe corresponding to each gene polymorphism, and then washed according to the Tm value of the probe. This is a method for detecting polymorphisms (hybrids are removed if there is a mismatch) [Clin. Chim. Acta, 189, 153-157 (1990)]. As the probe, a synthetic oligonucleotide of about 15 to 25 mer is usually used (labeling with a radioisotope, biotin or a fluorescent dye is necessary to obtain a signal). As the PCR primer, an oligonucleotide of 15 to 30 mer for amplifying a DNA fragment of about 0.05 to 4 kb containing a polymorphic site is preferable.
[0039]
(4) Direct sequence method
The direct sequencing method is a method in which a DNA fragment containing a gene polymorphism site is PCR-amplified and the nucleotide sequence of the amplified DNA is directly analyzed by the dideoxy method [Biotechniques, 11, 246-249 (1991)]. The PCR primer used in this method is preferably a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 4 kb containing a polymorphic site. Further, as the sequence primer, preferably, a 15 to 30-mer oligonucleotide corresponding to a position on the 5 'end side of about 50 to 300 nucleotides from the polymorphic site is used.
[0040]
(5) ARMS (Amplification Refraction Mutation System) method
In PCR, after a primer is annealed to a template DNA, a complementary strand DNA is synthesized from the 5 'end to the 3' end by a DNA polymerase. If there is a mismatch in the 3 ′ terminal nucleotide of the primer, the efficiency of PCR decreases, and detection by electrophoresis becomes impossible. The ARMS method is a method in which PCR is performed using primers designed so that the 3 ′ terminal nucleotide is complementary to the mutant nucleotide to be detected, and a gene polymorphism is detected based on the presence or absence of an amplification product [Nuc . Acids. Res. , 19, 3561-3567 (1991), Nuc. Acids. Res. , 20, 4831-4837 (1992)]. The PCR primers used in this method are preferably 15-30 mer oligonucleotides designed such that the polymorphic site is located at the 3 'end, and the other is preferably the polymorphic site. The oligonucleotide is a 15 to 30-mer oligonucleotide at a distance of about 0.05 to 2 kb from the oligonucleotide.
[0041]
(6) Denaturing Gradient Gel Electrophoresis (hereinafter referred to as "DGGE") method
The DGGE method is a method for detecting a gene polymorphism by utilizing the fact that a heteroduplex having a mismatch in a PCR product is easier to dissociate than a homoduplex [Biotechniques, 27, 1016-1018 (1999). ]. Since the mobility of the heteroduplex in gel electrophoresis decreases as the dissociation progresses, if the density gradient of urea and formamide is set in the polyacrylamide gel to be used, the difference in mobility from the homoduplex will further increase. The presence of the highlighted double-stranded DNA containing the mismatch, ie, the presence of the mutation, is detected. The PCR primer used in this method is usually a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 0.5 kb containing a polymorphic site.
[0042]
(7) RNase A cleavage method
RNase A (RNA degrading enzyme) has the property of degrading only single-stranded RNA without degrading double-stranded RNA or RNA / DNA complex. Therefore, after PCR-amplifying a DNA fragment containing a polymorphic site, an isotope-labeled RNA probe is hybridized with a denatured and single-stranded PCR product, treated with RNaseA, and then separated by electrophoresis. Since the RNA probe hybridized with DNA is cleaved at the mismatch site, it can be detected as two bands [DNA Cell. Biol. , 14, 87-94 (1995)]. As the RNA probe used in this method, an oligonucleotide of usually 15 to 30 mer containing a polymorphic site is preferable.
[0043]
(8) Chemical cutting method
After amplifying the DNA fragment containing the polymorphic site by PCR, the mismatch site of the double-stranded DNA, "c (cytosine)", is separately hydroxylamine, and "t (thymine)" is osmium tetraoxide. Modification and piperidine treatment cleaves the sugar. After forming a double strand using a labeled probe and performing the above-described treatment, electrophoresis is performed. If the size of the probe is short, mutation has been detected [Biotechniques, 21, 216-218 (1996) )]. The PCR primer used in this method is usually a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 4 kb containing a polymorphic site.
[0044]
(9) DOL (Dye-labeled Oligonucleotide Ligation) method
In the DOL method, after a DNA fragment containing a gene polymorphism is PCR-amplified, a dye primer containing up to a base immediately before the fluorescently labeled polymorphic site and a dye terminator labeled with a fluorescent dye specific to each allele are used. This is a method of ligation using a thermostable DNA ligase [Genome Res. , 8, 549-556 (1998)]. The PCR primer used in this method is usually a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 2 kb containing a polymorphic site.
[0045]
(10) TaqMan PCR method
The TaqMan PCR method is a method using PCR using a fluorescently labeled allele-specific oligonucleotide (TaqMan probe) and Taq DNA polymerase [Genet. Anal. , 14, 143-149 (1999), J. Am. Clin. Microbiol. , 34, 2933-2936 (1996)]. The PCR primer used in this method is usually a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 2 kb containing a polymorphic site. The TaqMan probe is an oligonucleotide of about 15 to 30 bases including a polymorphic site, the 5 ′ end is labeled with a fluorescent reporter dye, and the 3 ′ end is labeled with a quencher (quencher). . By using this probe, it is possible to detect a nucleotide change between a wild type and a mutant type.
[0046]
(11) Invader method
In the invader method, two types of non-fluorescently labeled oligonucleotides and one type of fluorescently labeled oligonucleotide are used. One of the non-fluorescent-labeled oligonucleotides has a sequence (hereinafter, referred to as “flap”) at the 5 ′ end thereof, which is unrelated to the genomic sequence (hereinafter, referred to as “template”) in which the polymorphic site to be detected exists. The 3 'end of the flap is a complementary sequence specific to the sequence at the 5' end from the polymorphic site of the template (hereinafter referred to as "allele probe"). That is, the 5 'end of the complementary sequence portion specific to the template of the allele probe corresponds to the polymorphic site of the template. The other non-fluorescently labeled oligonucleotide has a complementary sequence specific to the sequence at the 3 ′ end from the polymorphic site of the template (hereinafter referred to as “invader probe”). The 3 'end of the invader probe also corresponds to the polymorphic site of the template, but need not be complementary to the nucleotide at the polymorphic site of the template. Fluorescently labeled oligonucleotides (hereinafter referred to as “FRET (fluorescence resonance transfer) probes”) have a sequence complementary to the flap at the 3 ′ end and a palindromic sequence at the 5 ′ end, so that the probe itself is not conjugated. It forms a main chain. A portion near the 5 'end of the FRET probe is labeled with a fluorescent dye, and a quencher for canceling the fluorescence is bound to the 5' end. First, when the allele probe and the invader probe are hybridized to the template, if the allele probe and the template are complementary at the polymorphic site, the template, the 5 'end of the allele probe and the 3''Terminal forms a special structure. An enzyme that specifically recognizes this structure and exhibits endonuclease activity (hereinafter referred to as “Crybase”) separates the flap portion from the allele probe. The released flap hybridizes with the 3 'end of the FRET probe. At this time, since the flap and the FRET probe form a structure specifically recognized by Clebase at the position of the 5 ′ end where the quencher of the FRET probe is bonded, the 5 ′ to which the quencher is bonded is formed. The terminal nucleotide is cleaved from the FRET probe by the cleabase, and the fluorescent dye remaining on the FRET probe emits fluorescence. On the other hand, when a mutation is present in the template sequence, and the allele probe and the template are not complementary at the polymorphic site and are mismatched, a structure specifically recognized by Clebase is not formed. Not detached from the probe. At this time, the flap remaining on the allele probe without being detached can also hybridize with the 3 ′ end of the FRET probe, but the quencher binds as compared with the case where the flap detached from the allele probe is hybridized. Since the reaction efficiency at which the 5 ′ terminal nucleotide is cleaved from the FRET probe by cleabase is low, the fluorescence intensity emitted by the FRET probe is also low. Based on the above principle, polymorphism can be detected [Science, 5109, 778-783 (1993), J. Am. Biol. Chem. , 30, 21387-21394 (1999), Nat. Biotechnol. , 17, 292-296 (1999)].
[0047]
(12) MALDI-TOF / MS method (Matrix Assisted Laser Desorption-time of
Flight / Mass Spectrometry) method
The MALDI-TOF / MS method is a method of synthesizing a single-stranded oligonucleotide containing a different nucleotide corresponding to an allele of a gene polymorphism, measuring the mass thereof, and detecting the difference with a mass spectrometer for typing. [Genome Res. , 7, 378-388 (1997), Eur. J. Clin. Chem. Clin. Biochem. , 35, 545-548 (1997)]. In the MALDI-TOF / MS method, first, a DNA fragment containing a polymorphic site is PCR-amplified, and then a DNA extension reaction product specific to each allele is masked by an extension reaction using primers adjacent to the polymorphic site. Analyze based on spectrum. The PCR primer used at this time is usually a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 0.5 kb containing a polymorphic site. As a primer for detecting a polymorphism, an oligonucleotide of 15 to 30 mer adjacent to the polymorphic site is used.
[0048]
(13) TDI (Template-directed Dye-terminator Incorporation) method
In the TDI method, a DNA fragment containing a gene polymorphism is amplified by PCR, and then labeled with different fluorescent dyes corresponding to each allele by a primer extension reaction using a primer designed immediately before the polymorphic site. Is incorporated into a polymorphic site [Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)]. The primer extension product is analyzed using a DNA sequencer (ABI Prism 377, manufactured by Applied Biosystems) or the like. The PCR primer used at this time is usually a 15 to 30 mer oligonucleotide for amplifying a DNA fragment of about 0.05 to 1 kb containing a polymorphic site.
[0049]
(14) Molecular Beacons method
Molecular Beacons are oligonucleotides having a luminescence suppressor (Quencher) and a fluorescent material (Fluorophore) at both ends. Usually, it is a stem-loop structure having a stem portion of 5 to 7 nucleotides and a loop structure of 15 to 30 nucleotides. Therefore, the phosphor does not emit light even under the excitation light irradiation due to the function of the light emission suppressor. On the other hand, when the target DNA homologous to the base sequence in the loop structure and the base sequence in the loop structure hybridize, the phosphor is excited under the excitation light to emit fluorescence because the distance between the luminescence suppressor and the phosphor is large. [Nat. Biotechnol. , 1, p49-53 (1998), Gene Medicine, 4, p46-48 (2000)]. The base sequence of the stem part of Molecular Beacon is not complementary to the target DNA. The Tm value (melting temperature) of the stem structure of Molecular Beacons is designed to be 7-10 ° C. higher than the Tm values of the target DNA and the PCR primer. Then, when the target region is amplified by PCR, Molecular Beacons are put into the reaction solution, and excitation light is applied at the annealing temperature of PCR to measure fluorescence. If Molecular Beacons is a sequence completely homologous to the target gene, it is hybridized. Fluorescent. Conversely, if there is a mismatch, Molecular Beacons cannot hybridize to the target and cannot emit fluorescence. Genetic polymorphisms can be found by this method.
[0050]
(15) Dynamic Allele-Specific Hybridization (DASH) method
This is a method in which a target DNA (60-90 base pairs) is amplified from genomic DNA by PCR using primers whose ends are biotinylated [Nat. Biotechnol. , 1, p87-88, (1999), Gene Medicine, 4, p47-48 (2000)]. After completion of the PCR, the strand on the side of the biotinylated primer binds to a microtiter well coated with streptavidin. On the other hand, the unmodified Primer strand cannot be bonded and is removed by rinsing with an alkaline solution. Thereafter, probe DNA (15-21 nucleotides) is hybridized to the biotinylated primer strand. At this time, a fluorescent substance (Syber Green Idye) that specifically binds to the double-stranded DNA and emits fluorescence is incorporated together. Thereafter, denaturation is performed while observing the fluorescence intensity. When the target DNA and the probe DNA are not completely complementary and have a mismatch, the temperature at which the target DNA denatures and emits no light is lower than in the case where it is completely complementary. By observing this difference in temperature, a gene polymorphism can be detected.
[0051]
(16) Padlock Probe method
Lizardi et al. [Nat. Genet. , 3, p225-232 (1998), Gene Medicine, 4, p50-51 (2000)]. A probe designed to be circular when hybridized to a single-stranded target DNA is used. The probe is hybridized to the target DNA, and then ligated with DNA ligase to form a complete circular shape. Then, alkaline phosphatase (Calph intestinal alkaline phosphatase (CIAP)) is used. The phosphate group is removed with the phosphatase of (2). When the terminal cannot be circularized due to a mismatch at the end, the phosphate group is lost and the circularization cannot be performed. A primer (1) is designed for this circular DNA and replicated with a DNA polymerase to obtain a replicon multimer. A primer (2) is also designed for this replicon, and the two types of primers are mixed together and reacted with DNA polymerase to amplify double-stranded DNA. The primer (1) is designed on a portion not complementary to the target DNA, and the primer (2) is a terminal portion of a circular probe (Padlock Probe) (a sequence complementary to the target DNA (gene polymorphism at the 3 ′ end). And a primer with a single nucleotide substitution at the 3 ′ end is used for polymorphism detection.Rolling cycle-reaction (RCR) is performed on the circularized probe (Padlock Probe). After amplification using the improved Hyperbranched Rolling-Circle Amplification (HRCA) method, treatment with a restriction enzyme at a restriction enzyme recognition site in Padlock Probe was performed, and electrophoresis was performed. To confirm the presence or absence of command.
[0052]
(17) UCAN method
The UCAN method [refer to the Takara Shuzo Co., Ltd. homepage (http://www.takara.co.jp)] is based on the 3 ′ end of a DNA-RNA-DNA primer (DRD primer) of a type in which RNA is sandwiched between DNA on both sides. The DNA is chemically changed so that the replication of the template DNA by the DNA polymerase does not occur. Next, the DRD primer designed so that the RNA part binds to the base part where the single base conversion may have occurred is paired with the template DNA. Only when the DRD primer and the template completely match, the RNA portion of the paired DRD primer is cleaved by RNaseH. As a result of this cleavage, a new 3 ′ end appears, the extension reaction by the DNA polymerase proceeds, and the template DNA is amplified. On the other hand, when the DRD primer and the template DNA do not match at that position, that is, when an SNP is present, RNaseH does not cut the DRD primer, and thus DNA amplification does not occur. By detecting the presence or absence of this gene amplification, a gene polymorphism can be detected.
[0053]
(18) DNA chip or DNA microarray
DNA chips or DNA microarrays are obtained by immobilizing DNA probes containing various types of polymorphic sites on a glass substrate, and hybridizing a labeled nucleic acid sample to detect the presence or absence of the polymorphism by a fluorescent signal. In general, a method of placing cDNA on a glass substrate is called a DNA microarray, and a method of synthesizing DNA on a glass substrate is called a DNA chip (oligo DNA chip). The probe immobilized (or synthesized) on the substrate is usually an oligonucleotide of about 20 mer containing a polymorphic site in the case of an oligo DNA array, or a double-stranded DNA of about 100 to 1500 mer in the case of a cDNA microarray. .
[0054]
(19) ECA method
The ECA (Electrochemical Array) method is a genotyping method utilizing the electrochemical properties of an intercalator that binds to a double strand of DNA. That is, a region containing a polymorphism on the genome is amplified by a PCR method, and an intercalator is acted after hybridizing with a probe complementary to each allele immobilized on a substrate. At this time, the amount of the intercalator to be bound differs depending on whether the probe is completely complementary or incompletely complementary to the probe. Since the intercalator used in the ECA method contains a substance called ferrocene having electrochemical properties, the electrical signal differs in proportion to the amount of the intercalator bound. The ECA method is a method for detecting a gene polymorphism using this difference [Anal. Chem. , 72, p1334-1341, 2000].
[0055]
The above are typical polymorphism detection methods, but are not limited thereto. Other known gene polymorphism detection methods can be used for the determination method of the present invention. Further, in the method of the present invention, these gene polymorphism detection methods may be used alone or in combination of two or more. As a preferred embodiment of the present invention, a determination method using a PCR-SSCP method or a direct sequence method will be described in Examples described later.
[0056]
4. Diabetes risk assessment reagent or kit
In the determination method of the present invention, in order to detect a genetic polymorphism associated with the onset of diabetes, a probe or solid-phased sample for detecting a sequence at or near the site where the gene polymorphism to be detected is present, or Primers for specifically amplifying the sequence are used. These probes, primers and immobilized samples are useful as reagents or kits for performing the determination method of the present invention.
[0057]
That is, the present invention provides a probe, a primer, and an immobilized sample for detecting a gene polymorphism on the HIRIP5 gene or the GFAT1 gene, and a reagent or kit for determining the risk of developing diabetes, comprising at least one or more of these. I will provide a.
[0058]
(1) Probe
The probe used in the method of the present invention is a polynucleotide probe that specifically hybridizes to a site containing a novel polymorphism on the HIRIP5 gene or GFAT1 gene and detects the polymorphism site.
[0059]
Such a probe is selected from the following a) to j) on the HIRIP5 gene (for example, the nucleotide sequence represented by SEQ ID NOS: 1 to 9) or the GFAT1 gene (for example, the nucleotide sequence represented by SEQ ID NO: 10). It is designed as a continuous polynucleotide having a length of 16 to 500 bases, preferably 20 to 200 bases, more preferably 20 to 50 bases, including any one nucleotide.
a) Nucleotide at −34642 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 352 of SEQ ID NO: 1)
b) Nucleotide 26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2)
c) Nucleotide at position −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
d) Nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4)
e) The nucleotide at position 28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 in SEQ ID NO: 5)
f) The nucleotide at position 27915 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6)
g) Nucleotide at position 3270 of intron 3 of HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7)
h) Nucleotide 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8)
i) The -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9)
j) The 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10)
[0060]
(2) Primer
The primer used in the method of the present invention is an oligonucleotide primer for specifically amplifying a site containing a novel gene polymorphism on the HIRIP5 gene or the GFAT1 gene.
[0061]
Such a primer is selected from the preceding items a) to j) on the HIRIP5 gene (for example, the nucleotide sequence represented by SEQ ID NOS: 1 to 9) or the GFAT1 gene (for example, the nucleotide sequence represented by SEQ ID NO: 10). An oligonucleotide having a length of 15 to 30 bases, preferably about 18 to 25 bases, for hybridizing to a part of a continuous polynucleotide containing any one nucleotide or nucleotide sequence and specifically amplifying the polynucleotide. Designed as nucleotides.
[0062]
The length of the polynucleotide to be amplified is appropriately set depending on the detection method used, but is generally 16 to 1000 bases, preferably 20 to 500 bases, and more preferably 20 to 200 bases. .
[0063]
From the above, a polynucleotide (or oligonucleotide consisting of a continuous nucleotide sequence of 15 bases or more contained in the sequence near the polymorphic site (for example, the nucleotide sequence represented by any one of SEQ ID NOS: 1 to 10)) ) Can be used as the above primer or probe.
[0064]
The primers or probes of the present invention include those in which an appropriate label for detection, for example, a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin, or the like is added to the end of the sequence. .
[0065]
As the fluorescent dye used in the present invention, those generally used for labeling nucleotides and used for quantification and detection of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ', 4', 5 ', 7'-hexachloro-6-carboxyfluorescein, green fluorescent dye, fluorescein, NED (Applied Biosystems, trade name, yellow fluorescent dye), or 6-FAM (Applied Biosystems, trade name, yellow green) Fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but are not limited thereto. As a method for labeling a nucleotide with a fluorescent dye, an appropriate labeling method can be used among known labeling methods [see Nature Biotechnology, 14, p. 303-308 (1996)]. A commercially available fluorescent labeling kit can also be used (for example, oligonucleotide ECL 3'-oligolabeling system manufactured by Amersham Pharmacia).
[0066]
The primers of the present invention also include those to which a linker sequence for detecting a polymorphism is added at the end. Such a linker sequence includes, for example, a flap (a sequence completely unrelated to the sequence near the polymorphism) added to the 5′-terminal of the oligonucleotide used in the above-mentioned invader method.
[0067]
In the present specification, “polynucleotide” is synonymous with nucleic acid, and includes both DNA and RNA. It may be double-stranded or single-stranded, and thus, a polynucleotide having a certain sequence always includes a polynucleotide having a sequence complementary thereto (similarly, an oligonucleotide). . Furthermore, when the polynucleotide is RNA, the base symbol “t” shown in the sequence listing shall be read as “u”.
[0068]
Preferred examples of the above primers for detection using the SSCP method and the direct sequence method, which are one embodiment of the present invention, are shown below.
[0069]
i) For detecting a gene polymorphism at the −34642 nucleotide (nucleotide number 352 of SEQ ID NO: 1) in the 5 ′ upstream region of the HIRIP5 gene:
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 351 of SEQ ID NO: 1, more preferably in the nucleotide sequence represented by nucleotide numbers 152 to 351 of the same. 30 nucleotides) are selected. For example, the nucleotide sequence shown in SEQ ID NO: 11 can be mentioned.
[0070]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 353 to 720 of SEQ ID NO: 1), more preferably in the nucleotide sequence represented by nucleotide numbers 353 to 552 of SEQ ID NO: 1 An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 12 can be mentioned.
[0071]
ii) For detecting a gene polymorphism at nucleotide −26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 277 of SEQ ID NO: 2, more preferably the nucleotide sequence represented by nucleotide numbers 78 to 277 of SEQ ID NO: 2. 30 nucleotides) are selected. For example, the nucleotide sequence shown in SEQ ID NO: 13 can be mentioned.
[0072]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 279 to 660 of SEQ ID NO: 2, more preferably in the nucleotide sequence represented by nucleotide numbers 279 to 478 of SEQ ID NO: 2) An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 14 can be mentioned.
[0073]
iii) For detecting a gene polymorphism at nucleotide −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3):
As the sense strand primer, 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence shown in nucleotide numbers 1 to 310 of SEQ ID NO: 3, more preferably in the nucleotide sequence shown in nucleotide numbers 111 to 310 in SEQ ID NO: 3 30 nucleotides) are selected. For example, the nucleotide sequence shown in SEQ ID NO: 15 can be mentioned.
[0074]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 312 to 720 of SEQ ID NO: 3, more preferably in the nucleotide sequence represented by nucleotide numbers 312 to 511 of SEQ ID NO: 3) An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 16 can be mentioned.
[0075]
iv) For detecting a gene polymorphism at nucleotide 2721 of intron 4 of the HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence shown in nucleotide numbers 1 to 261 of SEQ ID NO: 4, more preferably in the nucleotide sequence shown in nucleotide numbers 62 to 261 in SEQ ID NO: 4. 30 nucleotides) are selected. An example is the nucleotide sequence shown in SEQ ID NO: 17.
[0076]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 263 to 720 of SEQ ID NO: 4), more preferably in the nucleotide sequence represented by nucleotide numbers 263 to 462 of SEQ ID NO: 4 An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 18 can be mentioned.
[0077]
v) For detecting a gene polymorphism at the nucleotide -28256 at the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 258 of SEQ ID NO: 5):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 257 of SEQ ID NO: 5, more preferably in the nucleotide sequence represented by nucleotide numbers 58 to 257 of the same. 30 nucleotides) are selected. For example, the nucleotide sequence shown in SEQ ID NO: 19 can be mentioned.
[0078]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 259 to 720 of SEQ ID NO: 5, more preferably in the nucleotide sequence represented by nucleotide numbers 259 to 458 of SEQ ID NO: 5) An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 20 can be mentioned.
[0079]
vi) For detecting a gene polymorphism at the nucleotide -27915 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 298 of SEQ ID NO: 6, more preferably in the nucleotide sequence represented by nucleotide numbers 99 to 298 of the same. 30 nucleotides) are selected. For example, the nucleotide sequence shown in SEQ ID NO: 21 can be mentioned.
[0080]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 300 to 720 of SEQ ID NO: 6, more preferably in the nucleotide sequence represented by nucleotide numbers 300 to 499 of SEQ ID NO: 6) An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 22 can be mentioned.
[0081]
vii) For detecting a gene polymorphism at nucleotide 3270 of intron 3 of HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 287 of SEQ ID NO: 7, more preferably in the nucleotide sequence represented by nucleotide numbers 88 to 287 of the same. 30 nucleotides) are selected. An example is the nucleotide sequence shown in SEQ ID NO: 23.
[0082]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 289 to 720 of SEQ ID NO: 7, more preferably in the nucleotide sequence represented by nucleotide numbers 289 to 488 of SEQ ID NO: 7) An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 24.
[0083]
viii) For detection of a gene polymorphism at nucleotide 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 309 of SEQ ID NO: 8, and more preferably in the nucleotide sequence represented by nucleotide numbers 110 to 309 of SEQ ID NO: 8. 30 nucleotides) are selected. For example, the nucleotide sequence shown in SEQ ID NO: 25 can be mentioned.
[0084]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 311 to 720 of SEQ ID NO: 8), more preferably in the nucleotide sequence represented by nucleotide numbers 311 to 510 of SEQ ID NO: 8 An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence set forth in SEQ ID NO: 26.
[0085]
ix) For detecting a gene polymorphism at nucleotide -168 of intron 3 of HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9):
The sense strand primer is preferably 15 nucleotides or more (preferably 15 nucleotides or more) in the nucleotide sequence represented by nucleotide numbers 1 to 312 of SEQ ID NO: 9, more preferably in the nucleotide sequence represented by nucleotide numbers 113 to 312 of the same. 30 nucleotides) are selected. An example is the nucleotide sequence shown in SEQ ID NO: 27.
[0086]
As the antisense strand primer, 15 nucleotides or more (preferably, in the nucleotide sequence represented by nucleotide numbers 314 to 720 of SEQ ID NO: 9), more preferably in the nucleotide sequence represented by nucleotide numbers 314 to 513 of SEQ ID NO: 9 An antisense sequence of a contiguous nucleotide sequence (15-30 nucleotides) is selected. For example, the nucleotide sequence shown in SEQ ID NO: 28.
[0087]
x) For detecting a gene polymorphism at the 36th nucleotide of intron 1 of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10):
The sense strand primer is preferably 15 nucleotides or 15 nucleotides in the nucleotide sequence shown in nucleotide numbers 132 to 631 of SEQ ID NO: 10 in the sequence listing, more preferably in the nucleotide sequence shown in nucleotide numbers 432 to 631 in the sequence listing. A continuous nucleotide sequence as described above (preferably 15 to 30 nucleotides), most preferably the nucleotide sequence shown in SEQ ID NO: 29 in the sequence listing;
As the antisense strand primer, preferably 15 nucleotides or 15 nucleotides in the nucleotide sequence represented by nucleotide numbers 633 to 782 of SEQ ID NO: 10 in the sequence listing, more preferably in the nucleotide sequence represented by nucleotide numbers 633 to 682 of the same. An antisense sequence of a continuous nucleotide sequence of more (preferably 15 to 30 nucleotides), most preferably the nucleotide sequence shown in SEQ ID NO: 30 in the sequence listing.
[0088]
The polynucleotide that can be used as the primer or the probe can be obtained by chemical synthesis based on a method well-known in the art. Polynucleotides used as these probes or primers can be used as reagents for determining the risk of developing diabetes.
[0089]
(3) Immobilized sample
The immobilized sample of the present invention is produced by immobilizing the polynucleotide used as the probe described in (1) on a solid phase such as a glass plate, a nylon membrane, a microbead, a silicon chip, or a capillary. Immobilization to a solid phase may be a method of placing a previously synthesized polynucleotide on a solid phase, or a method of synthesizing a target polynucleotide on a solid phase. For the immobilization method, for example, in the case of a DNA microarray, a method suitable for a target solid-phased sample such as using a commercially available spotter (manufactured by Amersham) is well known in the art. Examples of such an immobilized sample include a gene chip, a cDNA microarray, an oligo array, a membrane filter, and the like.
[0090]
(4) Kit
The kit of the present invention contains a polynucleotide that can be used as the probe or the primer, and at least one or more selected from a solid-phased sample. The kit of the present invention contains, as necessary, other reagents, instruments, and the like necessary for carrying out the determination method of the present invention, such as hybridization, labeling of a probe, detection of a label, and the like, as necessary. Is also good.
[0091]
5. Analysis and evaluation of detection results
From the results of the statistical analysis of the detection of the novel gene polymorphism in diabetic patients and healthy subjects, it has been found that the method of the present invention can determine the potential risk of developing diabetes with a high probability.
[0092]
For example, according to the method of the present invention, as a result of examining a gene polymorphism in a sample containing the isolated human genomic DNA, a subject who meets one or more of the following conditions has diabetes. It is possible to provide information that the risk of developing is relatively high.
a) A subject having a gene in which the -34642 nucleotide (nucleotide number 352 of SEQ ID NO: 1) in the 5 'upstream region of the HIRIP5 gene is c (cytosine).
b) A subject having a gene whose nucleotide at position 26376 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2) is g (guanine)
c) A subject having a gene in which t- (thymine) at nucleotide -350 of intron 3 of HIRIP5 gene (nucleotide number 311 in SEQ ID NO: 3) is repeated twice.
d) A subject having a gene which is c (cytosine) at nucleotide 2721 of intron 4 of the HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4).
[0093]
Also, by detecting a haplotype in which two or more gene polymorphisms are combined, it is possible to determine the potential risk of developing diabetes. For example, by detecting one or two or more of the following haplotypes of w) to z), the risk of developing a subject with diabetes can be determined.
[0094]
w) Present at the nucleotide -28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 of SEQ ID NO: 5) and at the nucleotide -27915 in the 5' upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6) Haplotype combining different genetic polymorphisms
x) A gene present at the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and the 3270th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7) Haplotype combining polymorphisms
y) The gene present at the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and the 3832th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8) Haplotype combining polymorphisms
z) It is present at the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and at the -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9). Haplotype combining genetic polymorphisms
[0095]
According to the present invention, a subject whose relative risk of developing diabetes is found to be relatively high based on the above-described criteria can be notified to that effect, and measures can be taken in advance to prevent the development of diabetes. Therefore, the present invention is extremely useful as a test method for preventing diabetes.
[0096]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.
[0097]
Example 1: Preparation of genomic DNA from peripheral blood
Leukocytes were isolated from peripheral blood of healthy Japanese people and type 2 diabetes patients. From this leukocyte, a genomic DNA sample of each subject was extracted using an automatic DNA extraction device (NA-1000: manufactured by Kurabo Industries). The absorbance at 260 nm of the obtained DNA samples was measured, and the DNA concentration of each sample was calculated. Thereafter, the genomic DNA was diluted to 50 ng / μl using sterile distilled water to obtain a PCR sample.
[0098]
Example 2: Amplification of DNA fragment containing polymorphism by PCR method
Using the genomic DNA sample of each subject obtained in Example 1 as a template, either of the 9 polymorphic sites on the HIRIP5 gene and the 1 polymorphic site on the GFAT1 gene was determined by the method described below. A DNA fragment containing either one was amplified.
[0099]
The 5 'end of each PCR primer used in this example was HEX (4,7,2', 4 ', 5', 7'-hexachloro-6-carboxyfluorescein, green fluorescent dye), NED (Applied BioSystems, trade name, yellow fluorescent dye, or 6-FAM (Applied Biosystems, trade name, yellow-green fluorescent dye) labeled with a fluorescent dye was ordered and purchased from Applied Biosystems.
[0100]
1) PCR for detecting whether the nucleotide at −34642 (nucleotide number 352 of SEQ ID NO: 1) in the 5 ′ upstream region of the HIRIP5 gene is t (thymine) or c (cytosine):
PCR was performed using the following primers and reaction conditions to amplify a 0.18 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 1: 5′-AAGATGGCGGGGAGGCCAG-3 ′ (SEQ ID NO: 11)
Primer No. 2: 5'-CGGCTCCATCAGATGCACTAC-3 '(SEQ ID NO: 12)
(Both the No. 1 and No. 2 labels the 5 'end with FAM)
[0101]
[Table 1]

PCR conditions: 94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0102]
2) PCR for detecting whether the nucleotide at position 26376 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2) is g (guanine) or c (cytosine):
PCR was performed using the following primers and reaction conditions to amplify a 0.10 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 3: 5′-CTTCTGGGAAAGAATTCATCCT-3 ′ (SEQ ID NO: 13);
Primer No. 4: 5′-ACAGTCTGAGTATCTGGGCT-3 ′ (SEQ ID NO: 14)
(In both No. 3 and No. 4, the 5 'end is labeled with HEX.)
[0103]
[Table 2]

PCR conditions: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0104]
3) PCR for detecting whether the repetition of the nucleotide at position −350 of intron 3 of the HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3) t (thymine) is once or twice:
PCR was performed using the following primers and reaction conditions to amplify a 0.14 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 5: 5'-ACCAGTAAATTGAGAAACTGA-3 '(SEQ ID NO: 15)
Primer No. 6: 5'-AAGCCAGTAAATATGCAGAC-3 '(SEQ ID NO: 16)
(Both No. 5 and No. 6 label the 5 'end with NED)
[0105]
[Table 3]

PCR conditions: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0106]
4) PCR for detecting whether nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4) is c (cytosine) or t (thymine):
PCR was performed using the following primers and reaction conditions to amplify a 0.15 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 7: 5'-ACACCCATGCACTCTGGTCA-3 '(SEQ ID NO: 17),
Primer No. 8: 5'-TTACCATTTACTGTTTCCTATTTT-3 '(SEQ ID NO: 18)
(Both No. 7 and No. 8 label the 5 'end with HEX.)
[0107]
[Table 4]

PCR conditions: 94 ° C for 30 seconds, 53 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0108]
5) PCR for detecting whether the -28256th nucleotide (nucleotide number 258 of SEQ ID NO: 5) in the 5 'upstream region of the HIRIP5 gene is g (guanine) or c (cytosine):
PCR was performed using the following primers and reaction conditions to amplify a 0.17 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 9: 5'-GTAGCCTGAATCAGGTTCCA-3 '(SEQ ID NO: 19)
Primer No. 10: 5'-ACCCTGTAGAGAACACGAGT-3 '(SEQ ID NO: 20)
(Both No. 9 and No. 10 label the 5 'end with NED)
[0109]
[Table 5]

PCR conditions: 94 ° C for 30 seconds, 59 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0110]
6) PCR for detecting whether the -27915 nucleotide (nucleotide number 299 of SEQ ID NO: 6) in the 5 'upstream region of the HIRIP5 gene is t (thymine) or c (cytosine):
PCR was performed with the following primers and reaction conditions to amplify a 0.16 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 11: 5'-GCATTATGCGTTAGCCAGAC-3 '(SEQ ID NO: 21)
Primer No. 12: 5'-GGAGCGAGATACACTGGAAA-3 '(SEQ ID NO: 22)
(No. 11 labels the 5 'end with FAM, No. 12 labels the 5' end with HEX)
[0111]
[Table 6]

PCR conditions: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0112]
7) PCR for detecting whether nucleotide 3270 of intron 3 of HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7) is g (guanine) or a (adenine):
PCR was performed using the following primers and reaction conditions to amplify a 0.19 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 13: 5'-GGTCTTCAAGTGATCCTCCT-3 '(SEQ ID NO: 23)
Primer No. 14: 5'-GCTCACACAATCTGCTGCCT-3 '(SEQ ID NO: 24)
(No. 13 labels the 5 'end with NED, No. 14 labels the 5' end with NED)
[0113]
[Table 7]

PCR conditions: 94 ° C for 30 seconds, 61 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0114]
8) PCR for detecting whether the nucleotide at position 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 in SEQ ID NO: 8) is c (cytosine) or t (thymine):
PCR was performed using the following primers and reaction conditions to amplify a 0.20 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 15: 5'-GTGGTTTACATTAGTTCATCTT-3 '(SEQ ID NO: 25)
Primer No. 16: 5'-CATACACATATGACAGTATCT-3 '(SEQ ID NO: 26)
(No. 15 labels the 5 'end with FAM, No. 16 labels the 5' end with FAM)
[0115]
[Table 8]

PCR conditions: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0116]
9) PCR for detecting whether the -168th nucleotide of intron 3 of HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9) is t (thymine) or a (adenine):
PCR was performed using the following primers and reaction conditions to amplify a 0.22 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 17: 5'-CCTAACATCATTAGCATATCA-3 '(SEQ ID NO: 27)
Primer No. 18: 5′-GCAAGTACGAGTATTAAAACA-3 ′ (SEQ ID NO: 28)
(No. 17 labels the 5 'end with FAM, No. 18 labels the 5' end with FAM)
[0117]
[Table 9]

PCR conditions: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, 37 cycles
[0118]
10) PCR for detecting whether the nucleotide at position 36 (nucleotide number 632 of SEQ ID NO: 10) of the first intron of the GFAT1 gene is c (cytosine) or t (thymine):
PCR was performed using the following primers and reaction conditions to amplify a 0.16 kbp DNA fragment containing a polymorphic site.
PCR primers:
Primer No. 19: 5'-CACCGAAGCTCGTGTGTGCA-3 '(SEQ ID NO: 29)
Primer No. 20: 5′-CGCCTTGGGCCTTAGCGGCA-3 ′ (SEQ ID NO: 30)
(No. 19 labels the 5 'end with HEX, No. 20 labels the 5' end with HEX)
[0119]
[Table 10]

PCR conditions: 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 2 minutes and 30 seconds, 40 cycles
[0120]
Example 3: Detection of polymorphisms in HIRIP5 gene and GFAT1 gene
1) Analysis using DNA sequencer
The nucleotide sequence for identifying each polymorphic site of the HIRIP5 gene and the GFAT1 gene was determined by a conventional method using a DNA sequencer (ABI PRISM 377: manufactured by Applied Biosystems).
[0121]
As a result, it was confirmed that each polymorphism exists at the following positions.
a) the nucleotide at −34642 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 352 in SEQ ID NO: 1);
b) the nucleotide at position 26376 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2);
c) the -350th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3);
d) nucleotide 2721 of intron 4 of the HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4);
e) the nucleotide at position 28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 of SEQ ID NO: 5);
f) the nucleotide at position 27915 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 299 of SEQ ID NO: 6);
g) nucleotide 3270 of intron 3 of the HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7);
h) the nucleotide at position 3832 of intron 3 of the HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8);
i) the -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9);
j) Position 36 of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10)
[0122]
2) SSCP method
One-fifth of each PCR product obtained in Example 2 was added to a 5: 1 mixture of formamide solution: loading buffer (a 0.05 M aqueous solution of EDTA containing 0.03 g / ml blue dextran) and mixed. After heat denaturation at 95 ° C. for 2 minutes, the mixture was ice-cooled to prepare a sample for electrophoresis. This sample was electrophoresed on a non-denaturing polyacrylamide gel, and a polymorphism was detected based on a difference in mobility of DNA fragments based on a difference in allele.
[0123]
The electrophoresis gel used was a 12% polyacrylamide gel to which 5% glycerol was added (gel thickness: 0.2 mm). In addition, in order to confirm that there is no change in polymorphism detection due to a difference in measurement conditions, 10% polyacrylamide and 5% glycerol were added, and 12% polyacrylamide gel was added with 10% glycerol. The procedure was performed under two conditions.
[0124]
The electrophoresis was carried out by using a DNA sequencer (ABI PRISM 377: manufactured by Applied Biosystems) and applying a current of 3 to 12 hours using an A filter at a constant power of 30 watts. The fluorescent band was detected using the detection function of the DNA sequencer. During electrophoresis, in order to maintain the gel plate temperature, water adjusted to 20 ° C. by a cooling device (RTE-III, manufactured by Neslab) was circulated outside the gel plate. The genotype of each DNA fragment was determined as a positive control using the DNA fragment for which the nucleotide sequence of the polymorphic site was determined in 1) above.
[0125]
Example 4: Association between HIRIP5 gene and GFAT1 gene polymorphism and diabetes
The association between the HIRIP5 gene and type 2 diabetes was analyzed by an association analysis method (association method). Chi-square method was used for significance test. (Fisher et al., Biostatistics, John Willey & Sons, 1993). Haplotype analysis was performed using the Arlequin program (statistical analysis software manufactured by Laurent Excoffer, URL: http://lgb.unige.ch/arlequin/). Exact test of population difference (PD test), which is an extension of Fisher's direct probability calculation method, was used for the significant difference test ("Basic Medical Statistics Revised 4th Edition", co-authored by Katsumi Kano and Hideto Takahashi, Nanedo, 1995) Year). The significance level was 5% risk, and those with less than 5% risk were judged to have significant difference.
[0126]
1) Relationship between type 2 diabetes and the nucleotide at -34642 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 352 in SEQ ID NO: 1)
The allele frequency at the -34642th nucleotide (nucleotide number 352 of SEQ ID NO: 1) in the 5 'upstream region of the HIRIP5 gene was calculated for 190 healthy Japanese people and 393 Japanese type 2 diabetes patients, and the association method was used. The association with type 2 diabetes was analyzed. Table 11 shows the results.
[0127]
[Table 11]

[0128]
In Table 11, the nucleotide at position -34642 (nucleotide number 352 of SEQ ID NO: 1) in the 5 'upstream region of the HIRIP5 gene is represented by "T" when it is thymine, or "C" when it is cytosine. From the results in Table 11, the allele frequency of the −34642 nucleotide (nucleotide number 352 of SEQ ID NO: 1) polymorphism in the 5 ′ upstream region of the HIRIP5 gene was statistically significantly higher between healthy individuals and patients with type 2 diabetes. It was shown to be different. This indicates that the relative risk of developing diabetes can be determined by examining the polymorphism at the −34642 nucleotide (nucleotide number 352 of SEQ ID NO: 1) in the 5 ′ upstream region of the HIRIP5 gene.
[0129]
2) Relationship between type 2 diabetes and the 26376th nucleotide (nucleotide number 278 of SEQ ID NO: 2) in the 5 'upstream region of the HIRIP5 gene
Allele frequency of type-2 diabetes and polymorphism at nucleotide -26376 at the 5 'upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2) in 191 Japanese healthy people and 390 Japanese type 2 diabetes patients Was calculated, and the association with type 2 diabetes was analyzed by the association method. Table 12 shows the results.
[0130]
[Table 12]

[0131]
In Table 12, the nucleotide at the 26376th nucleotide (nucleotide number 278 of SEQ ID NO: 2) in the 5 ′ upstream region of the HIRIP5 gene is represented by “G” when it is guanine, or “C” when it is cytosine. . From the results in Table 12, the allele frequency and the genotype frequency of the polymorphism at nucleotide −26,376 at the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2) between healthy individuals and type 2 diabetes patients are shown. It was shown to be statistically significantly different. This indicates that the relative risk of developing diabetes can be determined by examining the polymorphism at the nucleotide 26376 at the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2). .
[0132]
3) Relationship between type 2 diabetes and nucleotide -350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
The allele frequency at the -350th nucleotide of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3) was calculated for 190 healthy Japanese people and 394 Japanese type 2 diabetes patients, and the association method was used. The association with type 2 diabetes was analyzed. Table 13 shows the results.
[0133]
[Table 13]

[0134]
In Table 13, when the repetition of T at the -350th nucleotide (nucleotide number 311 of SEQ ID NO: 3) of intron 3 of the HIRIP5 gene is performed once, it is referred to as "T". Is represented. From the results in Table 13, the allele frequency of the -350th nucleotide (nucleotide number 311 in SEQ ID NO: 3) polymorphism of intron 3 of the HIRIP5 gene is statistically significantly different between healthy individuals and patients with type 2 diabetes. It was shown. This indicates that the relative risk of developing diabetes can be determined by examining the polymorphism at nucleotide −350 of intron 3 of the HIRIP5 gene (nucleotide number 311 in SEQ ID NO: 3).
[0135]
4) Relationship between type 2 diabetes and polymorphism at nucleotide 2721 (nucleotide number 262 of SEQ ID NO: 4) of intron 4 of HIRIP5 gene
For 191 healthy Japanese subjects and 388 Japanese patients with type 2 diabetes, the allele frequency of the 2271st nucleotide (nucleotide number 262 of SEQ ID NO: 4) of intron 4 of type 2 diabetes and HIRIP5 gene was calculated. The association with type 2 diabetes was analyzed by the association method. Table 14 shows the results.
[0136]
[Table 14]

[0137]
In Table 14, the site of nucleotide 2721 (nucleotide number 262 of SEQ ID NO: 4) of intron 4 of HIRIP5 gene is expressed as "C" when it is cytosine, or "T" when it is thymine. From the results shown in Table 14, the allele frequency of the 2721 nucleotide polymorphism of nucleotide 2721 (nucleotide number 262 of SEQ ID NO: 4) of intron 4 of the HIRIP5 gene is statistically significantly different between healthy individuals and patients with type 2 diabetes. It has been shown. This indicates that the relative risk of developing diabetes can be determined by examining the polymorphism at nucleotide 2721 of intron 4 of the HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4).
[0138]
5) Type 2 diabetes and the nucleotide at position 28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 in SEQ ID NO: 5) and the nucleotide at position 27915 in the 5' upstream region of the HIRIP5 gene (nucleotide in SEQ ID NO: 6) No. 299) Relationship with haplotype combining polymorphisms
For 191 Japanese healthy subjects and 386 Japanese type 2 diabetic patients, the −28256 nucleotide polymorphism (nucleotide number 258 of SEQ ID NO: 5) in the 5 ′ upstream region of the HIRIP5 gene and the 5 ′ position of the HIRIP5 gene The haplotype frequency was calculated by combining the −27915 nucleotide (nucleotide number 299 of SEQ ID NO: 6) polymorphism in the basin, and the association with type 2 diabetes was analyzed by PD test. Table 15 shows the results.
[0139]
[Table 15]

[0140]
In Table 15, subjects homozygous for a gene having a G at the -28256th nucleotide (nucleotide number 258 of SEQ ID NO: 5) in the 5 'upstream region of the HIRIP5 gene are "G / G", and the site is C (cytosine). A subject having a homozygous is expressed as “C / C”, and a subject having both genes as a heterogeneity is expressed as “G / C”. In Table 15, subjects homozygous for a gene in which the -27915th nucleotide (nucleotide number 299 of SEQ ID NO: 6) in the 5 'upstream region of the HIRIP5 gene is T are "T / T", and the site is C ( Subjects having a homozygous gene (cytosine) are represented as “C / C”, and subjects having both genes as a heterogeneity are represented as “T / C”. From the results of Table 15, it was shown that the frequency distribution of each haplotype was significantly different in the comparison between healthy subjects and type 2 diabetic patients. This means that the polymorphism at nucleotide -28,256 at the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 of SEQ ID NO: 5) and the nucleotide at the nucleotide -27915 at the 5' upstream region of the HIRIP5 gene (nucleotide number 299 of SEQ ID NO: 6) 2) It indicates that the relative risk of developing diabetes can be determined by examining haplotypes that combine polymorphisms.
[0141]
6) Polymorphism at nucleotide 3270 (nucleotide number 288 of SEQ ID NO: 7) of intron 3 of HIRIP5 gene and nucleotide 36 (nucleotide number 632 of SEQ ID NO: 10) of first intron of GFAT1 gene Relationship with haplotypes
For 185 Japanese healthy people and 357 Japanese type 2 diabetic patients, the 3270th nucleotide (nucleotide number 288 of SEQ ID NO: 7) of intron 3 of the HIRIP5 gene and the 36th nucleotide of the first intron of the GFAT1 gene were found. Of the nucleotide (nucleotide number 632 of SEQ ID NO: 10) polymorphism was calculated, and the association with type 2 diabetes was analyzed by PD test. Table 16 shows the results.
[0142]
[Table 16]

[0143]
In Table 16, subjects homozygous for the gene in which the 3270th nucleotide (nucleotide number 288 of SEQ ID NO: 7) of intron 3 of the HIRIP5 gene is G have the gene “G / G”, and the gene whose site is A (adenine). A subject having homozygosity is represented as “A / A”, and a subject having heterozygous both genes is represented as “G / A”. In Table 16, subjects homozygous for a gene in which the 36th nucleotide (nucleotide number 632 of SEQ ID NO: 10) of the first intron of the GFAT1 gene is T are “T / T”, and the site is C (cytosine). A subject having a homozygous is expressed as “C / C”, and a subject having a heterozygous both genes is expressed as “T / C”. From the results of Table 16, it was shown that the frequency distribution of each haplotype was significantly different in comparison between healthy subjects and type 2 diabetic patients. This indicates that the 3270th nucleotide (nucleotide number 288 of SEQ ID NO: 7) polymorphism of intron 3 of the HIRIP5 gene and the 36th nucleotide (nucleotide number 632 of SEQ ID NO: 10) of the first intron of the GFAT1 gene are combined. This indicates that the relative risk of developing diabetes can be determined by examining the haplotypes.
[0144]
7) Polymorphism at nucleotide 3832 of intron 3 of the HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8) and nucleotide polymorphism of nucleotide 36 of intron 1 of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10) in type 2 diabetes and HIRIP5 gene Relationship with haplotypes
About 185 Japanese healthy people and 382 Japanese type 2 diabetes patients, the polymorphism of nucleotide 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8) and the 36th nucleotide of intron 1 of GFAT1 gene were found. Of the nucleotide (nucleotide number 632 of SEQ ID NO: 10) polymorphism was calculated, and the association with type 2 diabetes was analyzed by PD test. Table 17 shows the results.
[0145]
[Table 17]

[0146]
In Table 17, subjects homozygous for a gene in which the nucleotide at position 3832 of intron 3 of the HIRIP5 gene (nucleotide number 310 in SEQ ID NO: 8) is C (cytosine) are “C / C”, and the site is T (thymine). Subjects who have a certain gene homozygously are indicated as "T / T", and subjects who have both genes heterozygously are indicated as "C / T". In Table 17, subjects homozygous for a gene in which the 36th nucleotide (nucleotide number 632 of SEQ ID NO: 10) of the first intron of the GFAT1 gene is T are “T / T”, and the site is C (cytosine). A subject having a homozygous is expressed as “C / C”, and a subject having a heterozygous both genes is expressed as “T / C”. From the results of Table 17, it was shown that the frequency distribution of each haplotype was significantly different in the comparison between healthy subjects and type 2 diabetic patients. This means that the polymorphism at nucleotide 3832 of intron 3 (nucleotide number 310 of SEQ ID NO: 8) of the HIRIP5 gene and the nucleotide polymorphism at nucleotide 36 (nucleotide number 632 of SEQ ID NO: 10) of the first intron of GFAT1 gene are combined. This indicates that the relative risk of developing diabetes can be determined by examining the haplotypes.
[0147]
8) Type 2 diabetes and a polymorphism at the -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9) and a polymorphism of the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10) Relationship with haplotypes combining types
For 186 Japanese healthy people and 385 Japanese type 2 diabetic patients, the polymorphism at nucleotide -168 of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9) and 36 of the first intron of the GFAT1 gene were determined. The haplotype frequency combining the second nucleotide (nucleotide number 632 of SEQ ID NO: 10) polymorphism was calculated, and the association with type 2 diabetes was analyzed by the association method. The results are shown in Table 18.
[0148]
[Table 18]

[0149]
In Table 18, subjects homozygous for a gene in which the -168th nucleotide (nucleotide number 313 of SEQ ID NO: 9) of intron 3 of the HIRIP5 gene is T (thymine), is "T / T", and the site is A (adenine). Are homozygously expressed as "A / A", and those having both genes heterozygically are expressed as "T / A". In Table 18, subjects homozygous for the gene in which the 36th nucleotide (nucleotide number 632 in SEQ ID NO: 10) of the first intron of the GFAT1 gene is T are “T / T”, and the site is C (cytosine). A subject having a homozygous is expressed as “C / C”, and a subject having a heterozygous both genes is expressed as “T / C”. From the results in Table 18, it was shown that the frequency distribution of each haplotype was significantly different in a comparison between a healthy person and a type 2 diabetic patient. This means that the polymorphism at nucleotide -168 of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9) and the nucleotide polymorphism at the nucleotide 36 of intron 1 (nucleotide number 632 of SEQ ID NO: 10) of the GFAT1 gene are different. This indicates that the relative risk of developing diabetes can be determined by examining the combined haplotypes.
[0150]
【The invention's effect】
According to the present invention, the relative risk of diabetes, particularly type 2 diabetes, can be easily determined. That is, the present invention provides a method for determining the risk of developing diabetes and materials for the method.
[0151]
[Sequence list]

[0152]
[Sequence List Free Text]
SEQ ID NO: 1—Description of mutation: single nucleotide polymorphism due to substitution of (T) and (C)
SEQ ID NO: 2 Description of mutation: Single nucleotide polymorphism due to substitution of (C) and (G)
SEQ ID NO: 3—Description of mutation: polymorphism due to single nucleotide insertion of (T)
SEQ ID NO: 4-Description of mutation: single nucleotide polymorphism due to substitution of (T) and (C)
SEQ ID NO: 5 Description of mutation: Single nucleotide polymorphism due to substitution of (C) and (G)
SEQ ID NO: 6 Description of mutation: Single nucleotide polymorphism due to substitution of (T) and (C)
SEQ ID NO: 7-Description of mutation: single nucleotide polymorphism by substitution of (A) and (G)
SEQ ID NO: 8-Description of mutation: single nucleotide polymorphism due to substitution of (C) and (T)
SEQ ID NO: 9-Description of mutation: single nucleotide polymorphism by substitution of (T) and (A)
SEQ ID NO: 10-Description of mutation: single nucleotide polymorphism due to substitution of (T) and (C)
SEQ ID NOS: 11-30-Description of Artificial Sequence: Primer

Claims (9)

In a sample containing human genomic DNA, the sample is provided by detecting a gene polymorphism present at any one or more positions selected from the following a) to j) and / or a haplotype comprising a combination thereof: For determining the risk of developing diabetes in an elderly person.
a) Nucleotide at −34642 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 352 of SEQ ID NO: 1)
b) Nucleotide 26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2)
c) Nucleotide at position −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
d) Nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4)
e) The nucleotide at position 28256 in the 5 'upstream region of the HIRIP5 gene (nucleotide number 258 in SEQ ID NO: 5)
f) The nucleotide at position 27915 in the 5 ′ upstream region of the HIRIP5 gene (nucleotide number 299 in SEQ ID NO: 6)
g) Nucleotide at position 3270 of intron 3 of HIRIP5 gene (nucleotide number 288 of SEQ ID NO: 7)
h) Nucleotide 3832 of intron 3 of HIRIP5 gene (nucleotide number 310 of SEQ ID NO: 8)
i) The -168th nucleotide of intron 3 of the HIRIP5 gene (nucleotide number 313 of SEQ ID NO: 9)
j) The 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10) 2. The method of claim 1, wherein the diabetes is type 2 diabetes. The detection target is any one or two or more selected from gene polymorphisms present at the following positions a) to d) and haplotypes represented by the following w) to z). The described method.
a) Nucleotide at −34642 in the 5 ′ upstream region of HIRIP5 gene (nucleotide number 352 of SEQ ID NO: 1)
b) Nucleotide 26376 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 278 of SEQ ID NO: 2)
c) Nucleotide at position −350 of intron 3 of HIRIP5 gene (nucleotide number 311 of SEQ ID NO: 3)
d) Nucleotide 2721 of intron 4 of HIRIP5 gene (nucleotide number 262 of SEQ ID NO: 4)
w) Present at nucleotide -28,256 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 258 of SEQ ID NO: 5) and at nucleotide 27,915 at the 5 ′ upstream region of HIRIP5 gene (nucleotide number 299 of SEQ ID NO: 6) X) The haplotype x) obtained by combining the gene polymorphisms, the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing), and the 3270th nucleotide of intron 3 of the HIRIP5 gene (nucleotide of SEQ ID NO: 7) Haplotype combining the genetic polymorphisms present at nucleotides 288) y) 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing) and 3832 of intron 3 of the HIRIP5 gene Haplotype z combining the genetic polymorphisms present at the nucleotides (nucleotide number 310 of SEQ ID NO: 8), the 36th nucleotide of the first intron of the GFAT1 gene (nucleotide number 632 of SEQ ID NO: 10 in the sequence listing), and HIRIP5 Haplotype combining gene polymorphisms present at nucleotide -168 of intron 3 of the gene (nucleotide number 313 of SEQ ID NO: 9) Detection includes RFLP, PCR-SSCP, ASO hybridization, direct sequencing, ARMS, denaturing gradient gel electrophoresis, RNaseA cleavage, chemical cleavage, DOL, TaqManｑPCR, Invader, MALDI A group consisting of the TOF / MS method, the TDI method, the molecular beacon method, the dynamic allele-specific hybridization method, the padlock probe method, the UCAN method, the nucleic acid hybridization method using a DNA chip or a DNA microarray, and the ECA method The method according to any one of claims 1 to 3, wherein the method is performed using one or more methods selected from the group consisting of: The method according to claim 4, wherein the detection is performed using a PCR-SSCP method or a direct sequencing method. Any one polynucleotide selected from the following a) to j), or a labeled product thereof;
a) On the nucleotide sequence represented by SEQ ID NO: 1, a continuous polynucleotide having a length of 16 to 500 bases including the nucleotide of nucleotide number 352 b) On the nucleotide sequence represented by SEQ ID NO: 2, containing the nucleotide of nucleotide number 278 A continuous polynucleotide having a length of 16 to 500 bases c) On the nucleotide sequence represented by SEQ ID NO: 3, a continuous polynucleotide having a length of 16 to 500 bases including nucleotide 311 d) A nucleotide sequence represented by SEQ ID NO: 4 Above, a continuous polynucleotide of 16-500 bases in length comprising nucleotides of nucleotide number 262 e) On the nucleotide sequence shown in SEQ ID NO: 5, 16-500 bases in length comprising nucleotides of nucleotide number 258 The following polynucleotide f) On the nucleotide sequence shown in SEQ ID NO: 6, a continuous polynucleotide having a length of 16 to 500 bases including the nucleotide of nucleotide number 299 g) On the nucleotide sequence shown in SEQ ID NO: 7, nucleotide number 288 H) On the nucleotide sequence represented by SEQ ID NO: 8, a continuous polynucleotide of 16-500 bases containing nucleotides of nucleotide No. 310 i) SEQ ID NO: 9 On the nucleotide sequence shown, a continuous polynucleotide having a length of 16 to 500 bases including the nucleotide of nucleotide No. 313 j) On the nucleotide sequence shown in SEQ ID No. 10, the nucleotide of nucleotide No. 632 Comprising 16 to 500 bases long contiguous polynucleotide (however, when the polynucleotide is RNA, the nucleotide symbol "t" in the sequence table is replaced with "u"). An oligonucleotide having a length of 15 to 30 bases or a labeled product thereof, which hybridizes to a part of any one of the polynucleotides selected from the following a) to j) and specifically amplifies the polynucleotide;
a) On the nucleotide sequence represented by SEQ ID NO: 1, a continuous polynucleotide having a length of 16 bases or more containing the nucleotide of nucleotide number 352 b) On the nucleotide sequence represented by SEQ ID NO: 2, containing 16 nucleotides containing the nucleotide of nucleotide number 278 A) a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide No. 311 d) a nucleotide having a length of 16 nucleotides or more including the nucleotide of nucleotide No. 311; A continuous polynucleotide having a length of 16 bases or more containing nucleotides of nucleotide number 262 e) On the nucleotide sequence represented by SEQ ID NO: 5, a continuous polynucleotide having a length of 16 bases or more containing nucleotides of nucleotide number 258 F) On the nucleotide sequence represented by SEQ ID NO: 6, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide number 299 g) On the nucleotide sequence represented by SEQ ID NO: 7, including the nucleotide of nucleotide number 288 A continuous polynucleotide having a length of 16 bases or more h) On the nucleotide sequence represented by SEQ ID NO: 8, a continuous polynucleotide having a length of 16 bases or more including the nucleotide of nucleotide No. 310 i) On a nucleotide sequence represented by SEQ ID NO: 9 A) a continuous polynucleotide having a length of 16 bases or more containing nucleotides of nucleotide number 313; j) a continuous polynucleotide having a length of 16 bases or more containing nucleotides of nucleotide number 632 on the nucleotide sequence represented by SEQ ID NO: 10; Reochido (However, if the polynucleotide is RNA, the nucleotide symbol "t" in the sequence table is replaced with "u"). A solid-phased sample on which the polynucleotide according to claim 6 is immobilized. A reagent or kit for determining the risk of developing diabetes, comprising at least one selected from the following 1) to 3):
1) The polynucleotide according to claim 6 or a labeled product thereof 2) The oligonucleotide according to claim 7 or a labeled product thereof 3) The immobilized sample according to claim 8