JPWO2004084797A1 - Type 2 diabetes-related genes and their use - Google Patents

Abstract

In order to identify a type 2 diabetes (DM) susceptibility gene, 2039 single nucleotide polymorphisms (SNPs) derived from 704 candidate genes were examined for association with type 2 diabetes using a case control test. As a result, 29 genes related to type 2 diabetes were successfully identified. By using the presence or absence of polymorphic mutations on these genes as an index, it is possible to test for type 2 diabetes.

Description

  The present invention relates to a gene associated with type 2 diabetes and a method for testing type 2 diabetes using the gene.

Type 2 diabetes (DM) is a heterogeneous glucose metabolism disorder characterized by both insulin resistance and pancreatic β-cell dysfunction. In developed countries, the disease has become a major health problem because of the high incidence (3% -10%) of vascular complications associated with DM and clinical severity. The hereditary pattern of DM suggests a polygenic feature of the disease and a complex interaction between genes and environmental factors (see Non-Patent Document 1). However, the genes associated with the general formation of DM are not known so far. Genome-wide studies using microsatellite markers and affected sibling pair analysis identified loci associated with DM in various genomic regions from different populations (see Non-Patent Documents 1-7). This difference in population-specific mapping suggests complex effects from various genomic regions in the formation of DM. Although intensive efforts have been made to identify DM-related genes at these loci, so far the calpain-10 gene from the NIDDM1 locus is the only DM-related gene reported for DM ( Non-patent document 8). In addition, only a few genes that increase the risk of disease have been identified by the candidate gene approach in the DM-related pathway (see Non-Patent Document 1).
DM is a multifactorial disease, affected by environmental factors, and the cause of disease among individuals is heterogeneous, and it develops with multiple causative genes (disease susceptibility genes), so the presence of genetic predisposition is certain. It is one of the diseases where it is difficult to identify the causative gene despite the fact that it is believed. In particular, in diabetes, it is difficult to clearly distinguish healthy individuals from those with a predisposition to diabetes, often because onset is in middle age or later, and it is difficult to obtain genetically important family data. Makes the identification of the causative gene more difficult.
According to conventional research, there are several genes causing special diabetes caused by abnormality of a single gene (5% or less of type 2 diabetes), candidate gene approaches, genes identified by disease model animals, and the like. The affected sibling pair analysis was performed all over the world, but the results differed by race / ethnicity, and the chromosomal regions that were found to be linked across the entire genome. It was difficult.
As mentioned above, few genes related to DM have been reported so far.
Barnett, A.M. H. Et al., “Diabetologia”, 1981, Vol. 20, p. 87-93 Knowler, W.M. C. Et al., “Am. J. Hum. Genet.”, 1988, Vol. 43, p. 520-526 Bell, G.M. I. Et al., “Proc. Natl. Acad. Sci. USA”, 1991, Vol. 88, p. 1484-1488 Van den Owenl and J.M. M.M. W. Et al., “Nature Genet.”, 1992, Vol. 1, p. 368-371 Vionnet, N.M. Et al., “Am. J. Hum. Genet.”, 2000, Vol. 67, p. 1470-1480 Lander, E .; S. And Kruglyak, L .; "Nature Genet.", 1995, Vol. 11, p. 241-247 Wiltshire, S .; Et al., “Am. J. Hum. Genet.”, 2001, Vol. 69, p. 553-569 Ghosh, S .; Et al., “Am. J. Hum. Genet.”, 2000, Vol. 67, p. 1174-1185

本発明は、被検者について、下記（１）〜（２９）のいずれかに記載の遺伝子における変異を検出することを特徴とする、２型糖尿病の検査方法を提供する。
（１）ＭＥＴ、（２）ＮＰＨＳ１、（３）ＳＬＣ１Ａ１、（４）ＳＥＲＰＩＮＢ２、（５）ＦＡＢＰ２、（６）ＺＮＦ２６３、（７）ＴＮＦ、（８）ＴＨＢＳ３、（９）ＡＢＣＡ１、（１０）ＳＥＲＰＩＮＢ１０、（１１）ＡＬＤＯＢ、（１２）ＣＴＳＤ、（１３）ＳＴＥ、（１４）ＡＢＣＣ８、（１５）ＳＥＲＰＩＮＡ３、（１６）ＲＣＶ１、（１７）ＴＦＲＣ、（１８）ＭＭＰ１９、（１９）ＭＹＬ２、（２０）ＶＬＤＬＲ、（２１）Ｐ２ＲＸ７、（２２）ＤＬＧ５、（２３）ＬＤＬＣ、（２４）ＡＱＰ８、（２５）ＬＡＲＧＥ、（２６）ＢＬＺＦ１、（２７）ＲＧＳ５、（２８）ＦＲＡＰ１、（２９）ＬＥＰ
なお、本発明の上記「いずれかに記載の遺伝子」とは、上記（１）〜（２９）のいずれか少なくとも１以上の遺伝子を意味する。即ち、上記（１）〜（２９）に記載の複数の遺伝子における変異を検出することによって２型糖尿病の検査を行う場合も、本発明に含まれる。
本発明において「２型糖尿病の検査」とは、被検者が、２型糖尿病に罹患する可能性が高いか低いか、およびすでにＤＭに罹患している場合にはその原因や病型の判定をするための検査が含まれる。本発明の方法においては、上記（１）〜（２９）の各遺伝子において変異が検出された場合に、被検者は２型糖尿病を罹患しやすい、あるいはしにくいものと判定される。２型糖尿病にすでに罹患した被検者であっても２型糖尿病を発症した原因および病型を判定することができ、治療方針の決定等に利用する事ができる。
本発明の上記方法における「変異」の位置について、予め規定することは困難であるが、通常、上記遺伝子のＯＲＦ中、あるいは上記遺伝子の発現を制御する領域（例えば、プロモーター領域、エンハンサー領域等）中である。また、この「変異」とは、上記遺伝子の発現量を変化させる、ｍＲＮＡの安定性等の性質を変化させる、あるいは上記遺伝子によってコードされるタンパク質の有する活性を変化させるような変異であれば、特に制限されず、例えば、塩基の付加、欠失、置換、挿入変異等を挙げることができる。
本発明者らは、ＤＭ患者における上記（１）〜（２９）の各遺伝子もしくは該遺伝子の近傍ＤＮＡ領域においてＤＭと有意に関連する多型変異を見出すことに成功した。従って、上記（１）〜（２９）の各遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位について変異の有無を指標とする（塩基種を決定する）ことにより、ＤＭの検査を行うことが可能である。尚、上記の「遺伝子の近傍ＤＮＡ領域」とは、通常、該遺伝子の近傍の染色体上に領域を指す。近傍とは、特に制限されるものではないが、通常、本発明の多型部位を含むＤＮＡ領域であり、好ましくは、遺伝子の末端部分から１０ｋｂ以内の領域を指す。
前後１０ｋｂすなわち２０ｋｂの範囲にある多型は、Ｇａｂｒｉｅｌらの報告の通り連鎖している可能性が高い（Ｇａｂｒｉｅｌ ＳＢ，Ｓｃｈａｆｆｎｅｒ ＳＦ，Ｎｇｕｙｅｎ Ｈ ｅｔ ａｌ．Ｔｈｅ ｓｔｒｕｃｔｕｒｅ ｏｆ ｈａｐｌｏｔｙｐｅ ｂｌｏｃｋｓ ｉｎ ｔｈｅ ｈｕｍａｎ ｇｅｎｏｍｅ．Ｓｃｉｅｎｃｅ ２９６，２２２５−９．２００２）。
上記（１）〜（２９）の各遺伝子および該遺伝子の近傍ＤＮＡ配列を、それぞれ配列番号：１〜２９に示す。（即ち、配列番号：１に記載の塩基配列は、ＭＥＴ遺伝子および該遺伝子の近傍のＤＮＡ配列を示す。）また、配列番号：３０には、配列番号：２８で示したＦＲＡＰ遺伝子および該遺伝子の近傍のＤＮＡ領域以外の、ＦＲＡＰ遺伝子および該遺伝子の近傍のＤＮＡ配列を示す。
本発明の好ましい態様においては、上記（１）〜（２９）のいずれかに記載の遺伝子における一塩基多型変異を検出することを特徴とする、２型糖尿病の検査方法である。
多型とは、遺伝学的には、人口中１％以上の頻度で存在している１遺伝子におけるある塩基の変化と一般的に定義されるが、本発明における「多型」は、この定義に制限されない。本発明における多型の種類としては、例えば、一塩基多型、一から数十塩基（時には数千塩基）が欠失あるいは挿入している多型等が挙げられるが、これらに特に限定されない。さらに、多型部位の数も、１個に限定されず、複数個の多型を有していてもよい。
また本発明の別の態様においては、上記（１）〜（２９）のいずれかに記載の遺伝子もしくは該遺伝子の近傍ＤＮＡ領域における多型部位の塩基種を決定することを特徴とする、２型糖尿病の検査方法である。
本発明の上記方法における「多型部位」は、上記（１）〜（２９）の各遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上に存在する多型であれば、特に制限されない。具体的には、本発明の検査方法に利用可能な多型部位として、上記（１）〜（２９）の各遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上に存在する以下の（１ａ）〜（２９ａ）多型部位を挙げることができる。（尚、本明細書においては、これらの多型部位を単に『本発明の多型部位』と記載する場合がある。）
（１ａ）ＭＥＴ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１に記載の塩基配列における７６５位、１３８３位、１８９０位、２０９９位、２８１４位、３２９３位、３７５２位、３７７４位、３９２７位、４６９０位、７４１９位、８２１４位、１０００１位、１０３１８位、１３７６６位、１３９８９位、１４２５８位、１５４２７位、１６２７５位、１６５９７位、１６９３２位、１９７６８位、１９８３５位、２００３２位、２０２４０位、２０５３７位、２０８８５位、２１５４６位、２４２０９位、２４２７１位、２４３７６位、２４６４２位、２６０２８位、２６２２６位、または３４２０９位のいずれかの多型部位
（２ａ）ＮＰＨＳ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２に記載の塩基配列における１７２位、１８６位、２１１位、２５５４位、２５５８位、１０００１位、１００６７位、１０１７６位、１１８４０位、１３７５９位、１４６４５位、１４９６７位、１６１９８位、１６４３９位、１７６３５位、１７６８５位、１７７９４位、１７８０５位、１７８４８位、１７８４９位、１８４９１位、１９９５５位、２００８８位、２０６１３位、２１５３３位、２１５８２位、２１８１１位、２１８１２位、２２１９０位、２２９６５位、２２９８２位、２３６６０位、２３７８０位、２４０４０位、２４１７２位、２５３３４位、または２６５８５位のいずれかの多型部位
（３ａ）ＳＬＣ１Ａ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：３に記載の塩基配列における１１４２位、２７３２位、２７３４位、７２２０位、１０００１位、１２２７６位、１２９２２位、または１８０４９位のいずれかの多型部位
（４ａ）ＳＥＲＰＩＮＢ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：４に記載の塩基配列における２８５３位、２８９１、５２１８位、５３１７位、７５６０位、８０５４位、８７７１位、１０００１位、１０３２９位、１１７８１位、１２０６３位、１２７０５位、１３１２５位、１３６９１位、１３９１３位、１３９１６位、１３９１７位、１４１２３位、１４１６９位、１４４５３位、１４４５６位、１４４８３位、１６５２６位、１６５５６位、１７６４３位、１７７２５位、１７８２４位、１８０７６位、１８２２７位、１８５００位、１８６８７位、１８７４１位、１８７４４位、１８８７５位、１９０４５位、１９１４１位、１９１４９位、１９１９０位、１９４２９位、１９５４８位、１９５６７位、１９６２４位、１９６４５位、１９８１４位、２００４９位、２００８２位、２００９７位、２０２４３位、２２１２２位、２２４９５位、２２５１６位、２２６９１位、２２８１６位、２３００７位、２３９０４位、２４００５位、２４４１８位、２４５７８位、２４８５５位、２４８８５位、２５５０５位、２５５３８位、２５５６４位、２５５８６位、２６０１７位、２６２３４位、２６５５７位、２６６２１位、３０１５５位、３０２６７位、または３０６０７位のいずれかの多型部位
（５ａ）ＦＡＢＰ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：５に記載の塩基配列における２９７０位、３１７４位、３１７５位、４５２５位、４５５６位、４５５８位、４６３６位、４６４６位、４７７４位、５７２６位、５７３６位、５８５２位、７２０３位、７２５３位、７３１０位、８２３９位、８２５１位、８２９６位、８３１２位、８９７４位、１０００１位、１０１０７位、１０４２４位、１０４６４位、１０４９１位、１０７９４位、１１９２２位、１１９５７位、１２８２０位、１６００６位、１６２５６位、１６３０９位、１６３１２位、１６３６０位、１６９２３位、１７８７７位、１８４０８位、１８６４９位、１８７１５位、１８７８３位、または１９８７８位のいずれかの多型部位
（６ａ）ＺＮＦ２６３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：６に記載の塩基配列における８７６位、１５５２位、３２１３位、３２６９位、６４０１位、６４２５位、６６０２位、９５９５位、１０００１位、１０２５２位、１０２６９位、１０２９４位、１１３７７位、１２４６９位、１３６４２位、１３６４３位、１３８７９位、１４３６７位、１６１３３位、１７０８４位、１７６５２位、または１９２６３位のいずれかの多型部位
（７ａ）ＴＮＦＡ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：７に記載の塩基配列における１７５位、２４０１位、２９７３位、４４５２位、４６７１位、４６７２位、５１８４位、５６０９位、５８２０位、５９４１位、５９４２位、５９４２位、６２４５位、６３１５位、６３１６位、６４８７位、６６０３位、６７３０位、６８６７位、６９３１位、６９３５位、６９５８位、７９５３位、８０２２位、８１３３位、８４８２位、８６２８位、８６５０位、８６５６位、８８２７位、８８２９位、８８６５位、８９３８位、８９４５位、８９９４位、９２０５位、９２６９位、９２７５位、１０００１位、１０２０２位、１０３６３位、１０３９９位、１０６８０位、１０７６５位、１０８１６位、１１０９２位、１１５６５位、１１９４１位、１２６６９位、１２９３６位、１２９６３位、１３０２４位、１３１２８位、１３１５４位、１３２８９位、１３５３８位、１３５３９位、１３５９４位、１３６３２位、１３６４６位、１３６８８位、１３８２２位、１３８５５位、１３８６３位、１３９６６位、１３９９３位、１４３７０位、１４５４２位、１４６２５位、１５０３０位、１５０６４位、１５５８１位、１５７３０位、１５９７５位、１６６２６位、１６７１３位、１６７３４位、１７２４０位、１７３６９位、１７６９８位、１７７１１位、または１９３４８位のいずれかの多型部位
（８ａ）ＴＨＢＳ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：８に記載の塩基配列における３０９８位、３１０３位、３１４３位、３１７８位、４５４３位、６９７７位、７１５８位、１０００１位、１０４４５位、１１７２８位、１３０１２位、１３５１４位、１６４０４位、１８０８８位、１８７０８位、１８７７６位、１８９８９位、１９４６５位、１９７６９位、または１９７８６位のいずれかの多型部位
（９ａ）ＡＢＣＡ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：９に記載の塩基配列における９９３位、１７７９位、１８１３位、３４５０位、４２８７位、４３８２位、７２７０位、７５２８位、８３８３位、８７８３位、８７９８位、９２５４位、１０００１位、１０９９９位、１１１２２位、１１７２０位、１２４７３位、１２５６０位、１２５９１位、１２６６４位、１２９１６位、１３４０３位、１３６０５位、１３６９１位、１４４５７位、１４７３７位、１５２７８位、１５３４５位、１５３４５位、１５６３６位、１５６３６位、１５８２４位、１６７９３位、１７０２２位、１７０３７位、１７２８３位、１７５７６位、１８１６７位、１８６１９位、１８６３２位、１８８４２位、１８８４３位、１８９２６位、１９４０４位、１９４０４位、１９８０９位、２０４４７位、２０５１４位、２０５１５位、２１９６３位、２２９８８位、２３０８１位、２３１５７位、２３５７７位、２３９５３位、２４３８９位、２５８１４位、２６１９８位、２６６５５位、２６９０２位、２７３６７位、２７５３５位、２７７０１位、２８１７５位、２８４６７位、２８５６４位、２８６６９位、２８７７４位、２８８３９位、２８９３７位、２８９４４位、２９０３７位、３００９１位、３０６１３位、３０９７５位、３２８０３位、３２８０３位、３３４３５位、３４２６３位、３４２６４位、３４８１２位、３５３０６位、３５３３９位、３５４７３位、３６２１７位、３７５９６位、または３７８５５位のいずれかの多型部位
（１０ａ）ＳＥＲＰＩＮＢ１０遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１０に記載の塩基配列における１４５位、４８４５位、６７５７位、６８５９位、７１５１位、７８２７位、８０６６位、１０００１位、１２８８５位、１３２２７位、１３４３２位、１３５６３位、１５４３９位、１５４４７位、１５７２９位、１５７５９位、１５８８５位、１６０９４位、１６３３０位、または１８５６５位のいずれかの多型部位
（１１ａ）ＡＬＤＯＢ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１１に記載の塩基配列における９９位、３８０位、３９６位、３９９位、７１２位、９３４位、１５３４位、１６１２位、１９４８位、２０７９位、２１０１位、４１６１位、４３１８位、４３１８位、５０９１位、６６２３位、６７１８位、７７９６位、７８５９位、８１９０位、８２０３位、９１０４位、９６２１位、９６４２位、１０００１位、１０４８９位、１１４４３位、１１５９２位、１１５９３位、１１６１６位、１１６１７位、１２２８１位、１２４５７位、１２８０２位、１３９９３位、１４０２６位、１４８００位、１６６５４位、１６８２２位、１７１３３位、１７２３９位、１８２１６位、１８７０９位、１９６１０位、または１９６１３位のいずれかの多型部位
（１２ａ）ＣＴＳＤ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１２に記載の塩基配列における８１３位、８１５位、８４４位、８６１位、９５４位、１０２６位、１１５９位、１２９９位、５４６４位、５６００位、７１４４位、７７０７位、７７２６位、７７３９位、７７７８位、７８０３位、７８４１位、７８４２位、７８８６位、７９３９位、７９６３位、８０５８位、８３３３位、９９４８位、１０００１位、１１９９０位、１２３４２位、１２８０７位、１６２６６位、または１６７７１位のいずれかの多型部位
（１３ａ）ＳＴＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１３に記載の塩基配列における５７位、４６８位、１８８１位、２９５５位、３４６０位、３９７６位、４５３７位、４５３８位、４７２２位、４８６７位、４８７３位、４９４７位、５０２５位、７０４３位、７６７６位、８５８１位、９０３５位、９３４７位、９６８６位、９８８２位、９８９４位、１０００１位、１０００３位、１２１４２位、１３５２０位、１３５５１位、１３７９７位、１４４９２位、１４７５３位、１４９３３位、１５６８４位、１７０２２位、１８２６４位、１８５６８位、１８９５８位、１９１９４位、１９３３０位、１９３５４位、１９３５５位、１９５６７位、１９７８５位、２０５１１位、２０６１２位、２０６２７位、２０９３６位、２０９６１位、２０９７１位、２１２１３位、２１３３６位、２１６０９位、２１９１４位、または２１９８３位のいずれかの多型部位
（１４ａ）ＡＢＣＣ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１４に記載の塩基配列における６９７８位、７３５８位、７３５９位、７６４９位、７７５５位、８０６８位、８２１４位、８２１５位、８３０８位、９６５８位、９７６１位、９８１１位、９８２８位、１０００１位、１００９５位、１０１２５位、１１６５８位、１１６７４位、１１７０８位、１１７６７位、１１８８３位、１１９９８位、１２１０９位、１２３０８位、１３６２０位、１３６６４位、１３９１２位、１４２９７位、１４３０９位、１４３２０位、１４３２３位、１４３６７位、１４３９３位、１４４７５位、１４５９１位、１４７２２位、１５２９４位、１５６０１位、１５６５７位、１６１６０位、１６２３０位、１６３６７位、１６４１７位、１７６９５位、１８２１３位、１８８１５位、１９０９３位、２００８２位、２０２１１位、２０４６１位、２１５４７位、２１９９５位、２２８２５位、または２３９０１位のいずれかの多型部位
（１５ａ）ＳＥＲＰＩＮＡ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１５に記載の塩基配列における９３７位、１１４５位、１８７８位、２３４５位、２５５９位、３２５０位、３５１３位、４４０２位、５７７６位、６６５９位、７４５３位、７９３４位、８０００位、８０４９位、８２０４位、８５４３位、８５５４位、８９３５位、９９２８位、１０００１位、１０２３３位、１０４７２位、１０４７９位、１０５２５位、１０５４４位、１０６２９位、１３３３９位、１３３４８位、１６２８７位、１６３８９位、１６６２６位、または１７８８０位のいずれかの多型部位
（１６ａ）ＲＣＶ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１６に記載の塩基配列における２５位、４９位、４６９位、５５７位、５９２位、２７３０位、２７５４位、５７６１位、６１５４位、６１８３位、８５１０位、８５２９位、８５４２位、８５９０位、９８３８位、１０００１位、１０１３２位、１０１３３位、１０６４９位、１０７０２位、１０８６６位、１２５６０位、１５６３２位、１５８２７位、１５８５３位、１５９９４位、１６６１７位、１６７９０位、１６８４１位、１７０１０位、１７３４８位、１７６５６位、１７６５６位、１７８１６位、１７８２０位、１７８８２位、１７８８２位、１８０６１位、１８１０１位、１９３０４位、または１９４８５位のいずれかの多型部位
（１７ａ）ＴＦＲＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１７に記載の塩基配列における８３６位、１０１９位、２５１１位、４０１６位、４０６８位、４１６９位、４２６９位、４４８１位、５３９８位、５４３６位、５４５０位、５７３０位、５９３７位、６０４４位、６３１３位、６７１３位、６７５９位、６７９４位、６８３３位、７５２８位、８０４７位、８０４８位、８０８０位、９２４８位、９３６１位、９４１７位、９７３４位、１０００１位、１１４６１位、１１８４５位、１１８５２位、１２０８６位、１２５９６位、１２８６４位、１２９３０位、１３９６９位、１３９６９位、１４１１９位、１４２８０位、１６８３３位、または１７０４７位のいずれかの多型部位
（１８ａ）ＭＭＰ１９遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１８に記載の塩基配列における６３２９位、７３２９位、７７１５位、８１３８位、８５０７位、８５５１位、８５７４位、１０００１位、１１１３８位、１１３４３位、１３１５１位、１３８６８位、または１８９２３位のいずれかの多型部位
（１９ａ）ＭＹＬ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１９に記載の塩基配列における３１４７位、３２１３位、４６８６位、７３４３位、７８９２位、１０００１位、１０１２３位、１１２６４位、１１９９２位、１５５３２位、１５６８１位、１５９５７位、１５９８８位、１６６４１位、１８３８６位、１８７８２位、２０３５４位、２１５３１位、２３２４５位、２７７４０位、３０６６９位、３０６７０位、３２０７７位、３２７３２位、３２９０７位、３２９５５位、４２５８９位、４３０２９位、４７３５４位、６４７７７位、７１８６９位、７１９６１位、７４２７４位、７５５５１位、７６３４３位、７６３４４位、７８２７０位、７８３３９位、７８６８９位、７８７４３位、７８８０６位、７８８０７位、７９２１５位、７９３１９位、７９３５７位、７９４６０位、７９４６３位、８００３４位、８００６２位、８００７６位、８０４０１位、８０４７５位、８０５９９位、８０７１５位、８０８６５位、８０９４３位、８０９５２位、８０９７４位、８０９９１位、８１１３０位、８１３７３位、８１４２８位、８１５４３位、８１６８２位、８２４２２位、８２５４５位、８２７９２位、８２９３８位、８３２６５位、８３２６６位、８３３９４位、８３４６３位、８３５００位、８３５７８位、８３６１２位、８３６５０位、８４７９０位、８４９２１位、８４９４５位、８５２１４位、８５３８３位、８５３８４位、８５５５０位、８５９８４位、８６１０９位、８６５３９位、８６５４２位、８７１６９位、８７１７０位、８７３５３位、８７６７１位、８７９５３位、８７９５４位、８８０５７位、８８１７８位、８８２４０位、８８２５８位、８８２７１位、８８２７２位、８８３０７位、８８５１２位、８８６９５位、８８７７８位、８８７９８位、８９１２４位、または９１０８１位のいずれかの多型部位
（２０ａ）ＶＬＤＬＲ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２０に記載の塩基配列における６２９７位、６７３３位、７９９４位、９２４２位、９９５２位、１０００１位、１１８３８位、１２９９５位、１３２２４位、１３２７７位、１８７０２位、１８８９６位、または１９２１４位のいずれかの多型部位
（２１ａ）Ｐ２ＲＸ７遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２１に記載の塩基配列における９９位、６９４位、２７６５位、２７６５位、３１０１位、３１１７位、３１２７位、３３０９位、３３４６位、３８０２位、３９１６位、４２０６位、４４３０位、５９３７位、６７０５位、７３７８位、８３１２位、８３９９位、９９２８位、１０００１位、１００４２位、１０２７７位、１０４４０位、１０４６１位、１０４７９位、１０５２７位、１０５３４位、１０５５０位、１０７０１位、１０９０９位、１１０６６位、１１１９８位、１１５７５位、１１６３１位、１１７６５位、１１９７６位、１２１１９位、１２１５８位、１２２０７位、１２２７２位、１２３６６位、１２４９８位、１２５６３位、１２７９１位、１２８５６位、１２８６０位、１３２０７位、１３４８３位、１３５９０位、１３８６０位、１３８６９位、１３８８０位、１３８８１位、１３９８４位、１４０４８位、１４０５９位、１４２１０位、１４８０６位、１４９２１位、１５３３１位、１５４８８位、１５５５８位、１５６５１位、１５６８６位、１５７９１位、１６０５１位、１６０８２位、１６１３７位、１６４９２位、１６６４９位、１６７８４位、１６７８８位、１７０７２位、１７０７６位、１７３７９位、１７５２７位、１７５９３位、１７６２３位、１７７１９位、１７９９８位、１８００５位、１８０４８位、１８２８８位、１８４１２位、１８５８８位、１８６４０位、１９３４０位、１９５４５位、１９６７６位、または１９６８５位のいずれかの多型部位
（２２ａ）ＤＬＧ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２２に記載の塩基配列における２位、１２１位、１４３８位、１６６９位、２１８０位、２４３６位、２５４３位、２６１９位、３７１５位、３７２７位、５２５６位、５３３５位、５３３９位、５６８８位、５７５２位、６３０４位、６６２７位、７１９５位、７３４２位、７６０３位、８２７３位、８９０５位、８９８８位、９０１３位、９０５７位、１０００１位、１０３７２位、１０４５３位、１０４６８位、１０４６８位、１０５１２位、１１０３８位、１１０５５位、１１５９７位、１１６８１位、１１７１９位、１１９２２位、１２０３４位、１２０４９位、１２１７６位、１２５５４位、１３４８１位、１３８６４位、１４３９０位、１５０４４位、１５５９８位、１６１７６位、１６８７０位、１７４７５位、１７７４９位、１８０７７位、１８５８５位、１８６１９位、１８６７７位、１９２２２位、１９７３１位、１９７５０位、２０７５８位、２０９６２位、２１０７３位、２１６８０位、２１８６７位、２２３８５位、２２８７４位、２３１９１位、２４０１６位、２４５２０位、２４９９３位、２５４６０位、２６１５５位、２６８０４位、２６９８０位、２７９８８位、２９２３４位、２９４６９位、３０２５４位、３０７４２位、３０７４２位、３０７５６位、３０７５６位、３１６７１位、３１８７４位、３１９４０位、３２６２６位、３３３９３位、３３５８１位、３４２４０位、３５２９４位、３５６６６位、３６５８７位、３６６５３位、３６９４１位、３７４０８位、３７５８３位、３７６１２位、３８４６２位、３８４７０位、３８４８５位、３９０６５位、３９１４６位、４０６１２位、４０６４１位、４１２４２位、４２２１７位、４２５３９位、４５９３４位、４６０３３位、または４６８９５位のいずれかの多型部位
（２３ａ）ＬＤＬＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２３に記載の塩基配列における１５８０位、１７５４位、１８８２位、３０６２位、３５０７位、４８００位、４９０５位、５５６９位、５６０８位、５６４３位、５６６５位、５６９３位、５８６４位、５８８４位、５９８１位、６１６９位、６８０８位、６８５８位、７１９４位、７２８３位、７７２８位、７８３５位、８０４９位、８０４９位、８２６５位、９２０９位、９４４５位、９６４６位、９６４７位、９７１６位、９７７５位、１０００１位、１００２９位、１００５４位、１０１９３位、１０２０８位、１０８２６位、１０９０８位、１０９９７位、１１３３６位、１１７５４位、１１８９５位、１２０３１位、１２３２７位、１２４６４位、１２５６１位、１４０５１位、１５３６６位、１５７７８位、１７７２３位、１７７７２位、１７７８３位、１７８１１位、１７８１５位、１７８６２位、１７９００位、１７９６２位、１８１８４位、１８１８９位、１８２９７位、１８４１１位、１８５１２位、１８５６３位、１９１２５位、１９１５２位、２００９２位、２１２２７位、２１５０６位、２１６１７位、２１８５５位、２３０９０位、２５７００位、２６４６８位、２６６３８位、２７６８２位、２７７５５位、２７８８８位、２７９７３位、２７９９５位、２８１２３位、２８１２７位、２８１４４位、２８１５３位、または２８２７１位のいずれかの多型部位
（２４ａ）ＡＱＰ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２４に記載の塩基配列における６７２２位、９６４３位、９９３４位、１０００１位、１３７８２位、１４６２９位、１５３７６位、１５８０８位、または１６０８２位のいずれかの多型部位
（２５ａ）ＬＡＲＧＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２５に記載の塩基配列における１８１６位、３１１６位、３１１７位、３１１８位、３１５４位、３１５７位、３１９１位、３１９７位、３２０５位、３２９１位、３６２２位、３６５２位、３７９２位、４４８８位、５０７６位、５４７４位、５４８１位、５４９２位、５５０３位、５６５４位、５６７２位、６５６３位、６８４３位、６９０８位、７１１９位、７５３８位、７５３８位、７７２１位、８４０３位、８６２６位、１０００１位、１２６３６位、１３１０３位、１４７９４位、１５３７０位、１５４４８位、１５５９９位、１５７６４位、１６０４５位、１６２６１位、１６２７７位、１６８６１位、１７３６０位、１７６１１位、１７７１４位、１７８０９位、１８２２１位、１８２２１位、１８３７６位、１８５３６位、１９０６３位、２２９４２位、２３４０４位、２３６１２位、２５１２５位、２５１３４位、２５６６２位、２５７０５位、２５７５１位、２７４０７位、２８１６６位、２８５６７位、３０３０２位、３０３１６位、３０３５３位、３０３５４位、３０３９１位、３３５９４位、３３６５０位、３３７５４位、３６６９０位、３７７５３位、３７８９５位、３７９００位、３９１０４位、３９７４５位、４１４０４位、４１６４６位、４１７１１位、４２０２０位、４２０８０位、４２９７４位、４３０５２位、４３２４０位、４３６２１位、４４７３４位、４４９５８位、４６９２０位、４７４３１位、４７５７３位、４８１０１位、４８１２７位、４８３０６位、４８８０７位、４９０４９位、４９８４８位、４９８６０位、４９９０３位、５０１１４位、５０２２３位、５２０５７位、５２５４３位、５２６９３位、または５３０６１位のいずれかの多型部位
（２６ａ）ＢＬＺＦ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２６に記載の塩基配列における１２８３位、４２８８位、７５２０位、７５９９位、７７４０位、８７４５位、９０５０位、９８６２位、１０００１位、１０１２４位、１０１６８位、１０３３９位、１１０１７位、１３７３６位、１３７４７位、１８３５４位、２００３１位、２０１７２位、２２０９４位、２４０８２位、２４３０５位、２４４７８位、２４５５２位、２５２７３位、２５３５１位、２９０９６位、２９１００位、２９７６４位のいずれかの多型部位
（２７ａ）ＲＧＳ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２７に記載の塩基配列における７３３位、８４８位、１０３９位、１３５１位、１５６９位、１８３１位、２０７８位、２７０７位、２７９４位、２９０４位、２９９５位、３１２２位、３５２０位、４３５１位、４８２４位、５１８９位、５２０５位、５２０６位、５５２５位、５７７９位、６４８７位、７７１２位、１０００１位、１０２０８位、１０２３５位、１０８６８位、１１０９２位、１１６１７位、１１６６６位、１２２１６位、１２３２６位、１２７４１位、１３３９６位、１３８９０位、１４７０２位、１５８２３位、１６７０５位、または１９０８６位のいずれかの多型部位
（２８ａ）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２８に記載の塩基配列における３４５５位、７８２６位、８７１５位、８７８３位、９０４２位、９３９０位、９４６２位、９７０９位、１０００１位、１０６３５位、１０７４８位、１２７９４位、１２８４９位、１２９１５位、１３１６９位、１７０２０位、１８５８６位、２１１２３位、または２１１２８位のいずれかの多型部位、
または、
（２８ａ−２）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：３０に記載の塩基配列における９５９８位、１０００１位、１０３０６位、１１１８１位、１１４８０位、１３５９３位、１３７２１位、１３７９２位、１５３１４位、１５６２３位、１５７８２位、１６７０８位、１６７４８位、または１９６３０位のいずれかの多型部位
（２９ａ）ＬＥＰ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２９に記載の塩基配列における２４４位、５７５位、６７５位、１４８７位、１６４４位、２３４３位、２７１５位、２７６３位、３０９８位、３５８９位、３８８０位、５７８７位、５８６５位、６４３６位、７３９２位、７９９５位、８１６１位、８５４４位、９２３０位、９７９５位、１０００１位、１０１９９位、１０５０３位、１１９７５位、１４９８１位、１５６８１位、または１９３００位のいずれかの多型部位
より詳細には、以下の表２〜３１に示す。
なお、ゲノムにおけるＤＮＡは、通常、互いに相補的な二本鎖ＤＮＡ構造を有している。従って、本明細書においては、便宜的に一方の鎖におけるＤＮＡ配列を示した場合であっても、当然の如く、当該配列（塩基）に相補的な配列も開示したものと解釈される。当業者にとって、一方のＤＮＡ配列（塩基）が判れば、該配列（塩基）に相補的な配列（塩基）は自明である。
〔ＭＥＴ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

尚、表中「Ｂａｓｅ１」および「Ｂａｓｅ２」のカラムにて掲載した塩基種は、当該部位の相補鎖側を記載しているものもあるが、当業者においては、掲載された多型バンクのアクセッション番号等を基に、当該部位についての塩基種の情報を、適宜取得することが可能である。
〔ＮＰＨＳ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＳＬＣ１Ａ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＳＥＲＰＩＮＢ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＦＡＢＰ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＺＮＦ２６３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＴＮＦＡ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＴＨＢＳ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＡＢＣＡ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

上記表１０−２中、＊印はアッセイ済みＳＮＰ、＊＊印は単ＳＮＰ有意であるものを表し、二重網掛けの範囲は連鎖不平衡ブロック、点網掛けの範囲は連鎖不平衡が予測される領域を表す。
〔ＳＥＲＰＩＮＢ１０遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＡＬＤＯＢ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＣＴＳＤ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＳＴＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＡＢＣＣ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＳＥＲＰＩＮＡ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＲＣＶ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＴＦＲＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＭＭＰ１９遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＭＹＬ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＶＬＤＬＲ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔Ｐ２ＲＸ７遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＤＬＧ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＬＤＬＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＡＱＰ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＬＡＲＧＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＢＬＺＦ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＲＧＳ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

上記表中に記載の「配列番号中の通しＮｏ．」は、配列番号：２８の通し番号に相当する。
〔ＬＥＰ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

〔ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位〕

上記表中に記載の「配列番号中の通しＮｏ．」は、配列番号：３０の通し番号に相当する。
尚、上記表中、ＳＮＰＩＤ＊欄の記載内容は、先頭にｒｓが付くものはｄｂＳＮＰデータベースの登録ＩＤ、ＩＭＳ−ＪＳＴが付くものはＪＳＮＰデータベースの登録ＩＤである。また、ｄｂＳＮＰデータベースはウェブサイト（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ＳＮＰ／ｉｎｄｅｘ．ｈｔｍｌ）に、ＪＳＮＰデータベースはウェブサイト（ｈｔｔｐ：／／ｓｎｐ．ｉｍｓ．ｕ−ｔｏｋｙｏ．ａｃ．ｊｐ）に公開されており、ＳＮＰＩＤ＊欄に記載された登録ＩＤ番号を用いてウェブサイト上で検索することにより、各塩基配列におけるＳＮＰｓの詳細な情報（例えば、染色体上の位置、多型部位の塩基の種類、前後の配列等）が入手できる。これらの情報を用いた場合、当業者においては、本発明に記載する検査を容易に行うことができる。上記表中に示した多型部位の塩基種は配列表に示した配列に対して相補鎖側にある塩基種を示している場合があるが、ｄｂＳＮＰおよびＪＳＮＰデータベースにて公開される前後配列を用いれば異同を確認する事は当業者にとって容易であり、検査を行うにあたってはプラス鎖とマイナス鎖のどちらを調べても必然的にもう一方の結果を決定する事ができる。
また、当業者においては、通常、本明細書において開示された多型に付与された登録ＩＤ番号、例えばｄｂＳＮＰデータベースにおけるｒｓ番号、ＪＳＮＰデータベースにおけるＩＭＳ−ＪＳＴ番号、ｓｓｊ番号によって、本発明の多型部位について実際のゲノム上の位置および前後の配列等を容易に知ることができる。これによって、知ることができない場合であっても、当業者においては、配列番号：１〜３０で示される塩基配列および多型部位等に関する情報から、適宜、該多型部位に相当する実際のゲノム上の位置を知ることは容易である。例えば、公開されているゲノムデータベース等と照会することにより、本発明の多型部位のゲノム上の位置を知ることができる。即ち、配列表に掲げた塩基配列とゲノム上の実際の塩基配列との間に若干の塩基配列の相違がみられた場合であっても、配列表に掲げた塩基配列を基にゲノム配列と相同検索等を行うことにより、本発明の多型部位について、実際のゲノム上の位置を正確に知ることが可能である。また、ゲノム上の位置が特定できない場合でも、本明細書に記載の配列表および多型部位の情報から本発明に記載する検査を行うことは容易である。
本発明の多型部位の前後の（５’側および３’側の）配列の一例として、一部の多型部位について、その前後の配列を以下の表３２および表３３に例示する。

本発明の検査方法においては、以下（１ｂ）〜（２９ｂ）のいずれかに記載の多型部位について塩基種の決定を行うことが好ましい。
（１ｂ）ＭＥＴ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１に記載の塩基配列における１４２５８位、または１９７６８位の部位
（２ｂ）ＮＰＨＳ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２に記載の塩基配列における１０００１位、または１７８４８位の部位
（３ｂ）ＳＬＣ１Ａ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：３に記載の塩基配列における１０００１位の部位
（４ｂ）ＳＥＲＰＩＮＢ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：４に記載の塩基配列における１０００１位の部位
（５ｂ）ＦＡＢＰ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：５に記載の塩基配列における１０００１位の部位
（６ｂ）ＺＮＦ２６３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：６に記載の塩基配列における１０００１位の部位
（７ｂ）ＴＮＦＡ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：７に記載の塩基配列における１０００１位の部位
（８ｂ）ＴＨＢＳ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：８に記載の塩基配列における１０００１位の部位
（９ｂ）ＡＢＣＡ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：９に記載の塩基配列における１７２８３位の部位
（１０ｂ）ＳＥＲＰＩＮＢ１０遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１０に記載の塩基配列における１０００１位の部位
（１１ｂ）ＡＬＤＯＢ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１１に記載の塩基配列における１０００１位の部位
（１２ｂ）ＣＴＳＤ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１２に記載の塩基配列における１１９９０位、または１６７７１位の部位
（１３ｂ）ＳＴＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１３に記載の塩基配列における１８９５８位、または２１２１３位の部位
（１４ｂ）ＡＢＣＣ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１４に記載の塩基配列における１４４７５位の部位
（１５ｂ）ＳＥＲＰＩＮＡ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１５に記載の塩基配列における１０００１位の部位
（１６ｂ）ＲＣＶ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１６に記載の塩基配列における１０００１位の部位
（１７ｂ）ＴＦＲＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１７に記載の塩基配列における１０００１位の部位
（１８ｂ）ＭＭＰ１９遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１８に記載の塩基配列における１０００１位の部位
（１９ｂ）ＭＹＬ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１９に記載の塩基配列における１０００１位の部位
（２０ｂ）ＶＬＤＬＲ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２０に記載の塩基配列における１０００１位の部位
（２１ｂ）Ｐ２ＲＸ７遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２１に記載の塩基配列における１０００１位の部位
（２２ｂ）ＤＬＧ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２２に記載の塩基配列における１０００１位、または３７５８３位の部位
（２３ｂ）ＬＤＬＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２３に記載の塩基配列における１８５６３位の部位
（２４ｂ）ＡＱＰ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２４に記載の塩基配列における１０００１位の部位
（２５ｂ）ＬＡＲＧＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２５に記載の塩基配列における１０００１位、１３１０３位、または１５４４８位の部位
（２６ｂ）ＢＬＺＦ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２６に記載の塩基配列における２００３１位の部位
（２７ｂ）ＲＧＳ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２７に記載の塩基配列における１０００１位の部位
（２８ｂ）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２８に記載の塩基配列における１７０２０位の部位
（２８ｂ−２）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：３０に記載の塩基配列における１０００１位の部位
（２９ｂ）ＬＥＰ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２９に記載の塩基配列における１０５０３位の部位
また本発明の好ましい態様においては、上記（１ｂ）〜（２９ｂ）に記載の多型部位における塩基種がそれぞれ以下の（１ｃ）〜（２９ｃ）である場合に、２型糖尿病を罹患しているものと判定される。２型糖尿病に罹患していない被検者であっても２型糖尿病を発症する疑いがある、あるいは２型糖尿病に罹患し易いものと判定される。
（１ｃ）ＭＥＴ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１に記載の塩基配列における１４２５８位の塩基種がＴ、または１９７６８位の塩基種がＴ
（２ｃ）ＮＰＨＳ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２に記載の塩基配列における１０００１位の塩基種がＡ、または１７８４８位の塩基種がＡ
（３ｃ）ＳＬＣ１Ａ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：３に記載の塩基配列における１０００１位の塩基種がＧ
（４ｃ）ＳＥＲＰＩＮＢ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：４に記載の塩基配列における２４５７８位の塩基種がＡ
（５ｃ）ＦＡＢＰ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：５に記載の塩基配列における１０００１位の塩基種がＡ
（６ｃ）ＺＮＦ２６３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：６に記載の塩基配列における１０００１位の塩基種がＧ
（７ｃ）ＴＮＦＡ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：７に記載の塩基配列における１０００１位の塩基種がＡ
（８ｃ）ＴＨＢＳ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：８に記載の塩基配列における１０００１位の塩基種がＣ
（９ｃ）ＡＢＣＡ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：９に記載の塩基配列における１７２８３位の塩基種がＴ
（１０ｃ）ＳＥＲＰＩＮＢ１０遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１０に記載の塩基配列における１０００１位の塩基種がＧ
（１１ｃ）ＡＬＤＯＢ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１１に記載の塩基配列における１０００１位の塩基種がＴ
（１２ｃ）ＣＴＳＤ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１２に記載の塩基配列における１１９９０位の塩基種がＡ、または１６７７１位の塩基種がＣ
（１３ｃ）ＳＴＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１３に記載の塩基配列における１８９５８位の塩基種がＧ、または２１２１３位の塩基種がＣ
（１４ｃ）ＡＢＣＣ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１４に記載の塩基配列における１４４７５位の塩基種がＡ
（１５ｃ）ＳＥＲＰＩＮＡ３遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１５に記載の塩基配列における１０００１位の塩基種がＡ
（１６ｃ）ＲＣＶ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１６に記載の塩基配列における１０００１位の塩基種がＴ
（１７ｃ）ＴＦＲＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１７に記載の塩基配列における１０００１位の塩基種がＧ
（１８ｃ）ＭＭＰ１９遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１８に記載の塩基配列における１０００１位の塩基種がＡ
（１９ｃ）ＭＹＬ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１９に記載の塩基配列における１０００１位の塩基種がＴ
（２０ｃ）ＶＬＤＬＲ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２０に記載の塩基配列における１０００１位の塩基種がＴ
（２１ｃ）Ｐ２ＲＸ７遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２１に記載の塩基配列における１０００１位の塩基種がＣ
（２２ｃ）ＤＬＧ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２２に記載の塩基配列における１０００１位の塩基種がＧ、または３７５８３位の塩基種がＣ
（２３ｃ）ＬＤＬＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２３に記載の塩基配列における１８５６３位の塩基種がＧ
（２４ｃ）ＡＱＰ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２４に記載の塩基配列における１０００１位の塩基種がＧ
（２５ｃ）ＬＡＲＧＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２５に記載の塩基配列における１０００１位の塩基種がＴ、１３１０３位の塩基種がＧ、または１５４４８位の塩基種がＧ
（２６ｃ）ＢＬＺＦ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２６に記載の塩基配列における２００３１位の塩基種がＣ
（２７ｃ）ＲＧＳ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２７に記載の塩基配列における１０００１位の塩基種がＧ
（２８ｃ）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２８に記載の塩基配列における１７０２０位の塩基種がＧ
（２８ｃ−２）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：３０に記載の塩基配列における１０００１位の塩基種がＧ
（２９ｃ）ＬＥＰ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２９に記載の塩基配列における１０５０３位の塩基種がＧ
本発明においては、上記多型部位以外であっても、該多型部位とその周辺のＤＮＡ領域は強く連鎖しているものと考えられることから、上記多型部位の近傍の多型部位について塩基種を決定することによっても、ＤＭの検査が可能である。即ち、多型部位の塩基種が上記（１ｃ）〜（２９ｃ）に記載の塩基種であるようなＤＭ患者を含むヒトの小集団について、この「近傍の多型部位」（例えば、表２〜３１に記載の多型部位）における塩基種を予め決定する。次いで、この「近傍の多型部位」について被検者における塩基種を決定し、予め決定された前記塩基種と比較することにより、２型糖尿病の検査を行うことができる。予め決定された塩基種と同一の塩基種である場合に、被検者は２型糖尿病を罹患し易い／しにくいと判定される。２型糖尿病にすでに罹患している被検者の場合には２型糖尿病を発症した原因や病型を判定することができ、治療方針の決定等に利用することができる。
以下、ＭＥＴ遺伝子の場合を例にとって説明する。まず、例えば、ＭＥＴ遺伝子上の配列番号：１に記載の塩基配列における１０００１位の多型部位の塩基種がＧであるＤＭ患者を含むヒトの小集団について、近傍の多型部位、例えば８２１４位の多型部位の塩基種を決定する。この部位の塩基種がＤＭ患者群においてＴであったとすると、被検者について８２１４位の多型部位の塩基種を調べ、この部位の塩基種が同様にＴであった場合には、被検者は２型糖尿病に罹患し易いものと判定される。
以上のように、本発明によりＤＭ関連遺伝子上の領域が明らかになった後には、当業者に過度の負担を強いる事なくＤＭに関する検査を行うことができる。
また、ヒトゲノムの解析が進み全塩基配列やＳＮＰ、マイクロサテライト、ＶＮＴＲ、ＲＦＬＰｓなどの多型情報も充実してきた。ゲノムの塩基配列について詳細が明らかになりつつある現在、最大の関心事は遺伝子あるいは特定の配列と機能（疾患・薬剤応答性などの表現型）との関連を解析する事である。特に糖尿病、高血圧等の生活習慣病では、複数の疾患感受性遺伝子と環境因子により発症するため解析は非常に困難である。これを解決するための有力な手法の一つが連鎖不平衡を用いた遺伝統計学的解析である。
ヒトの染色体は２本１組で存在し、それぞれ父親と母親から由来している。ハプロタイプとは、その一方に関する個体の遺伝子型の組み合わせをいい、それぞれ父母由来の１本の染色体上に遺伝子座がどのように並んでいるかを示すものである。染色体を父母から１本ずつ受け継ぐので、配偶子形成の際に組み換えが起きないとすれば１本の染色体上にのっている遺伝子は必ず一緒に子に伝えられる、すなわち連鎖する事になる。しかし、実際は減数分裂の際に組み換えが起きるため、１本の染色体上にのっている遺伝子であっても必ずしも連鎖しているわけではない。しかし逆に、遺伝的組み換えが起きた場合であっても同一染色体上の距離が近い遺伝子座は強く連鎖する。
このような現象を集団において観察し、アリルの非独立が認められる事を連鎖不平衡という。例えば、３つの遺伝子座を観察した場合、これらの間に連鎖不平衡がないとすると存在するハプロタイプは２^３通りと予測され、それぞれの頻度は各遺伝子座の頻度から予測される値となるが、連鎖不平衡がある場合には２^３通りより少ないハプロタイプしか存在せず、その頻度も予測と異なる値を示す結果となる。
近年、ハプロタイプが連鎖不平衡解析に有用である事が示されており（Ｇｅｎｅｔｉｃ Ｅｐｉｄｅｍｉｏｌｏｇｙ ２３：２２１−２３３）研究が行われているが、ゲノム上には組換えが起きやすい部位と起きにくい部位があり、１つの領域として先祖から子孫へと伝えられる部分（ハプロタイプ）は人種を越えて共通性がある事が明らかになっている（Ｓｃｉｅｎｃｅ ２２６，５５７６：２２２５−２２２９）。
つまり、２型糖尿病と関連するハプロタイプが見出されれば、該ハプロタイプを検出することにより、２型糖尿病の検査が可能となる。本発明者らは、鋭意研究により、２型糖尿病と関連するハプロタイプを見出すことに成功した。
従って、本発明は、以下の（１）〜（１４）のいずれかに記載の遺伝子および／または該遺伝子の近傍ＤＮＡ領域に存在する２型糖尿病と関連するハプロタイプを検出することを特徴とする、２型糖尿病の検査方法を提供する。
（１）ＭＥＴ、（２）ＮＰＨＳ１、（３）ＳＥＲＰＩＮＢ２、（４）ＡＢＣＡ１、（５）ＣＴＳＤ、（６）ＳＴＥ、（７）ＡＢＣＣ８、（８）ＭＹＬ２、（９）ＤＬＧ５、（１０）ＬＤＬＣ、（１１）ＬＡＲＧＥ、（１２）ＢＬＺＦ１、（１３）ＦＲＡＰ１、（１４）ＬＥＰ
本方法においては、被検者について、「２型糖尿病と関連するハプロタイプ」が検出された場合に、２型糖尿病を罹患し易い／しにくいものと判定される。２型糖尿病にすでに罹患している被検者の場合には、発症した原因および病型を判定することができ、治療方針の決定等に利用するとができる。
上記「２型糖尿病と関連するハプロタイプ」とは、具体的には、以下のようなハプロタイプを示すことができる。なお、「感受性（ｓｕｓｃｅｐｔｉｂｌｅ）」とは２型糖尿病に罹患し易い、「抵抗性（ｐｒｏｔｅｃｔｉｖｅ）」とは２型糖尿病に罹患しにくいことを指す。
（１’）ＭＥＴ遺伝子上の多型部位であって、配列番号：１に記載の塩基配列における１０００１位、１４２５８位、および１９７６８位の多型部位の塩基種が、それぞれＧ、Ｔ、およびＴであるハプロタイプ、
ＭＥＴ遺伝子上の多型部位であって、配列番号：１に記載の塩基配列における１０００１位、１４２５８位、および１９７６８位の多型部位の塩基種が、それぞれ、Ｇ、Ｃ、およびＣであるハプロタイプ、または、
ＭＥＴ遺伝子上の多型部位であって、配列番号：１に記載の塩基配列における１９７６８位および２４２０９位の多型部位の塩基種が、それぞれＴおよびＴであるハプロタイプ
より詳しくは、ＭＥＴ遺伝子上の多型部位であって、配列番号：１に記載の塩基配列における１０００１位、１４２５８位、および１９７６８位の多型部位の塩基種が、それぞれＧ、Ｔ、およびＴであるハプロタイプは２型糖尿病感受性であり、該塩基種がそれぞれＧ、Ｃ、およびＣであるハプロタイプは２型糖尿病抵抗性である。また、ＭＥＴ遺伝子上の多型部位であって、配列番号：１に記載の塩基配列における１９７６８位および２４２０９位の多型部位の塩基種が、それぞれＴおよびＴであるハプロタイプは２型糖尿病感受性である。
（２’）ＮＰＨＳ１遺伝子上の多型部位であって、配列番号：２に記載の塩基配列における１０００１位、１６１９８位、および１７８４８位の多型部位の塩基種が、それぞれＡ、Ｃ、およびＡであるハプロタイプ、
ＮＰＨＳ１遺伝子上の多型部位であって、配列番号：２に記載の塩基配列における１０００１位、１６１９８位、および１７８４８位の多型部位の塩基種が、それぞれＧ、Ｇ、およびＧであるハプロタイプ、または、
ＮＰＨＳ１遺伝子上の多型部位であって、配列番号：２に記載の塩基配列における１０００１位、１６１９８位、および１７８４８位の多型部位の塩基種が、それぞれＧ、Ｃ、およびＧであるハプロタイプ
より詳しくは、ＮＰＨＳ１遺伝子上の多型部位であって、配列番号：２に記載の塩基配列における１０００１位、１６１９８位、および１７８４８位の多型部位の塩基種が、それぞれＧ、Ｇ、およびＧであるハプロタイプは２型糖尿病感受性であり、該塩基種がＧ、Ｃ、およびＧであるハプロタイプは２型糖尿病感受性であり、該塩基種がＡ、Ｃ、およびＡであるハプロタイプは２型糖尿病抵抗性である。
（３’）ＳＥＲＰＩＮＢ２遺伝子上の多型部位であって、配列番号：４に記載の塩基配列における１０００１位、および２４５７８位の多型部位の塩基種が、それぞれＣおよびＡであるハプロタイプ、または
ＳＥＲＰＩＮＢ２遺伝子上の多型部位であって、配列番号：４に記載の塩基配列における１０００１位、および２４５７８位の多型部位の塩基種が、それぞれＴおよびＴであるハプロタイプ
より詳しくは、ＳＥＲＰＩＮＢ２遺伝子上の多型部位であって、配列番号：４に記載の塩基配列における１０００１位、および２４５７８位の多型部位の塩基種が、それぞれＣおよびＡであるハプロタイプは２型糖尿病感受性であり、該塩基種がＴおよびＴであるハプロタイプは２型糖尿病抵抗性である。
（４’）ＡＢＣＡ１遺伝子上の多型部位であって、配列番号：９に記載の塩基配列における１０００１位、１５８２４位、１７２８３位、２０５１５位、および２８５６４位の多型部位の塩基種が、それぞれＣ、Ａ、Ｃ、Ｔ、およびＧであるハプロタイプ、または
ＡＢＣＡ１遺伝子上の多型部位であって、配列番号：７１に記載の塩基配列における１０００１位および１１０００位の多型部位の塩基種が、それぞれＧおよびＣであるハプロタイプ
より詳しくは、ＡＢＣＡ１遺伝子上の多型部位であって、配列番号：９に記載の塩基配列における１０００１位、１５８２４位、１７２８３位、２０５１５位、および２８５６４位の多型部位の塩基種が、それぞれＣ、Ａ、Ｃ、Ｔ、およびＧであるハプロタイプは２型糖尿病抵抗性であり、ＡＢＣＡ１遺伝子上の多型部位であって、配列番号：７１に記載の塩基配列における１０００１位および１１０００位の多型部位の塩基種が、それぞれＧおよびＣであるハプロタイプは２型糖尿病感受性である。
（５’）ＣＴＳＤ遺伝子上の多型部位であって、配列番号：１２に記載の塩基配列における１０００１位、１１９９０位、および１６７７１位の多型部位の塩基種が、それぞれＣ、Ｇ、およびＴであるハプロタイプ
より詳しくは、ＣＴＳＤ遺伝子上の多型部位であって、配列番号：１２に記載の塩基配列における１０００１位、１１９９０位、および１６７７１位の多型部位の塩基種が、それぞれＣ、Ｇ、およびＴであるハプロタイプは２型糖尿病抵抗性である。
（６’）ＳＴＥ遺伝子上の多型部位であって、配列番号：１３に記載の塩基配列における１０００１位、１８９５８位、１９３５４位、１９５６７位、および２０９７１位の多型部位の塩基種が、それぞれＡ、Ｇ、Ｔ、Ｔ、およびＣであるハプロタイプ、または
ＳＴＥ遺伝子上の多型部位であって、配列番号：１３に記載の塩基配列における２０９７１位、および２１２１３位の多型部位の塩基種が、それぞれＣおよびＣであるハプロタイプ
より詳しくは、ＳＴＥ遺伝子上の多型部位であって、配列番号：１３に記載の塩基配列における１０００１位、１８９５８位、１９３５４位、１９５６７位、および２０９７１位の多型部位の塩基種が、それぞれＡ、Ｇ、Ｔ、Ｔ、およびＣであるハプロタイプは２型糖尿病感受性である。また、ＳＴＥ遺伝子上の多型部位であって、配列番号：１３に記載の塩基配列における２０９７１位、および２１２１３位の多型部位の塩基種が、それぞれＣおよびＣであるハプロタイプは２型糖尿病感受性である。
（７’）ＡＢＣＣ８遺伝子上の多型部位であって、配列番号：１４に記載の塩基配列における１４４７５位の多型部位の塩基種がＡであるハプロタイプ
より詳しくは、ＡＢＣＣ８遺伝子上の多型部位であって、配列番号：１４に記載の塩基配列における１４４７５位の多型部位の塩基種がＡであるハプロタイプは２型糖尿病感受性である。
（８’）ＭＹＬ２遺伝子上の多型部位であって、配列番号：１９に記載の塩基配列における１０００１位、１８３８６位、４２５８９位、および８３５００位の多型部位の塩基種が、それぞれＣ、Ｔ、Ｔ、およびＣであるハプロタイプ
より詳しくは、ＭＹＬ２遺伝子上の多型部位であって、配列番号：１９に記載の塩基配列における１０００１位、１８３８６位、４２５８９位、および８３５００位の多型部位の塩基種が、それぞれＣ、Ｔ、Ｔ、およびＣであるハプロタイプは２型糖尿病感受性である。
（９’）ＤＬＧ５遺伝子上の多型部位であって、配列番号：２２に記載の塩基配列における１０００１位、２４０１６位、２９４６９位、および３７５８３位の多型部位の塩基種が、それぞれＧ、Ｃ、Ｇ、およびＣであるハプロタイプ
ＤＬＧ５遺伝子上の多型部位であって、配列番号：２２に記載の塩基配列における２４０１６位、２９４６９位、および３７５８３位の多型部位の塩基種が、それぞれＣ、Ｇ、およびＣであるハプロタイプ、または
ＤＬＧ５遺伝子上の多型部位であって、配列番号：２２に記載の塩基配列における２４０１６位、２９４６９位、および３７５８３位の多型部位の塩基種が、それぞれＣ、Ｇ、およびＴであるハプロタイプ
より詳しくは、ＤＬＧ５遺伝子上の多型部位であって、配列番号：２２に記載の塩基配列における２４０１６位、２９４６９位、および３７５８３位の多型部位の塩基種が、それぞれＣ、Ｇ、およびＣであるハプロタイプは２型糖尿病感受性であり、該塩基種がそれぞれＣ、Ｇ、およびＴであるハプロタイプは２型糖尿病抵抗性である。
（１０’）ＬＤＬＣ遺伝子上の多型部位であって、配列番号：２３に記載の塩基配列における１０００１位、および１８５６３位の多型部位の塩基種が、それぞれＧおよびＡであるハプロタイプ、または
ＬＤＬＣ遺伝子上の多型部位であって、配列番号：２３に記載の塩基配列における１０００１位、および１８５６３位の多型部位の塩基種が、それぞれＧおよびＧであるハプロタイプ
より詳しくは、ＬＤＬＣ遺伝子上の多型部位であって、配列番号：２３に記載の塩基配列における１０００１位、および１８５６３位の多型部位の塩基種が、それぞれＧおよびＧであるハプロタイプは２型糖尿病感受性であり、該塩基種がそれぞれＧおよびＡであるハプロタイプは２型糖尿病抵抗性である。
（１１’）ＬＡＲＧＥ遺伝子上の多型部位であって、配列番号：２５に記載の塩基配列における１０００１位、１３１０３位、１５４４８位、３６６９０位、および４３２４０位の多型部位の塩基種が、それぞれＴ、Ｇ、Ｇ、Ｔ、およびＡであるハプロタイプ
より詳しくは、ＬＡＲＧＥ遺伝子上の多型部位であって、配列番号：２５に記載の塩基配列における１０００１位、１３１０３位、１５４４８位、３６６９０位、および４３２４０位の多型部位の塩基種が、それぞれＴ、Ｇ、Ｇ、Ｔ、およびＡであるハプロタイプは２型糖尿病感受性である。
（１２’）ＢＬＺＦ１遺伝子上の多型部位であって、配列番号：２６に記載の塩基配列における１０００１位および２００３１位の多型部位の塩基種が、それぞれＧおよびＴであるハプロタイプ
より詳しくは、ＢＬＺＦ１遺伝子上の多型部位であって、配列番号：２６に記載の塩基配列における１０００１位および２００３１位の多型部位の塩基種が、それぞれＧおよびＴであるハプロタイプは２型糖尿病抵抗性である。
（１３’）ＦＲＡＰ１遺伝子上の多型部位であって、配列番号：２８に記載の塩基配列における１７０２０位の多型部位の塩基種がＧであるハプロタイプ
より詳しくは、ＦＲＡＰ１遺伝子上の多型部位であって、配列番号：２８に記載の塩基配列における１７０２０位の多型部位の塩基種がＧであるハプロタイプは２型糖尿病感受性である。
（１４’）ＬＥＰ遺伝子上の多型部位であって、配列番号：２９に記載の塩基配列における１０００１位および１０５０３位の多型部位の塩基種が、それぞれＧおよびＡであるハプロタイプ
より詳しくは、ＬＥＰ遺伝子上の多型部位であって、配列番号：２９に記載の塩基配列における１０００１位および１０５０３位の多型部位の塩基種が、それぞれＧおよびＡであるハプロタイプは２型糖尿病感受性である。
上記方法の好ましい態様においては、以下の工程（ａ）および（ｂ）を含む、２型糖尿病の検査方法である。
（ａ） 被検者における上記（１）〜（１４）のいずれかに記載の遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の多型部位について、塩基種を決定する工程、
（ｂ） 工程（ａ）により決定された塩基種を、上記（１’）〜（１４’）のいずれかに記載のハプロタイプを示す前記遺伝子もしくは該遺伝子の近傍ＤＮＡ領域における多型部位の塩基種と、比較する工程
上記工程（ａ）における多型部位としては、好ましくは、以下の多型部位を示すことができる。
（１）ＭＥＴ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１に記載の塩基配列における７６５位、１３８３位、１８９０位、２０９９位、２８１４位、３２９３位、３７５２位、３７７４位、３９２７位、４６９０位、７４１９位、８２１４位、１０００１位、１０３１８位、１３７６６位、１３９８９位、１４２５８位、１５４２７位、１６２７５位、１６５９７位、１６９３２位、１９７６８位、１９８３５位、２００３２位、２０２４０位、２０５３７位、２０８８５位、２１５４６位、２４２０９位、２４２７１位、２４３７６位、２４６４２位、２６０２８位、２６２２６位、または３４２０９位のいずれかの多型部位
（２）ＮＰＨＳ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２に記載の塩基配列における１７２位、１８６位、２１１位、２５５４位、２５５８位、１０００１位、１００６７位、１０１７６位、１１８４０位、１３７５９位、１４６４５位、１４９６７位、１６１９８位、１６４３９位、１７６３５位、１７６８５位、１７７９４位、１７８０５位、１７８４８位、１７８４９位、１８４９１位、１９９５５位、２００８８位、２０６１３位、２１５３３位、２１５８２位、２１８１１位、２１８１２位、２２１９０位、２２９６５位、２２９８２位、２３６６０位、２３７８０位、２４０４０位、２４１７２位、２５３３４位、または２６５８５位のいずれかの多型部位
（３）ＳＥＲＰＩＮＢ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：４に記載の塩基配列における２８５３位、２８９１、５２１８位、５３１７位、７５６０位、８０５４位、８７７１位、１０００１位、１０３２９位、１１７８１位、１２０６３位、１２７０５位、１３１２５位、１３６９１位、１３９１３位、１３９１６位、１３９１７位、１４１２３位、１４１６９位、１４４５３位、１４４５６位、１４４８３位、１６５２６位、１６５５６位、１７６４３位、１７７２５位、１７８２４位、１８０７６位、１８２２７位、１８５００位、１８６８７位、１８７４１位、１８７４４位、１８８７５位、１９０４５位、１９１４１位、１９１４９位、１９１９０位、１９４２９位、１９５４８位、１９５６７位、１９６２４位、１９６４５位、１９８１４位、２００４９位、２００８２位、２００９７位、２０２４３位、２２１２２位、２２４９５位、２２５１６位、２２６９１位、２２８１６位、２３００７位、２３９０４位、２４００５位、２４４１８位、２４５７８位、２４８５５位、２４８８５位、２５５０５位、２５５３８位、２５５６４位、２５５８６位、２６０１７位、２６２３４位、２６５５７位、２６６２１位、３０１５５位、３０２６７位、または３０６０７位のいずれかの多型部位
（４）ＡＢＣＡ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：９に記載の塩基配列における９９３位、１７７９位、１８１３位、３４５０位、４２８７位、４３８２位、７２７０位、７５２８位、８３８３位、８７８３位、８７９８位、９２５４位、１０００１位、１０９９９位、１１１２２位、１１７２０位、１２４７３位、１２５６０位、１２５９１位、１２６６４位、１２９１６位、１３４０３位、１３６０５位、１３６９１位、１４４５７位、１４７３７位、１５２７８位、１５３４５位、１５３４５位、１５６３６位、１５６３６位、１５８２４位、１６７９３位、１７０２２位、１７０３７位、１７２８３位、１７５７６位、１８１６７位、１８６１９位、１８６３２位、１８８４２位、１８８４３位、１８９２６位、１９４０４位、１９４０４位、１９８０９位、２０４４７位、２０５１４位、２０５１５位、２１９６３位、２２９８８位、２３０８１位、２３１５７位、２３５７７位、２３９５３位、２４３８９位、２５８１４位、２６１９８位、２６６５５位、２６９０２位、２７３６７位、２７５３５位、２７７０１位、２８１７５位、２８４６７位、２８５６４位、２８６６９位、２８７７４位、２８８３９位、２８９３７位、２８９４４位、２９０３７位、３００９１位、３０６１３位、３０９７５位、３２８０３位、３２８０３位、３３４３５位、３４２６３位、３４２６４位、３４８１２位、３５３０６位、３５３３９位、３５４７３位、３６２１７位、３７５９６位、または３７８５５位のいずれかの多型部位、またはＡＢＣＡ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：７１に記載の塩基配列における１２０６位、１９０５位、１９７９位、２００８位、２０５４位、２２８６位、２３０３位、２８０８位、３１７６位、３４１３位、３４１５位、３５４８位、３６６９位、３７５６位、３９８３位、４５２１位、４５４４位、４５７７位、４６０９位、５２２６位、５８３３位、５８６８位、６４２２位、６５３２位、６５８３位、６６９６位、７０４３位、７１９８位、７３２４位、７３９５位、７４８４位、７７１６位、７７８７位、８１９７位、８３５０位、９５４１位、９６４３位、９７９３位、１０００１位、１０３４８位、１０４１５位、１０７４１位、１０８２６位、１１０００位、１１０２４位、１１１２９位、１１１８５位、１１２８６位、１１４５２位、１１４７１位、１１６５３位、１１７４６位、１１８５８位、１２１１４位、１２２２５位、１２５２０位、１２７２５位、１２８９６位、１２８９９位、１２９０５位、１３１１８位、１３４９２位、１３５８９位、１３８３６位、１４２２６位、１４３９１位、１４４８９位、１４５３６位、１４８１３位、１５０７１位、１５２１５位、１５３７７位、１５３９９位、１５６４４位、１５６８７位、１５８３０位、１６１０３位、１６１１２位、１６１３９位、１６７５４位、１７３０３位、１８２３７位、１８３７９位、１８６１８位、１８９０３位、１８９２１位、１９０１４位、１９０３４位、１９０７４位、１９１４９位、１９１６５位、１９１６７位、１９３６５位、１９３７３位、１９３７６位、１９３８６位、１９４８７位または１９６５９位のいずれかの多型部位
（５）ＣＴＳＤ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１２に記載の塩基配列における８１３位、８１５位、８４４位、８６１位、９５４位、１０２６位、１１５９位、１２９９位、５４６４位、５６００位、７１４４位、７７０７位、７７２６位、７７３９位、７７７８位、７８０３位、７８４１位、７８４２位、７８８６位、７９３９位、７９６３位、８０５８位、８３３３位、９９４８位、１０００１位、１１９９０位、１２３４２位、１２８０７位、１６２６６位、または１６７７１位のいずれかの多型部位
（６）ＳＴＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１３に記載の塩基配列における５７位、４６８位、１８８１位、２９５５位、３４６０位、３９７６位、４５３７位、４５３８位、４７２２位、４８６７位、４８７３位、４９４７位、５０２５位、７０４３位、７６７６位、８５８１位、９０３５位、９３４７位、９６８６位、９８８２位、９８９４位、１０００１位、１０００３位、１２１４２位、１３５２０位、１３５５１位、１３７９７位、１４４９２位、１４７５３位、１４９３３位、１５６８４位、１７０２２位、１８２６４位、１８５６８位、１８９５８位、１９１９４位、１９３３０位、１９３５４位、１９３５５位、１９５６７位、１９７８５位、２０５１１位、２０６１２位、２０６２７位、２０９３６位、２０９６１位、２０９７１位、２１２１３位、２１３３６位、２１６０９位、２１９１４位、または２１９８３位のいずれかの多型部位
（７）ＡＢＣＣ８遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１４に記載の塩基配列における６９７８位、７３５８位、７３５９位、７６４９位、７７５５位、８０６８位、８２１４位、８２１５位、８３０８位、９６５８位、９７６１位、９８１１位、９８２８位、１０００１位、１００９５位、１０１２５位、１１６５８位、１１６７４位、１１７０８位、１１７６７位、１１８８３位、１１９９８位、１２１０９位、１２３０８位、１３６２０位、１３６６４位、１３９１２位、１４２９７位、１４３０９位、１４３２０位、１４３２３位、１４３６７位、１４３９３位、１４４７５位、１４５９１位、１４７２２位、１５２９４位、１５６０１位、１５６５７位、１６１６０位、１６２３０位、１６３６７位、１６４１７位、１７６９５位、１８２１３位、１８８１５位、１９０９３位、２００８２位、２０２１１位、２０４６１位、２１５４７位、２１９９５位、２２８２５位、または２３９０１位のいずれかの多型部位
（８）ＭＹＬ２遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：１９に記載の塩基配列における３１４７位、３２１３位、４６８６位、７３４３位、７８９２位、１０００１位、１０１２３位、１１２６４位、１１９９２位、１５５３２位、１５６８１位、１５９５７位、１５９８８位、１６６４１位、１８３８６位、１８７８２位、２０３５４位、２１５３１位、２３２４５位、２７７４０位、３０６６９位、３０６７０位、３２０７７位、３２７３２位、３２９０７位、３２９５５位、４２５８９位、４３０２９位、４７３５４位、６４７７７位、７１８６９位、７１９６１位、７４２７４位、７５５５１位、７６３４３位、７６３４４位、７８２７０位、７８３３９位、７８６８９位、７８７４３位、７８８０６位、７８８０７位、７９２１５位、７９３１９位、７９３５７位、７９４６０位、７９４６３位、８００３４位、８００６２位、８００７６位、８０４０１位、８０４７５位、８０５９９位、８０７１５位、８０８６５位、８０９４３位、８０９５２位、８０９７４位、８０９９１位、８１１３０位、８１３７３位、８１４２８位、８１５４３位、８１６８２位、８２４２２位、８２５４５位、８２７９２位、８２９３８位、８３２６５位、８３２６６位、８３３９４位、８３４６３位、８３５００位、８３５７８位、８３６１２位、８３６５０位、８４７９０位、８４９２１位、８４９４５位、８５２１４位、８５３８３位、８５３８４位、８５５５０位、８５９８４位、８６１０９位、８６５３９位、８６５４２位、８７１６９位、８７１７０位、８７３５３位、８７６７１位、８７９５３位、８７９５４位、８８０５７位、８８１７８位、８８２４０位、８８２５８位、８８２７１位、８８２７２位、８８３０７位、８８５１２位、８８６９５位、８８７７８位、８８７９８位、８９１２４位、または９１０８１位のいずれかの多型部位
（９）ＤＬＧ５遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２２に記載の塩基配列における２位、１２１位、１４３８位、１６６９位、２１８０位、２４３６位、２５４３位、２６１９位、３７１５位、３７２７位、５２５６位、５３３５位、５３３９位、５６８８位、５７５２位、６３０４位、６６２７位、７１９５位、７３４２位、７６０３位、８２７３位、８９０５位、８９８８位、９０１３位、９０５７位、１０００１位、１０３７２位、１０４５３位、１０４６８位、１０４６８位、１０５１２位、１１０３８位、１１０５５位、１１５９７位、１１６８１位、１１７１９位、１１９２２位、１２０３４位、１２０４９位、１２１７６位、１２５５４位、１３４８１位、１３８６４位、１４３９０位、１５０４４位、１５５９８位、１６１７６位、１６８７０位、１７４７５位、１７７４９位、１８０７７位、１８５８５位、１８６１９位、１８６７７位、１９２２２位、１９７３１位、１９７５０位、２０７５８位、２０９６２位、２１０７３位、２１６８０位、２１８６７位、２２３８５位、２２８７４位、２３１９１位、２４０１６位、２４５２０位、２４９９３位、２５４６０位、２６１５５位、２６８０４位、２６９８０位、２７９８８位、２９２３４位、２９４６９位、３０２５４位、３０７４２位、３０７４２位、３０７５６位、３０７５６位、３１６７１位、３１８７４位、３１９４０位、３２６２６位、３３３９３位、３３５８１位、３４２４０位、３５２９４位、３５６６６位、３６５８７位、３６６５３位、３６９４１位、３７４０８位、３７５８３位、３７６１２位、３８４６２位、３８４７０位、３８４８５位、３９０６５位、３９１４６位、４０６１２位、４０６４１位、４１２４２位、４２２１７位、４２５３９位、４５９３４位、４６０３３位、または４６８９５位のいずれかの多型部位
（１０）ＬＤＬＣ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２３に記載の塩基配列における１５８０位、１７５４位、１８８２位、３０６２位、３５０７位、４８００位、４９０５位、５５６９位、５６０８位、５６４３位、５６６５位、５６９３位、５８６４位、５８８４位、５９８１位、６１６９位、６８０８位、６８５８位、７１９４位、７２８３位、７７２８位、７８３５位、８０４９位、８０４９位、８２６５位、９２０９位、９４４５位、９６４６位、９６４７位、９７１６位、９７７５位、１０００１位、１００２９位、１００５４位、１０１９３位、１０２０８位、１０８２６位、１０９０８位、１０９９７位、１１３３６位、１１７５４位、１１８９５位、１２０３１位、１２３２７位、１２４６４位、１２５６１位、１４０５１位、１５３６６位、１５７７８位、１７７２３位、１７７７２位、１７７８３位、１７８１１位、１７８１５位、１７８６２位、１７９００位、１７９６２位、１８１８４位、１８１８９位、１８２９７位、１８４１１位、１８５１２位、１８５６３位、１９１２５位、１９１５２位、２００９２位、２１２２７位、２１５０６位、２１６１７位、２１８５５位、２３０９０位、２５７００位、２６４６８位、２６６３８位、２７６８２位、２７７５５位、２７８８８位、２７９７３位、２７９９５位、２８１２３位、２８１２７位、２８１４４位、２８１５３位、または２８２７１位のいずれかの多型部位
（１１）ＬＡＲＧＥ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２５に記載の塩基配列における１８１６位、３１１６位、３１１７位、３１１８位、３１５４位、３１５７位、３１９１位、３１９７位、３２０５位、３２９１位、３６２２位、３６５２位、３７９２位、４４８８位、５０７６位、５４７４位、５４８１位、５４９２位、５５０３位、５６５４位、５６７２位、６５６３位、６８４３位、６９０８位、７１１９位、７５３８位、７５３８位、７７２１位、８４０３位、８６２６位、１０００１位、１２６３６位、１３１０３位、１４７９４位、１５３７０位、１５４４８位、１５５９９位、１５７６４位、１６０４５位、１６２６１位、１６２７７位、１６８６１位、１７３６０位、１７６１１位、１７７１４位、１７８０９位、１８２２１位、１８２２１位、１８３７６位、１８５３６位、１９０６３位、２２９４２位、２３４０４位、２３６１２位、２５１２５位、２５１３４位、２５６６２位、２５７０５位、２５７５１位、２７４０７位、２８１６６位、２８５６７位、３０３０２位、３０３１６位、３０３５３位、３０３５４位、３０３９１位、３３５９４位、３３６５０位、３３７５４位、３６６９０位、３７７５３位、３７８９５位、３７９００位、３９１０４位、３９７４５位、４１４０４位、４１６４６位、４１７１１位、４２０２０位、４２０８０位、４２９７４位、４３０５２位、４３２４０位、４３６２１位、４４７３４位、４４９５８位、４６９２０位、４７４３１位、４７５７３位、４８１０１位、４８１２７位、４８３０６位、４８８０７位、４９０４９位、４９８４８位、４９８６０位、４９９０３位、５０１１４位、５０２２３位、５２０５７位、５２５４３位、５２６９３位、または５３０６１位のいずれかの多型部位
（１２）ＢＬＺＦ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２６に記載の塩基配列における１２８３位、４２８８位、７５２０位、７５９９位、７７４０位、８７４５位、９０５０位、９８６２位、１０００１位、１０１２４位、１０１６８位、１０３３９位、１１０１７位、１３７３６位、１３７４７位、１８３５４位、２００３１位、２０１７２位、２２０９４位、２４０８２位、２４３０５位、２４４７８位、２４５５２位、２５２７３位、２５３５１位、２９０９６位、２９１００位、２９７６４位のいずれかの多型部位
（１３）ＦＲＡＰ１遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２８に記載の塩基配列における３４５５位、７８２６位、８７１５位、８７８３位、９０４２位、９３９０位、９４６２位、９７０９位、１０００１位、１０６３５位、１０７４８位、１２７９４位、１２８４９位、１２９１５位、１３１６９位、１７０２０位、１８５８６位、２１１２３位、または２１１２８位のいずれかの多型部位
（１４）ＬＥＰ遺伝子もしくは該遺伝子の近傍ＤＮＡ領域上の部位であって、配列番号：２９に記載の塩基配列における２４４位、５７５位、６７５位、１４８７位、１６４４位、２３４３位、２７１５位、２７６３位、３０９８位、３５８９位、３８８０位、５７８７位、５８６５位、６４３６位、７３９２位、７９９５位、８１６１位、８５４４位、９２３０位、９７９５位、１０００１位、１０１９９位、１０５０３位、１１９７５位、１４９８１位、１５６８１位、または１９３００位のいずれかの多型部位
さらに好ましくは、上記工程（ａ）の多型部位として、以下の多型部位を示すことができる。
（１）ＭＥＴ遺伝子上の多型部位であって、配列番号：１に記載の塩基配列における１０００１位、１４２５８位、１９７６８位、または２４２０９位の多型部位
（２）ＮＰＨＳ１遺伝子上の多型部位であって、配列番号：２に記載の塩基配列における１０００１位、１６１９８位または１７８４８位の多型部位
（３）ＳＥＲＰＩＮＢ２遺伝子上の多型部位であって、配列番号：４に記載の塩基配列における１０００１位、または２４５７８位の多型部位
（４）ＡＢＣＡ１遺伝子上の多型部位であって、配列番号：９に記載の塩基配列における１０００１位、１５８２４位、１７２８３位、２０５１５位、または２８５６４位の多型部位、または
ＡＢＣＡ１遺伝子上の多型部位であって、配列番号：７１に記載の塩基配列における１０００１位および１１０００位の多型部位
（５）ＣＴＳＤ遺伝子上の多型部位であって、配列番号：１２に記載の塩基配列における１０００１位、１１９９０位、または１６７７１位の多型部位
（６）ＳＴＥ遺伝子上の多型部位であって、配列番号：１３に記載の塩基配列における１０００１位、１８９５８位、１９３５４位、１９５６７位、２０９７１位、または２１２１３位の多型部位
（７）ＡＢＣＣ８遺伝子上の多型部位であって、配列番号：１４に記載の塩基配列における１０００１位、または１４４７５位の多型部位
（８）ＭＹＬ２遺伝子上の多型部位であって、配列番号：１９に記載の塩基配列における１０００１位、１８３８６位、４２５８９位、または８３５００位の多型部位
（９）ＤＬＧ５遺伝子上の多型部位であって、配列番号：２２に記載の塩基配列における１０００１位、２４０１６位、２９４６９位、または３７５８３位の多型部位
（１０）ＬＤＬＣ遺伝子上の多型部位であって、配列番号：２３に記載の塩基配列における１０００１位、または１８５６３位の多型部位
（１１）ＬＡＲＧＥ遺伝子上の多型部位であって、配列番号：２５に記載の塩基配列における１０００１位、１３１０３位、１５４４８位、３６６９０位、または４３２４０位の多型部位
（１２）ＢＬＺＦ１遺伝子上の多型部位であって、配列番号：２６に記載の塩基配列における１０００１位、または２００３１位の多型部位
（１３）ＦＲＡＰ１遺伝子上の多型部位であって、配列番号：２８に記載の塩基配列における１０００１位または１７０２０位の多型部位
（１４）ＬＥＰ遺伝子上の多型部位であって、配列番号：２９に記載の塩基配列における１０００１位、または１０５０３位の多型部位
本発明の多型部位における塩基種の決定は、当業者においては種々の方法によって行うことができる。一例を示せば、本発明の多型部位を含むＤＮＡの塩基配列を直接決定することによって行うことができる。この方法においては、まず、被検者からＤＮＡ試料を調製する。本発明においてＤＮＡ試料は、例えば被検者の血液、皮膚、口腔粘膜、手術により採取あるいは切除した組織または細胞、検査等の目的で採取された体液等から抽出した染色体ＤＮＡ、あるいはＲＮＡを基に調製することができる。
本方法においては、次いで、本発明の多型部位を含むＤＮＡを単離する。該ＤＮＡの単離は、本発明の多型部位を含むＤＮＡにハイブリダイズするプライマーを用いて、染色体ＤＮＡ、あるいはＲＮＡを鋳型としたＰＣＲ等によって行うことも可能である。
本方法においては、次いで、単離したＤＮＡの塩基配列を決定する。単離したＤＮＡの塩基配列の決定は、当業者においては、ＤＮＡシークエンサー等を用いて容易に実施することができる。
本発明の多型部位は、通常、その部位の塩基種のバリエーションが既に明らかになっている。本発明における「塩基種の決定」とは、必ずしもその多型部位についてＡ、Ｇ、Ｔ、Ｃのいずれかの塩基種であるかを判別することを意味するものではない。例えば、ある多型部位について塩基種のバリエーションがＡまたはＧであることが判明している場合には、その部位の塩基種が「Ａでない」もしくは「Ｇでない」ことが判明すれば充分である。
予め塩基のバリエーションが明らかにされている多型部位について、その塩基種を決定するための様々な方法が公知である。本発明の塩基種の決定のための方法は、特に限定されない。例えば、ＰＣＲ法を応用した解析方法として、ＴａｑＭａｎ ＰＣＲ法、Ａｃｙｃｌｏ Ｐｒｉｍｅ法、およびＭＡＬＤＩ−ＴＯＦ／ＭＳ法等が実用化されている。またＰＣＲに依存しない塩基種の決定法としてＩｎｖａｄｅｒ法やＲＣＡ法が知られている。更にＤＮＡアレイを使って塩基種を決定することもできる。以下にこれらの方法について簡単に述べる。ここに述べた方法は、いずれも本発明における多型部位の塩基種の決定に応用できる。
［ＴａｑＭａｎ ＰＣＲ法］
ＴａｑＭａｎ ＰＣＲ法の原理は次のとおりである。ＴａｑＭａｎ ＰＣＲ法は、アレルを含む領域を増幅することができるプライマーセットと、ＴａｑＭａｎプローブを利用した解析方法である。ＴａｑＭａｎプローブは、このプライマーセットによって増幅されるアレルを含む領域にハイブリダイズするように設計される。
ＴａｑＭａｎプローブのＴｍに近い条件で標的塩基配列にハイブリダイズさせれば、１塩基の相違によってＴａｑＭａｎプローブのハイブリダイズ効率は著しく低下する。ＴａｑＭａｎプローブの存在下でＰＣＲ法を行うと、プライマーからの伸長反応は、いずれハイブリダイズしたＴａｑＭａｎプローブに到達する。このときＤＮＡポリメラーゼの５’−３’エキソヌクレアーゼ活性によって、ＴａｑＭａｎプローブはその５’末端から分解される。ＴａｑＭａｎプローブをレポーター色素とクエンチャーで標識しておけば、ＴａｑＭａｎプローブの分解を、蛍光シグナルの変化として追跡することができる。つまり、ＴａｑＭａｎプローブの分解が起きれば、レポーター色素が遊離してクエンチャーとの距離が離れることによって蛍光シグナルが生成する。１塩基の相違のためにＴａｑＭａｎプローブのハイブリダイズが低下すればＴａｑＭａｎプローブの分解が進まず蛍光シグナルは生成されない。
多型に対応するＴａｑＭａｎプローブをデザインし、更に各プローブの分解によって異なるシグナルが生成されるようにすれば、同時に塩基種の判定を行うこともできる。例えば、レポーター色素として、あるアレルのアレルＡのＴａｑＭａｎプローブに６−ｃａｒｂｏｘｙ−ｆｌｕｏｒｅｓｃｅｉｎ（ＦＡＭ）を、アレルＢのプローブにＶＩＣを用いる。プローブが分解されない状態では、クエンチャーによってレポーター色素の蛍光シグナル生成は抑制されている。各プローブが対応するアレルにハイブリダイズすれば、ハイブリダイズに応じた蛍光シグナルが観察される。すなわち、ＦＡＭまたはＶＩＣのいずれかのシグナルが他方よりも強い場合には、アレルＡまたはアレルＢのホモであることが判明する。他方、アレルをヘテロで有する場合には、両者のシグナルがほぼ同じレベルで検出されることになる。ＴａｑＭａｎ ＰＣＲ法の利用によって、ゲル上での分離のような時間のかかる工程無しで、ゲノムを解析対象としてＰＣＲと塩基種の決定を同時に行うことができる。そのため、ＴａｑＭａｎ ＰＣＲ法は、多くの被検者についての塩基種を決定できる方法として有用である。
［Ａｃｙｃｌｏ Ｐｒｉｍｅ法］
ＰＣＲ法を利用した塩基種を決定する方法として、Ａｃｙｃｌｏ Ｐｒｉｍｅ法も実用化されている。Ａｃｙｃｌｏ Ｐｒｉｍｅ法では、ゲノム増幅用のプライマー１組と、多型検出用の１つのプライマーを用いる。まず、ゲノムの多型部位を含む領域をＰＣＲで増幅する。この工程は、通常のゲノムＰＣＲと同じである。次に、得られたＰＣＲ産物に対して、多型検出用のプライマーをアニールさせ、伸長反応を行う。多型検出用のプライマーは、検出対象となっている多型部位に隣接する領域にアニールするようにデザインされている。
このとき、伸長反応のためのヌクレオチド基質として、蛍光偏光色素でラベルし、かつ３’−ＯＨをブロックしたヌクレオチド誘導体（ターミネータ）を用いる。その結果、多型部位に相当する位置の塩基に相補的な塩基が１塩基だけ取りこまれて伸長反応が停止する。ヌクレオチド誘導体のプライマーへの取りこみは、分子量の増大による蛍光偏光（Ｆｌｕｏｒｅｓｃｅｎｃｅ ｐｏｌａｒｉｚａｔｉｏｎ；ＦＰ）の増加によって検出することができる。蛍光偏光色素に波長の異なる２種類のラベルを用いれば、特定のＳＮＰｓが２種類の塩基のうちのいずれであるのかを特定することができる。蛍光偏光のレベルは定量することができるので、１度の解析でアレルがホモかヘテロかを判定することもできる。
［ＭＡＬＤＩ−ＴＯＦ／ＭＳ法］
ＰＣＲ産物をＭＡＬＤＩ−ＴＯＦ／ＭＳで解析することによって塩基種の決定を行うこともできる。ＭＡＬＤＩ−ＴＯＦ／ＭＳは、分子量をきわめて正確に知ることができるため、タンパク質のアミノ酸配列や、ＤＮＡの塩基配列のわずかな相違を明瞭に識別することができる解析手法として様々な分野で利用されている。ＭＡＬＤＩ−ＴＯＦ／ＭＳによる塩基種の決定のためには、まず解析対象であるアレルを含む領域をＰＣＲで増幅する。次いで増幅産物を単離してＭＡＬＤＩ−ＴＯＦ／ＭＳによってその分子量を測定する。アレルの塩基配列は予めわかっているので、分子量に基づいて増幅産物の塩基配列は一義的に決定される。
ＭＡＬＤＩ−ＴＯＦ／ＭＳを利用した塩基種の決定には、ＰＣＲ産物の分離工程などが必要となる。しかし標識プライマーや標識プローブを使わないで、正確な塩基種の決定が期待できる。また複数の場所の多型の同時検出にも応用することができる。
［ＩＩｓ型制限酵素を利用したＳＮＰｓ特異的な標識方法］
ＰＣＲ法を利用した更に高速な塩基種の決定が可能な方法も報告されている。例えば、ＩＩｓ型制限酵素を利用して多型部位の塩基種の決定が行われている。この方法においては、ＰＣＲにあたり、ＩＩｓ型制限酵素の認識配列を有するプライマーが用いられる。遺伝子組み換えに利用される一般的な制限酵素（ＩＩ型）は、特定の塩基配列を認識して、その塩基配列中の特定部位を切断する。これに対してＩＩｓ型の制限酵素は、特定の塩基配列を認識して、認識塩基配列から離れた部位を切断する。酵素によって、認識配列と切断個所の間の塩基数は決まっている。従って、この塩基数の分だけ離れた位置にＩＩｓ型制限酵素の認識配列を含むプライマーがアニールするようにすれば、ＩＩｓ型制限酵素によってちょうど多型部位で増幅産物を切断することができる。
ＩＩｓ型制限酵素で切断された増幅産物の末端には、ＳＮＰｓの塩基を含む付着末端（ｃｏｎｈｅｓｉｖｅ ｅｎｄ）が形成される。ここで、増幅産物の付着末端に対応する塩基配列からなるアダプターをライゲーションする。アダプターは、多型変異に対応する塩基を含む異なる塩基配列からなり、それぞれ異なる蛍光色素で標識しておくことができる。最終的に、増幅産物は多型部位の塩基に対応する蛍光色素で標識される。
前記ＩＩｓ型制限酵素認識配列を含むプライマーに、捕捉プライマー（ｃａｐｔｕｒｅ ｐｒｉｍｅｒ）を組み合せてＰＣＲ法を行えば、増幅産物は蛍光標識されるとともに、捕捉プライマーを利用して固相化することができる。例えばビオチン標識プライマーを捕捉プライマーとして用いれば、増幅産物はアビジン結合ビーズに捕捉することができる。こうして捕捉された増幅産物の蛍光色素を追跡することにより、塩基種を決定することができる。
［磁気蛍光ビーズを使った多型部位における塩基種の決定］
複数のアレルを単一の反応系で並行して解析することができる技術も公知である。複数のアレルを並行して解析することは、多重化と呼ばれている。一般に蛍光シグナルを利用したタイピング方法では、多重化のために異なる蛍光波長を有する蛍光成分が必要である。しかし実際の解析に利用することができる蛍光成分は、それほど多くない。これに対して、樹脂等に複数種の蛍光成分を混合した場合には、限られた種類の蛍光成分であっても、相互に識別可能な多様な蛍光シグナルを得ることができる。更に、樹脂中に磁気で吸着される成分を加えれば蛍光を発するとともに、磁気によって分離可能なビーズとすることができる。このような磁気蛍光ビーズを利用した、多重化多型タイピングが考え出された（バイオサイエンスとバイオインダストリー，Ｖｏｌ．６０ Ｎｏ．１２，８２１−８２４）。
磁気蛍光ビーズを利用した多重化多型タイピングにおいては、各アレルの多型部位に相補的な塩基を末端に有するプローブが磁気蛍光ビーズに固定化される。各アレルにそれぞれ固有の蛍光シグナルを有する磁気蛍光ビーズが対応するように、両者は組み合せられる。一方、磁気蛍光ビーズに固定されたプローブが相補配列にハイブリダイズしたときに、当該アレル上で隣接する領域に相補的な塩基配列を有する蛍光標識オリゴＤＮＡを調製する。
アレルを含む領域を非対称ＰＣＲによって増幅し、上記の磁気蛍光ビーズ固定化プローブと蛍光標識オリゴＤＮＡをハイブリダイズさせ、更に両者をライゲーションする。磁気蛍光ビーズ固定化プローブの末端が、多型部位の塩基に相補的な塩基配列であった場合には効率的にライゲーションされる。逆にもしも多型のために末端の塩基が異なれば、両者のライゲーション効率は低下する。その結果、各磁気蛍光ビーズには、試料が当該磁気蛍光ビーズに相補的な塩基種であった場合に限り、蛍光標識オリゴＤＮＡが結合する。
磁気によって磁気蛍光ビーズを回収し、更に各磁気蛍光ビーズ上の蛍光標識オリゴＤＮＡの存在を検出することにより、塩基種が決定される。磁気蛍光ビーズは、フローサイトメーターでビーズ毎に蛍光シグナルを解析できるので、多種類の磁気蛍光ビーズが混合されていてもシグナルの分離は容易である。つまり、多種類の多型部位について、単一の反応容器で並行して解析する「多重化」が達成される。
［Ｉｎｖａｄｅｒ法］
ＰＣＲ法に依存しないジェノタイピングのための方法も実用化されている。例えば、Ｉｎｖａｄｅｒ法では、アレルプローブ、インベーダープローブ、およびＦＲＥＴプローブの３種類のオリゴヌクレオチドと、ｃｌｅａｖａｓｅと呼ばれる特殊なヌクレアーゼのみで、塩基種の決定を実現している。これらのプローブのうち標識が必要なのはＦＲＥＴプローブのみである。
アレルプローブは、検出すべきアレルに隣接する領域にハイブリダイズするようにデザインされる。アレルプローブの５’側には、ハイブリダイズに無関係な塩基配列からなるフラップが連結されている。アレルプローブは多型部位の３’側にハイブリダイズし、多型部位の上でフラップに連結する構造を有する。
一方インベーダープローブは、多型部位の５’側にハイブリダイズする塩基配列からなっている。インベーダープローブの塩基配列は、ハイブリダイズによって３’末端が多型部位に相当するようにデザインされている。インベーダープローブにおける多型部位に相当する位置の塩基は任意で良い。つまり、多型部位を挟んでインベーダープローブとアレルプローブとが隣接してハイブリダイズするように両者の塩基配列はデザインされている。
多型部位がアレルプローブの塩基配列に相補的な塩基であった場合には、インベーダープローブとアレルプローブの両者がアレルにハイブリダイズすると、アレルプローブの多型部位に相当する塩基にインベーダープローブが侵入（ｉｎｖａｓｉｏｎ）した構造が形成される。ｃｌｅａｖａｓｅは、このようにして形成された侵入構造を形成したオリゴヌクレオチドのうち、侵入された側の鎖を切断する。切断は侵入構造の上で起きるので、結果としてアレルプローブのフラップが切り離されることになる。一方、もしも多型部位の塩基がアレルプローブの塩基に相補的でなかった場合には、多型部位におけるインベーダープローブとアレルプローブの競合は無く、侵入構造は形成されない。したがってｃｌｅａｖａｓｅによるフラップの切断が起こらない。
ＦＲＥＴプローブは、こうして切り離されたフラップを検出するためのプローブである。ＦＲＥＴプローブは５’末端側に自己相補配列を有し、３’末端側に１本鎖部分が配置されたヘアピンループを構成している。ＦＲＥＴプローブの３’末端側に配置された１本鎖部分は、フラップに相補的な塩基配列からなっていて、ここにフラップがハイブリダイズすることができる。フラップがＦＲＥＴプローブにハイブリダイズすると、ＦＲＥＴプローブの自己相補配列の５’末端部分にフラップの３’末端が侵入した構造が形成されるように両者の塩基配列がデザインされている。ｃｌｅａｖａｓｅは侵入構造を認識して切断する。ＦＲＥＴプローブのｃｌｅａｖａｓｅによって切断される部分を挟んで、ＴａｑＭａｎ ＰＣＲと同様のレポーター色素とクエンチャーで標識しておけば、ＦＲＥＴプローブの切断を蛍光シグナルの変化として検知することができる。
なお、理論的には、フラップは切断されない状態でもＦＲＥＴプローブにハイブリダイズするはずである。しかし実際には、切断されたフラップとアレルプローブの状態で存在しているフラップとでは、ＦＲＥＴに対する結合効率に大きな差が有る。そのため、ＦＲＥＴプローブを利用して、切断されたフラップを特異的に検出することは可能である。
Ｉｎｖａｄｅｒ法に基づいて塩基種を決定するためには、アレルＡとアレルＢのそれぞれに相補的な塩基配列を含む、２種類のアレルプローブを用意すれば良い。このとき両者のフラップの塩基配列は異なる塩基配列とする。フラップを検出するためのＦＲＥＴプローブも２種類を用意し、それぞれのレポーター色素を識別可能なものとしておけば、ＴａｃＭａｎ ＰＣＲ法と同様の考え方によって、塩基種を決定することができる。
Ｉｎｖａｄｅｒ法の利点は、標識の必要なオリゴヌクレオチドがＦＲＥＴプローブのみであることである。ＦＲＥＴプローブは検出対象の塩基配列とは無関係に、同一のオリゴヌクレオチドを利用することができる。従って、大量生産が可能である。一方アレルプローブとインベーダープローブは標識する必要が無いので、結局、ジェノタイピングのための試薬を安価に製造することができる。
［ＲＣＡ法］
ＰＣＲ法に依存しない塩基種の決定方法として、ＲＣＡ法を挙げることができる。鎖置換作用を有するＤＮＡポリメラーゼが、環状の１本鎖ＤＮＡを鋳型として、長い相補鎖を合成する反応に基づくＤＮＡの増幅方法が、Ｒｏｌｌｉｎｇ Ｃｉｒｃｌｅ Ａｍｐｌｉｆｉｃａｔｉｏｎ（ＲＣＡ）法である（Ｌｉｚａｒｄｒｉ ＰＭ ｅｔ ａｌ．，Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ １９，２２５，１９９８）。ＲＣＡ法においては、環状ＤＮＡにアニールして相補鎖合成を開始するプライマーと、このプライマーによって生成する長い相補鎖にアニールする第２のプライマーを利用して、増幅反応を構成している。
ＲＣＡ法には、鎖置換作用を有するＤＮＡポリメラーゼが利用されている。そのため、相補鎖合成によって２本鎖となった部分は、より５’側にアニールした別のプライマーから開始した相補鎖合成反応によって置換される。例えば、環状ＤＮＡを鋳型とする相補鎖合成反応は、１周分では終了しない。先に合成した相補鎖を置換しながら相補鎖合成は継続し、長い１本鎖ＤＮＡが生成される。一方、環状ＤＮＡを鋳型として生成した長い１本鎖ＤＮＡには、第２のプライマーがアニールして相補鎖合成が開始する。ＲＣＡ法において生成される１本鎖ＤＮＡは、環状のＤＮＡを鋳型としていることから、その塩基配列は同じ塩基配列の繰り返しである。従って、長い１本鎖の連続的な生成は、第２のプライマーの連続的なアニールをもたらす。その結果、変性工程を経ることなく、プライマーがアニールすることができる１本鎖部分が連続的に生成される。こうして、ＤＮＡの増幅が達成される。
ＲＣＡ法に必要な環状１本鎖ＤＮＡが多型部位の塩基種に応じて生成されれば、ＲＣＡ法を利用して塩基種の決定をすることができる。そのために、直鎖状で１本鎖のパドロックプローブが利用される。パドロックプローブは、５’末端と３’末端に検出すべき多型部位の両側に相補的な塩基配列を有している。これらの塩基配列は、バックボーンと呼ばれる特殊な塩基配列からなる部分で連結されている。多型部位がパドロックプローブの末端に相補的な塩基配列であれば、アレルにハイブリダイズしたパドロックプローブの末端をＤＮＡリガーゼによってライゲーションすることができる。その結果、直鎖状のパドロックプローブが環状化され、ＲＣＡ法の反応がトリガーされる。ＤＮＡリガーゼの反応は、ライゲーションすべき末端部分が完全に相補的でない場合には反応効率が著しく低下する。従って、ライゲーションの有無をＲＣＡ法で確認することによって、多型部位の塩基種の決定が可能である。
ＲＣＡ法は、ＤＮＡを増幅することはできるが、そのままではシグナルを生成しない。また増幅の有無のみを指標とするのでは、アレル毎に反応を行わなければ、通常、塩基種を決定することができない。これらの点を塩基種の決定のために改良した方法が公知である。例えば、モレキュラービーコンを利用して、ＲＣＡ法に基づいて１チューブで塩基種の決定を行うことができる。モレキュラービーコンは、ＴａｑＭａｎ法と同様に、蛍光色素とクエンチャーを利用したシグナル生成用プローブである。モレキュラービーコンの５’末端と３’末端は相補的な塩基配列で構成されており、単独ではヘアピン構造を形成する。両端付近を蛍光色素とクエンチャーで標識しておけば、ヘアピン構造を形成している状態では蛍光シグナルが検出できない。モレキュラービーコンの一部を、ＲＣＡ法の増幅産物に相補的な塩基配列としておけば、モレキュラービーコンはＲＣＡ法の増幅産物にハイブリダイズする。ハイブリダイズによってヘアピン構造が解消されるため、蛍光シグナルが生成される。
モレキュラービーコンの利点は、パドロックプローブのバックボーン部分の塩基配列を利用することによって、検出対象とは無関係にモレキュラービーコンの塩基配列を共通にできる点である。アレル毎にバックボーンの塩基配列を変え、蛍光波長が異なる２種類のモレキュラービーコンを組み合せれば、１チューブで塩基種の決定が可能である。蛍光標識プローブの合成コストは高いので、測定対象に関わらず共通のプローブを利用できることは、経済的なメリットである。
これらの方法はいずれも多量のサンプルを高速にジェノタイピングするために開発された方法である。ＭＡＬＤＩ−ＴＯＦ／ＭＳを除けば、通常、いずれの方法にも何らかの形で標識プローブなどを用意する必要がある。これに対して、標識プローブなどに頼らない塩基種決定法も古くから行われている。このような方法の一つとして、例えば、制限酵素断片長多型（Ｒｅｓｔｒｉｃｔｉｏｎ Ｆｒａｇｍｅｎｔ Ｌｅｎｇｔｈ Ｐｏｌｙｍｏｒｐｈｉｓｍ／ＲＦＬＰ）を利用した方法やＰＣＲ−ＲＦＬＰ法等が挙げられる。
ＲＦＬＰは、制限酵素の認識部位の変異、あるいは制限酵素処理によって生じるＤＮＡ断片内における塩基の挿入または欠失が、制限酵素処理後に生じる断片の大きさの変化として検出できることを利用している。検出対象となる多型を含む塩基配列を認識する制限酵素が存在すれば、ＲＦＬＰの原理によって多型部位の塩基を知ることができる。
標識プローブを必要としない方法として、ＤＮＡの二次構造の変化を指標として塩基の違いを検出する方法も公知である。ＰＣＲ−ＳＳＣＰでは、１本鎖ＤＮＡの二次構造がその塩基配列の相違を反映することを利用している（Ｃｌｏｎｉｎｇ ａｎｄ ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ−ｓｉｎｇｌｅ−ｓｔｒａｎｄ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍ ａｎａｌｙｓｉｓ ｏｆ ａｎｏｎｙｍｏｕｓ Ａｌｕ ｒｅｐｅａｔｓ ｏｎ ｃｈｒｏｍｏｓｏｍｅ １１．Ｇｅｎｏｍｉｃｓ．１９９２ Ｊａｎ １；１２（１）：１３９−１４６．、Ｄｅｔｅｃｔｉｏｎ ｏｆ ｐ５３ ｇｅｎｅ ｍｕｔａｔｉｏｎｓ ｉｎ ｈｕｍａｎ ｂｒａｉｎ ｔｕｍｏｒｓ ｂｙ ｓｉｎｇｌｅ−ｓｔｒａｎｄ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍ ａｎａｌｙｓｉｓ ｏｆ ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ ｐｒｏｄｕｃｔｓ．Ｏｎｃｏｇｅｎｅ．１９９１ Ａｕｇ １；６（８）：１３１３−１３１８．、Ｍｕｌｔｉｐｌｅ ｆｌｕｏｒｅｓｃｅｎｃｅ−ｂａｓｅｄ ＰＣＲ−ＳＳＣＰ ａｎａｌｙｓｉｓ ｗｉｔｈ ｐｏｓｔｌａｂｅｌｉｎｇ．、ＰＣＲ Ｍｅｔｈｏｄｓ Ａｐｐｌ．１９９５ Ａｐｒ １；４（５）：２７５−２８２．）。ＰＣＲ−ＳＳＣＰ法は、ＰＣＲ産物を１本鎖ＤＮＡに解離させ、非変性ゲル上で分離する工程により実施される。ゲル上の移動度は、１本鎖ＤＮＡの二次構造によって変動するので、もしも多型部位における塩基の相違があれば、移動度の違いとして検出することができる。
その他、標識プローブを必要としない方法として、例えば、変性剤濃度勾配ゲル（ｄｅｎａｔｕｒａｎｔ ｇｒａｄｉｅｎｔ ｇｅｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ：ＤＧＧＥ法）等を例示することができる。ＤＧＧＥ法は、変性剤の濃度勾配のあるポリアクリルアミドゲル中で、ＤＮＡ断片の混合物を泳動し、それぞれの不安定性の違いによってＤＮＡ断片を分離する方法である。ミスマッチのある不安定なＤＮＡ断片が、ゲル中のある変性剤濃度の部分まで移動すると、ミスマッチ周辺のＤＮＡ配列はその不安定さのために、部分的に１本鎖へと解離する。部分的に解離したＤＮＡ断片の移動度は、非常に遅くなり、解離部分のない完全な二本鎖ＤＮＡの移動度と差がつくことから、両者を分離することができる。
具体的には、まずＰＣＲ法等によって多型部位を含む領域を増幅する。増幅産物に、塩基配列がわかっているプローブＤＮＡをハイブリダイズさせて２本鎖とする。これを尿素などの変性剤の濃度が移動するに従って徐々に高くなっているポリアクリルアミドゲル中で電気泳動し、対照と比較する。プローブＤＮＡとのハイブリダイズによってミスマッチを生じたＤＮＡ断片では、より低い変性剤濃度位置でＤＮＡ断片が一本鎖になり、極端に移動速度が遅くなる。こうして生じた移動度の差を検出することによりミスマッチの有無を検出することができる。
更にＤＮＡアレイを使って塩基種を決定することもできる（細胞工学別冊「ＤＮＡマイクロアレイと最新ＰＣＲ法」，秀潤社，２０００．４／２０発行，ｐｐ９７−１０３「オリゴＤＮＡチップによるＳＮＰの解析」，梶江慎一）。ＤＮＡアレイは、同一平面上に配置した多数のプローブに対してサンプルＤＮＡ（あるいはＲＮＡ）をハイブリダイズさせ、当該平面をスキャンすることによって、各プローブに対するハイブリダイズが検出される。多くのプローブに対する反応を同時に観察することができることから、例えば、多数の多型部位について同時に解析するには、ＤＮＡアレイは有用である。
一般にＤＮＡアレイは、高密度に基板にプリントされた何千ものヌクレオチドで構成されている。通常これらのＤＮＡは非透過性（ｎｏｎ−ｐｏｒｏｕｓ）の基板の表層にプリントされる。基板の表層は、一般的にはガラスであるが、透過性（ｐｏｒｏｕｓ）の膜、例えばニトロセルロースメンブレムを使用することもできる。
本発明において、ヌクレオチドの固定（アレイ）方法として、Ａｆｆｙｍｅｔｒｉｘ社開発によるオリゴヌクレオチドを基本としたアレイが例示できる。オリゴヌクレオチドのアレイにおいて、オリゴヌクレオチドは通常インビトロ（ｉｎ ｖｉｔｒｏ）で合成される。例えば、ｐｈｏｔｏｌｉｔｈｏｇｒａｐｈｉｃの技術（Ａｆｆｙｍｅｔｒｉｘ社）、および化学物質を固定させるためのインクジェット（Ｒｏｓｅｔｔａ Ｉｎｐｈａｒｍａｔｉｃｓ社）技術等によるオリゴヌクレオチドのインサイチュ合成法が既に知られており、いずれの技術も本発明の基板の作製に利用することができる。
オリゴヌクレオチドは、検出すべきＳＮＰｓを含む領域に相補的な塩基配列で構成される。基板に結合させるヌクレオチドプローブの長さは、オリゴヌクレオチドを固定する場合は、通常１０〜１００ベースであり、好ましくは１０〜５０ベースであり、さらに好ましくは１５〜２５ベースである。更に、一般にＤＮＡアレイ法においては、クロスハイブリダイゼーション（非特異的ハイブリダイゼーション）による誤差を避けるために、ミスマッチ（ＭＭ）プローブが用いられる。ミスマッチプローブは、標的塩基配列と完全に相補的な塩基配列からなるオリゴヌクレオチドとのペアを構成している。ミスマッチプローブに対して、完全に相補的な塩基配列からなるオリゴヌクレオチドはパーフェクトマッチ（ＰＭ）プローブと呼ばれる。データ解析の過程で、ミスマッチプローブで観察されたシグナルを消去することによって、クロスハイブリダイゼーションの影響を小さくすることができる。
ＤＮＡアレイ法によるジェノタイピングのための試料は、被検者から採取された生物学的試料をもとに当業者に周知の方法で調製することができる。生物学的試料は特に限定されない。例えば被検者の末梢血白血球、皮膚、口腔粘膜等の組織または細胞、涙、唾液、尿、糞便または毛髪から抽出した染色体ＤＮＡから、ＤＮＡ試料を調製することができる。判定すべき多型部位を含む領域を増幅するためのプライマーを用いて、染色体ＤＮＡの特定の領域が増幅される。このとき、マルチプレックスＰＣＲ法によって複数の領域を同時に増幅することができる。マルチプレックスＰＣＲ法とは、複数組のプライマーセットを、同じ反応液中で用いるＰＣＲ法である。複数の多型部位を解析するときには、マルチプレックスＰＣＲ法が有用である。
一般にＤＮＡアレイ法においては、ＰＣＲ法によってＤＮＡ試料を増幅するとともに、増幅産物が標識される。増幅産物の標識には、標識を付したプライマーが利用される。例えば、まず多型部位を含む領域に特異的なプライマーセットによるＰＣＲ法でゲノムＤＮＡを増幅する。次に、ビオチンラベルしたプライマーを使ったラベリングＰＣＲ法によって、ビオチンラベルされたＤＮＡを合成する。こうして合成されたビオチンラベルＤＮＡを、チップ上のオリゴヌクレオチドプローブにハイブリダイズさせる。ハイブリダイゼーションの反応液および反応条件は、基板に固定するヌクレオチドプローブの長さや反応温度等の条件に応じて、適宜調整することができる。当業者は、適切なハイブリダイゼーションの条件をデザインすることができる。ハイブリダイズしたＤＮＡを検出するために、蛍光色素で標識したアビジンが添加される。アレイをスキャナで解析し、蛍光を指標としてハイブリダイズの有無を確認する。
上記方法をより具体的に示せば、被検者から調製した本発明の多型部位を含むＤＮＡ、およびヌクレオチドプローブが固定された固相、を取得した後、次いで、該ＤＮＡと該固相を接触させる。さらに、固相に固定されたヌクレオチドプローブにハイブリダイズしたＤＮＡを検出することにより、本発明の多型部位の塩基種を決定する。
本発明において「固相」とは、ヌクレオチドを固定することが可能な材料を意味する。本発明の固相は、ヌクレオチドを固定することが可能であれば特に制限はないが、具体的には、マイクロプレートウェル、プラスチックビーズ、磁性粒子、基板などを含む固相等を例示することができる。本発明の「固相」としては、一般にＤＮＡアレイ技術で使用される基板を好適に用いることができる。本発明において「基板」とは、ヌクレオチドを固定することが可能な板状の材料を意味する。また、本発明においてヌクレオチドには、オリゴヌクレオチドおよびポリヌクレオチドが含まれる。
上記の方法以外にも、特定部位の塩基を検出するために、アレル特異的オリゴヌクレオチド（Ａｌｌｅｌｅ Ｓｐｅｃｉｆｉｃ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ／ＡＳＯ）ハイブリダイゼーション法が利用できる。アレル特異的オリゴヌクレオチド（ＡＳＯ）は、検出すべき多型部位が存在する領域にハイブリダイズする塩基配列で構成される。ＡＳＯを試料ＤＮＡにハイブリダイズさせるとき、多型によって多型部位にミスマッチが生じるとハイブリッド形成の効率が低下する。ミスマッチは、サザンブロット法や、特殊な蛍光試薬がハイブリッドのギャップにインターカレーションすることにより消光する性質を利用した方法等によって検出することができる。また、リボヌクレアーゼＡミスマッチ切断法によって、ミスマッチを検出することもできる。
本発明はまた、本発明の多型部位を含むＤＮＡにハイブリダイズし、少なくとも１５ヌクレオチドの鎖長を有するオリゴヌクレオチドを含む、２型糖尿病の検査薬を提供する。これらはそれぞれ遺伝子発現を指標とする検査、または遺伝子多型を指標とする検査に使用される。
該オリゴヌクレオチドは、本発明の多型部位を含むＤＮＡに特異的にハイブリダイズするものである。ここで「特異的にハイブリダイズする」とは、通常のハイブリダイゼーション条件下、好ましくはストリンジェントなハイブリダイゼーション条件下（例えば、サムブルックら，Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｕｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ，ＵＳＡ，第２版 １９８９に記載の条件）において、他のタンパク質をコードするＤＮＡとクロスハイブリダイゼーションを有意に生じないことを意味する。特異的なハイブリダイズが可能であれば、該オリゴヌクレオチドは、検出する上記（１）〜（２９）記載の各塩基配列に対し、完全に相補的である必要はない。
該オリゴヌクレオチドは、上記本発明の検査方法におけるプローブやプライマーとして用いることができる。該オリゴヌクレオチドをプライマーとして用いる場合、その長さは、通常１５ｂｐ〜１００ｂｐであり、好ましくは１７ｂｐ〜３０ｂｐである。プライマーは、本発明の多型部位を含むＤＮＡの少なくとも一部を増幅しうるものであれば、特に制限されない。
本発明は、本発明の多型部位を含む領域を増幅するためのプライマー、および多型部位を含むＤＮＡ領域にハイブリダイズするプロープを提供する。本発明において、多型部位を含む領域を増幅するためのプライマーには、多型部位を含むＤＮＡを鋳型として、多型部位に向かって相補鎖合成を開始することができるプライマーも含まれる。該プライマーは、多型部位を含むＤＮＡにおける、多型部位の３’側に複製開始点を与えるためのプライマーと表現することもできる。プライマーがハイブリダイズする領域と多型部位との間隔は、任意である。両者の間隔は、多型部位の塩基の解析手法に応じて、好適な塩基数を選択することができる。たとえば、ＤＮＡチップによる解析のためのプライマーであれば、多型部位を含む領域として、２０〜５００、通常５０〜２００塩基の長さの増幅産物が得られるようにプライマーをデザインすることができる。当業者においては、多型部位を含む周辺ＤＮＡ領域についての塩基配列情報を基に、解析手法に応じたプライマーをデザインすることができる。本発明のプライマーを構成する塩基配列は、ゲノムの塩基配列に対して完全に相補的な塩基配列のみならず、適宜改変することができる。
本発明のプライマーには、ゲノムの塩基配列に相補的な塩基配列に加え、任意の塩基配列を付加することができる。例えば、ＩＩｓ型の制限酵素を利用した多型の解析方法のためのプライマーにおいては、ＩＩｓ型制限酵素の認識配列を付加したプライマーが利用される。このような、塩基配列を修飾したプライマーは、本発明のプライマーに含まれる。更に、本発明のプライマーは、修飾することができる。例えば、蛍光物質や、ビオチンまたはジゴキシンのような結合親和性物質で標識したプライマーが各種のジェノタイピング方法において利用される。これらの修飾を有するプライマーも本発明に含まれる。
一方本発明において、多型部位を含む領域にハイブリダイズするプローブとは、多型部位を含む領域の塩基配列を有するポリヌクレオチドとハイブリダイズすることができるプローブを言う。より具体的には、プローブの塩基配列中に多型部位を含むプローブは本発明のプローブとして好ましい。あるいは、多型部位における塩基の解析方法によっては、プローブの末端が多型部位に隣接する塩基に対応するように、デザインされる場合もある。従って、プローブ自身の塩基配列には多型部位が含まれないが、多型部位に隣接する領域に相補的な塩基配列を含むプローブも、本発明における望ましいプローブとして示すことができる。
言いかえれば、ゲノムＤＮＡ上の本発明の多型部位、または多型部位に隣接する部位にハイブリダイズすることができるプローブは、本発明のプローブとして好ましい。本発明のプローブには、プライマーと同様に、塩基配列の改変、塩基配列の付加、あるいは修飾が許される。例えば、Ｉｎｖａｄｅｒ法に用いるプローブは、フラップを構成するゲノムとは無関係な塩基配列が付加される。このようなプローブも、多型部位を含む領域にハイブリダイズする限り、本発明のプロープに含まれる。本発明のプローブを構成する塩基配列は、ゲノムにおける本発明の多型部位の周辺ＤＮＡ領域の塩基配列をもとに、解析方法に応じてデザインすることができる。
本発明のプライマー、またはプローブは、それを構成する塩基配列をもとに、任意の方法によって合成することができる。本発明のプライマーまたはプローブの、ゲノムＤＮＡに相補的な塩基配列の長さは、通常１５〜１００、一般に１５〜５０、通常１５〜３０である。与えられた塩基配列に基づいて、当該塩基配列を有するオリゴヌクレオチドを合成する手法は公知である。更に、オリゴヌクレオチドの合成において、蛍光色素やビオチンなどで修飾されたヌクレオチド誘導体を利用して、オリゴヌクレオチドに任意の修飾を導入することもできる。あるいは、合成されたオリゴヌクレオチドに、蛍光色素などを結合する方法も公知である。
本発明はまた、本発明の検査方法に使用するための試薬を提供する。本発明の試薬は、前記本発明のプライマーおよび／またはプローブを含む。本発明の試薬には、塩基種の決定方法に応じて、各種の酵素、酵素基質、および緩衝液などを組み合せることができる。酵素としては、ＤＮＡポリメラーゼ、ＤＮＡリガーゼ、あるいはＩＩｓ制限酵素などの、上記の塩基種決定方法として例示した各種の解析方法に必要な酵素を示すことができる。緩衝液は、これらの解析に用いる酵素の活性の維持に好適な緩衝液が、適宜選択される。更に、酵素基質としては、例えば、相補鎖合成用の基質等が用いられる。
更に本発明の試薬には、多型部位における塩基が明らかな対照を添付することができる。対照は、予め多型部位の塩基種が明らかなゲノム、あるいはゲノムの断片を用いることができる。ゲノムは、細胞から抽出されたものでもよいし、細胞あるいは細胞の分画を用いることもできる。細胞を対照として用いれば、対照の結果によってゲノムＤＮＡの抽出操作が正しく行われたことを証明することができる。あるいは、多型部位を含む塩基配列からなるＤＮＡを対照として用いることもできる。具体的には、本発明の多型部位における塩基種が明らかにされたゲノム由来のＤＮＡを含むＹＡＣベクターやＢＡＣベクターは、対照として有用である。あるいは多型部位に相当する数百ベースのみを切り出して挿入したベクターを対照として用いることもできる。
さらに、本発明における検査薬の別の態様は、本発明の多型部位を含むＤＮＡとハイブリダイズするヌクレオチドプローブが固定された固相からなる２型糖尿病の検査薬である。これらは本発明の多型部位を指標とする検査に使用される。これらの調製方法に関しては、上述の通りである。The present invention has been made in view of such a situation, and an object thereof is to find a gene associated with type 2 diabetes and to identify a polymorphism associated with type 2 diabetes existing on the gene. Therefore, it is providing the inspection method of type 2 diabetes.
The present inventors have intensively studied to solve the above problems. First, in order to identify a type 2 diabetes (DM) -related gene, the present inventors used 2039 single nucleotide polymorphisms (SNPs) derived from 704 candidate genes using a case control test, and related to DM. I investigated. The test group consists of 148 cases and 227 control subjects. As a result, 29 genes were found to be significantly related to DM. Although these genes and the base sequences of these genes are already known, the present inventors have shown for the first time that they are related to DM. In addition, the present inventors have succeeded in finding polymorphic sites (SNPs) significantly associated with DM and haplotypes associated with DM containing these polymorphic sites. By using these SNPs or haplotypes as indicators, it is possible to examine the cause or the disease type if the possibility of being affected by DM is high or low, and if it is already affected by DM.
As described above, the identification of genes related to DM by the present inventors is highly expected to elucidate the onset mechanism of DM and to develop drugs for the prevention or treatment of DM.
As described above, the present inventors have succeeded in identifying 29 genes related to DM, SNPs related to DM, and haplotypes, and completed a method for testing DM using SNPs or haplotypes on the genes as indices. I let you. That is, the present invention relates to a gene associated with type 2 diabetes, and a method for examining type 2 diabetes using a polymorphic site or haplotype on the gene as an index, more specifically,
As described above, the present inventors have succeeded in identifying 29 genes related to DM, SNPs related to DM, and haplotypes, and completed a method for testing DM using SNPs or haplotypes on the genes as indices. I let you. That is, the present invention relates to a gene associated with type 2 diabetes, and a method for examining type 2 diabetes using a polymorphic site or haplotype on the gene as an index, more specifically,
[1] A test method for type 2 diabetes, comprising detecting a mutation in a gene according to any one of the following (1) to (29) for a subject:
(1) MET, (2) NPHS1, (3) SLC1A1, (4) SERPINB2, (5) FABP2, (6) ZNF263, (7) TNFA, (8) THBS3, (9) ABCA1, (10) SERPINB10, (11) ALDOB, (12) CTSD, (13) STE, (14) ABCC8, (15) SERPINA3, (16) RCV1, (17) TFRC, (18) MMP19, (19) MYL2, (20) VLDLR, (21) P2RX7, (22) DLG5, (23) LDLC, (24) AQP8, (25) LARGE, (26) BLZF1, (27) RGS5, (28) FRAP1, (29) LEP
The test method detects, for example, a mutation in the gene according to any one of (1) to (29) described above with respect to a test sample obtained from a subject, and the test subject has type 2 diabetes. It is a test method of whether or not there is a predisposition to sensitivity or resistance.
[2] The testing method according to [1], wherein the mutation is a single nucleotide polymorphism mutation (SNP),
[3] The subject is characterized by determining the base type of the polymorphic site in the gene according to any one of (1) to (29) in [1] or a DNA region in the vicinity of the gene. Type diabetes testing method,
For example, for the test sample obtained from the subject, the test method determines the base type of the polymorphic site in the gene according to any one of (1) to (29) above or a DNA region in the vicinity of the gene. And whether or not the subject has a predisposition to susceptibility or resistance to type 2 diabetes.
[4] The method according to [3], wherein the polymorphic site is a polymorphic site described in the following (1a) to (29a),
(1a) a MET gene or a site on a DNA region in the vicinity of the gene, the positions 765, 1383, 1890, 2099, 2814, 3293, 3752 in the nucleotide sequence set forth in SEQ ID NO: 1; 3774, 3927, 4690, 7419, 8219, 8201, 10001, 10318, 13766, 13789, 14258, 15427, 16275, 16597, 16932, 19768, 19835, 230032 , 20240, 20537, 20885, 21546, 24209, 24271, 24376, 24642, 26028, 26226, or 34209
(2a) the NPHS1 gene or a site on the DNA region in the vicinity of the gene, the positions 172, 186, 211, 2554, 2558, 2558, 10001, 10001 in the nucleotide sequence set forth in SEQ ID NO: 2; 10176th, 11840th, 13759th, 14645th, 14967th, 16198th, 16439th, 17635th, 17685th, 17794th, 17805th, 17848th, 17849th, 18491th, 19955th, 20955th, 20613th 21533, 21582, 21811, 21812, 22190, 22965, 22982, 23660, 23780, 24040, 24172, 25334, or 26585 polymorphic sites
(3a) SLC1A1 gene or a site on the DNA region in the vicinity of the gene, and positions 1142, 2732, 2734, 7220, 7001, 12001, 12276, 12922 in the nucleotide sequence set forth in SEQ ID NO: 3. Or any polymorphic site at position 18049
(4a) SERPINB2 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence of SEQ ID NO: 4, positions 2853, 2891, 5218, 5317, 7560, 8054, 8771, 10001 , 10329, 11781, 12063, 12705, 13125, 13691, 13913, 13916, 13917, 14123, 14169, 14453, 14456, 14483, 16526, 16556, 17643, 17725, 17824, 18076, 18227, 18500, 18687, 18741, 18744, 18875, 19045, 19141, 19149, 19190, 19429, 19548, 195 7th, 19624th, 19645th, 19814th, 200049th, 20082th, 20097th, 20243th, 22243th, 22122th, 22516th, 22561th, 22816th, 23007th, 23904th, 24005th, 24418th Any of the polymorphic sites at positions 24578, 24855, 24855, 25505, 25538, 25564, 25586, 26017, 26234, 26557, 26621, 30155, 30267, or 30607
(5a) the FABP2 gene or a site on the DNA region in the vicinity of the gene, and the positions 2970, 3174, 3175, 4525, 4556, 4556, 4558, 4636 in the nucleotide sequence set forth in SEQ ID NO: 5; 4646, 4774, 5726, 5736, 5852, 7203, 7253, 7310, 8239, 8251, 8296, 8312, 8974, 10001, 10107, 10424, 10464 , 10491, 10794, 11922, 11957, 12820, 16006, 16256, 16309, 1631, 16360, 16923, 17877, 18408, 18649, 18715, 18783, or Any polymorphic part in 19878
(6a) a ZNF263 gene or a site on the DNA region in the vicinity of the gene, and positions 876, 1552, 3213, 3269, 6401, 6425, 6602 in the nucleotide sequence set forth in SEQ ID NO: 6; 9595th, 10001th, 10252th, 10269th, 10294th, 11377th, 12469th, 13642th, 13643th, 13879th, 14367th, 16133th, 17084th, 1765th, or 19263 Mold part
(7a) a TNFA gene or a site on the DNA region in the vicinity of the gene, the positions 175, 2401, 2773, 4452, 4671, 4672, 5184 in the nucleotide sequence set forth in SEQ ID NO: 7; 5609, 5820, 5941, 5942, 5942, 6245, 6315, 6316, 6487, 6603, 6730, 6867, 6931, 6935, 6958, 7953, 8022 , 8133, 8482, 8628, 8650, 8656, 8827, 8829, 8865, 8938, 8945, 8994, 9205, 9269, 9275, 10001, 10002, 1023, 10363 , 10399, 10680, 10765, 10816, 110 2nd, 11565, 11194, 12669, 12936, 12963, 13024, 13128, 13154, 13289, 13538, 13539, 13594, 13632, 13646, 13688, 13822 13855, 13863, 13966, 13993, 14370, 14542, 14625, 15030, 15064, 15581, 15730, 15975, 16626, 16713, 16734, 17240, 17369 Position, polymorphic site at positions 17698, 17711, or 19348
(8a) THBS3 gene or a site on the DNA region in the vicinity of the gene, which is 3098, 3103, 3143, 3178, 4543, 6977, 7158 in the nucleotide sequence set forth in SEQ ID NO: 8; Polymorphic site at any of the positions 10001, 10445, 11728, 13012, 13514, 16404, 18088, 18708, 18776, 18989, 19465, 19769, or 19786
(9a) ABCA1 gene or a site on the DNA region in the vicinity of the gene, wherein the positions 993, 1779, 1813, 3450, 4287, 4287, 4382, 7270 in the nucleotide sequence set forth in SEQ ID NO: 9; 7528, 8383, 8783, 8798, 9798, 9254, 10001, 10999, 11122, 11720, 12473, 12560, 12591, 12664, 12916, 13403, 13605, 13691 14457, 14737, 15278, 15345, 15345, 15636, 15636, 15824, 16793, 17022, 17037, 17283, 17576, 18167, 18619, 18632, 18842 Rank, 1884 18926, 19404, 19404, 19809, 20447, 20514, 20514, 20515, 21963, 22888, 23081, 23157, 23777, 23533, 24389, 25814, 26198, 26655, 26902, 27367, 27535, 27701, 28175, 28467, 28564, 28669, 28774, 28839, 28937, 28944, 29037, 30091, 30613, 30975 , 32803, 32803, 33435, 34263, 34264, 34812, 35306, 35339, 35473, 36217, 37596, or 37855
(10a) SERPINB10 gene or a site on the DNA region in the vicinity of the gene, and positions 145, 4845, 6757, 6859, 7151, 7827, 8066 in the nucleotide sequence set forth in SEQ ID NO: 10; Any polymorphic site at positions 10001, 12885, 13227, 13432, 13563, 15439, 15447, 15729, 15759, 15585, 16094, 16330, or 18565
(11a) ALDOB gene or a site on a DNA region in the vicinity of the gene, wherein the nucleotide position is 99, 380, 396, 399, 712, 934, 1534 in the nucleotide sequence of SEQ ID NO: 11. 1612, 1948, 2079, 2101, 4161, 4318, 4318, 4318, 5091, 6623, 6718, 7796, 7859, 8190, 8203, 9104, 9621, 9642 , 10001, 10489, 11443, 11592, 11593, 11616, 11616, 11617, 12281, 12457, 12802, 13993, 14026, 14800, 16654, 16822, 17133, 17239 , 18216, 18709, 19610 One of the polymorphic sites, or 19,613 positions
(12a) a site on the CTSD gene or a nearby DNA region of the gene, and positions 813, 815, 844, 861, 954, 1026, 1159 in the nucleotide sequence set forth in SEQ ID NO: 12; 1299, 5464, 5600, 7144, 7707, 7726, 7739, 7778, 7803, 7803, 7841, 7842, 7886, 7939, 7963, 8058, 8333, 9948 , 10001, 11990, 12342, 12807, 16266, or 16771
(13a) A site on the STE gene or a DNA region in the vicinity of the gene, and in positions 57, 468, 1881, 2955, 3460, 3460, 3976, 4537 in the nucleotide sequence set forth in SEQ ID NO: 13. 4538, 4722, 4867, 4873, 4947, 5025, 7043, 7676, 8581, 9035, 9347, 9686, 9882, 9894, 10001, 10003, 12142 , 13520, 13551, 13797, 14492, 14753, 14933, 15684, 17022, 18264, 18568, 18958, 19194, 19330, 19354, 19355, 19567, 19785 Rank, 20511th, 20612th, 2 627 of, 20,936 positions, 20,961 positions, 20,971 positions, 21213-position, 21336 positions, 21,609 positions, or polymorphic sites 21914 position, or 21,983 of
(14a) ABCC8 gene or a site on the DNA region in the vicinity of the gene, and positions 6978, 7358, 7359, 7649, 7755, 7755, 8068, 8214 in the nucleotide sequence set forth in SEQ ID NO: 14. 8215, 8308, 9658, 9761, 9861, 9811, 9828, 10001, 10095, 10125, 11658, 11684, 11708, 11767, 11883, 111998, 12109, 12308 13620th, 13664th, 13912th, 14297th, 14309th, 14320th, 14323th, 14367th, 14393th, 14475th, 1459th, 14722th, 15294th, 15601th, 15657th, 16160th, 16230 Rank, 16 67 position, 16417 positions, 17,695 positions, 18213-position, 18815 positions, 19,093 positions, 20082-position, 20211-position, 20461 positions, 21,547 positions, 21995-position, or polymorphic sites 22825 position, or 23,901 of
(15a) SERPINA3 gene or a site on the DNA region in the vicinity of the gene, which is a position 937, 1145, 1878, 2345, 2559, 3559, 3250, 3513 in the nucleotide sequence set forth in SEQ ID NO: 15. 4402, 5776, 6659, 7453, 7934, 7934, 8000, 8049, 8204, 8543, 8554, 8935, 9928, 10001, 10233, 10472, 10479, 10525 , 10544, 10629, 13339, 13348, 16287, 16389, 16626, or 17880
(16a) the RCV1 gene or a site on the DNA region in the vicinity of the gene, and the 25th position, 49th position, 469th position, 557th position, 592th position, 2730th position, 2754th position in the nucleotide sequence set forth in SEQ ID NO: 16; 5761, 6154, 6183, 8510, 8529, 8542, 8590, 9838, 10001, 10132, 10133, 10649, 10702, 10866, 12560, 15632, 15527 , 15853, 15994, 16617, 16790, 16841, 17010, 17348, 17656, 17656, 17816, 17820, 17882, 17882, 18061, 18101, 18304, Any polymorphic site at position 19485
(17a) a site on a TFRC gene or a DNA region in the vicinity of the gene, and positions 836, 1019, 2511, 4016, 4068, 4169, 4169, 4269 in the nucleotide sequence set forth in SEQ ID NO: 17; 4481, 5398, 5436, 5450, 5730, 5937, 6044, 6313, 6713, 6759, 6794, 6833, 7528, 8047, 8048, 8080, 9248 , 9361, 9417, 9734, 10001, 11461, 11845, 11852, 12086, 12596, 12864, 12930, 13969, 13969, 14119, 14280, 16833, or Any polymorphic site at position 17047
(18a) a position on the MMP19 gene or a DNA region in the vicinity of the gene, the positions 6329, 7329, 7715, 8138, 8507, 8551, 8551, 8574 in the nucleotide sequence set forth in SEQ ID NO: 18. Any polymorphic site at positions 10001, 11138, 11343, 13151, 13868, or 18923
(19a) MYL2 gene or a site on the DNA region in the vicinity of the gene, and the nucleotide sequence described in SEQ ID NO: 19 is position 3147, position 3213, position 4686, position 7343, position 7789, position 7801, position 10001, position 10123, 11264th, 11992th, 15532th, 15681, 15957, 15988, 16641, 18386, 18782, 20354, 21531, 23245, 27740, 30669, 30670, 32077, 32732 32907, 32955, 42589, 43029, 47354, 64777, 71869, 71196, 74274, 75551, 76343, 76344, 78270, 78339, 78689, 78743, 78 06, 78807, 79215, 79319, 79357, 79460, 79463, 790034, 80034, 80062, 800076, 80401, 80475, 80599, 80715, 80865, 80943, 80952 80974, 80991, 81130, 81373, 81428, 81543, 81682, 82422, 82545, 82792, 82929, 83265, 83266, 83394, 83463, 83500, 83578 , 83612, 83650, 84790, 8492, 84945, 85214, 85383, 85384, 85550, 85984, 86109, 86539, 86542, 87169 87170, 87353, 87671, 87953, 89554, 88057, 88178, 88240, 88258, 88271, 88272, 88307, 88512, 88695, 88778, 88798, 89124 Or any polymorphic position at position 91081
(20a) a VLDLR gene or a site on a DNA region in the vicinity of the gene, and positions 6297, 6733, 7994, 9242, 9952, 9901, 11801, 11838 in the nucleotide sequence set forth in SEQ ID NO: 20; Any polymorphic site at positions 12995, 13224, 13277, 18702, 18896, or 19214
(21a) the P2RX7 gene or a site on the DNA region in the vicinity of the gene, and the 99th position, the 694th position, the 2765th position, the 2765th position, the 3101st position, the 3117th position, the 3127th position, 3309th, 3346th, 3802th, 3916th, 4206th, 4430th, 5937th, 6705th, 7378th, 8312th, 8399th, 9928th, 10001, 100th, 10042th, 10277th, 10440th, 10461th , 10479, 10527, 10534, 10550, 10701, 10909, 11066, 11198, 11575, 11631, 11765, 11976, 12119, 12158, 12207, 12272, 12272 Rank, 12498, 1256 , 12791, 12856, 12860, 13207, 13383, 13590, 13860, 13869, 13880, 13881, 13984, 14048, 14059, 14210, 14806, 14921, 15331, 15488, 15558, 15651, 15651, 15686, 15791, 16051, 16082, 16137, 16492, 16649, 16784, 16788, 17072, 17076, 17379, 17527 17593, 17623, 17719, 17998, 18005, 18048, 18288, 18412, 18588, 18640, 19340, 19545, 19676, or 19685 One of the polymorphic sites of
(22a) a DLG5 gene or a site on the DNA region in the vicinity of the gene, and the 2nd position, 121st position, 1438th position, 1669th position, 2180th position, 2436th position, 2543th position in the nucleotide sequence set forth in SEQ ID NO: 22; 2619, 3715, 3727, 5327, 5256, 5335, 5339, 5688, 5752, 6304, 6627, 7195, 7342, 7603, 8273, 8905, 8988, 9013 , 9057, 10001, 10372, 10453, 10468, 10468, 10512, 11038, 11055, 11597, 11681, 11719, 11922, 12034, 12049, 12176, 12554 , 13481, 13864, 14390 15044, 15598, 16176, 16870, 17475, 17649, 18077, 18585, 18619, 18677, 19222, 19731, 19750, 20758, 20962, 21073, 21680 , 21867, 22385, 22874, 23191, 24016, 24520, 24993, 25460, 26155, 26804, 26980, 27882, 29234, 29469, 30254, 30742, 30742, 30756, 30756, 31671, 31874, 31940, 32626, 33393, 33581, 34240, 35294, 35666, 36587, 36653, 36 41st, 37408th, 37583, 37612, 38462, 38470, 38470, 38485, 39146, 39146, 40612, 40641, 41242, 42217, 42539, 45934, 46033, or 46895 Any polymorphic site
(23a) a site on the LDLC gene or a nearby DNA region of the gene, and in the base sequence described in SEQ ID NO: 23, positions 1580, 1754, 1882, 3062, 3507, 4507, 4800, 4905, 5569, 5608, 5643, 5665, 5593, 5864, 5884, 5981, 6169, 6808, 6858, 7194, 7283, 7728, 7835, 8049, 8049 , 8265, 9209, 9445, 9646, 9647, 9716, 9716, 9775, 10001, 10027, 10054, 10193, 10208, 10826, 10908, 10997, 11336, 11754 , 11895, 12031, 12327, 2464, 12561, 14051, 15366, 15778, 17723, 17772, 17783, 17811, 17815, 17862, 17900, 17962, 18184, 18189, 18297, 18411 , 18512, 18563, 19125, 19152, 200092, 21227, 21506, 21617, 21855, 23090, 25700, 26468, 26638, 27682, 27755, 27888, 27973 Position, 27995, 28123, 28127, 28144, 28153, or 28271 polymorphic site
(24a) AQP8 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence set forth in SEQ ID NO: 24, positions 6722, 9643, 9934, 10001, 10002, 13682, 14629, 15376, Polymorphic site at either position 15808 or 16082
(25a) a LARGE gene or a site on a DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 25, positions 1816, 3116, 3117, 3118, 3154, 3157, 3191, 3197, 3205, 3291, 3622, 3652, 3652, 3792, 4488, 5076, 5474, 5481, 5492, 5503, 5654, 5672, 6563, 6843, 6908 7119, 7538, 7538, 7721, 8403, 8626, 10001, 12636, 13103, 14794, 15370, 15448, 15599, 15564, 16045, 16261, 16277 Rank, 16861, 17360, 17611 17714, 17809, 18221, 18221, 18376, 18536, 19063, 22294, 23404, 23612, 25125, 25134, 25662, 25705, 25751, 27407, 28166 , 28567, 30302, 30316, 30353, 30354, 30391, 33594, 33650, 33754, 36690, 37753, 37895, 37900, 39104, 39745, 41404, 41646, 41711, 42020, 42080, 42974, 43052, 43240, 43621, 44734, 44958, 46920, 47431, 47573, 48101, 48 Position 27, 48306-position, 48,807 positions, 49049 positions, 49,848 positions, 49,860 positions, 49,903 positions, 50,114 positions, 50,223 positions, 52,057 positions, 52,543 positions, one of the polymorphic sites of 52,693 positions, or 53,061 of
(26a) a BLZF1 gene or a site on the DNA region in the vicinity of the gene, and positions 1283, 4288, 7520, 7599, 7740, 7745, 8745 and 9050 in the nucleotide sequence set forth in SEQ ID NO: 26; 9862, 10001, 10124, 10168, 10168, 10339, 11017, 13737, 13747, 18354, 20031, 20072, 22094, 24082, 24305, 24478, 24552, 25273 , 25351, 29096, 29100, 2976
(27a) RGS5 gene or a site on the DNA region in the vicinity of the gene, which is a position of 733, 848, 1039, 1351, 1569, 1831, 2078 in the nucleotide sequence set forth in SEQ ID NO: 27; 2707, 2794, 2904, 2995, 2995, 3122, 3520, 4351, 4824, 5189, 5205, 5206, 5525, 5779, 6487, 7712, 10001, 10208 , 10235, 10868, 11092, 11617, 11666, 12216, 12326, 12741, 13396, 13890, 14702, 15823, 16705, or 19086
(28a) the FRAP1 gene or a site on the DNA region in the vicinity of the gene, and positions 3455, 7826, 8715, 8783, 9042, 9390, 9462 in the nucleotide sequence set forth in SEQ ID NO: 28; 9709, 10001, 10635, 10748, 12,794, 12849, 12,915, 13,169, 17020, 18586, 21586, 21123, or 21128 polymorphic sites,
Or
(28a-2) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence described in SEQ ID NO: 30, positions 9598, 10001, 10306, 11181, 11480, 13593, 13721 Position, 13792, 15314, 15623, 15782, 16708, 16748, or 19630 polymorphic sites
(29a) a LEP gene or a site on a DNA region in the vicinity of the gene, the positions 244, 575, 675, 1487, 1644, 2343, 2715 in the nucleotide sequence set forth in SEQ ID NO: 29; 2,763, 3098, 3589, 3880, 5787, 5865, 6436, 7392, 7995, 8161, 8544, 9230, 9795, 10001, 10199, 10503, 111975 , 14981, 15681, or 19300
[5] The method according to [3], wherein the polymorphic site is a polymorphic site described in the following (1b) to (29b),
(1b) A site on the MET gene or a nearby DNA region of the gene, and the site at position 14258 or 19768 in the nucleotide sequence set forth in SEQ ID NO: 1.
(2b) a site on the NPHS1 gene or a DNA region in the vicinity of the gene, the 10001 position or the 17848 position in the nucleotide sequence set forth in SEQ ID NO: 2
(3b) A site on the SLC1A1 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 3
(4b) A site on the SERPINB2 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 4
(5b) a site on the FABP2 gene or a nearby DNA region of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 5
(6b) A site on the ZNF263 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 6
(7b) A site on the TNFA gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 7
(8b) a site on the THBS3 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 8
(9b) A site on the ABCA1 gene or a nearby DNA region of the gene, and the site at position 17283 in the base sequence set forth in SEQ ID NO: 9
(10b) a site on the SERPINB10 gene or a nearby DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 10
(11b) A site on the ALDOB gene or a nearby DNA region of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 11
(12b) a site on the CTSD gene or a DNA region in the vicinity of the gene, which is located at position 11990 or position 16771 in the nucleotide sequence set forth in SEQ ID NO: 12
(13b) a site on the STE gene or a DNA region in the vicinity of the STE gene, and a site at position 18958 or 21213 in the nucleotide sequence set forth in SEQ ID NO: 13
(14b) a site on the ABCC8 gene or a DNA region in the vicinity of the gene, which is located at position 14475 in the base sequence set forth in SEQ ID NO: 14
(15b) a site on the SERPINA3 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 15
(16b) A site on the RCV1 gene or a DNA region in the vicinity of the gene, the 10001 position in the nucleotide sequence set forth in SEQ ID NO: 16
(17b) A site on the TFRC gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 17
(18b) a site on the MMP19 gene or in the vicinity DNA region of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 18
(19b) A site on the MYL2 gene or a nearby DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 19
(20b) a site on the VLDLR gene or a DNA region in the vicinity of the gene, the 10001 position in the nucleotide sequence set forth in SEQ ID NO: 20
(21b) a site on the P2RX7 gene or a nearby DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 21
(22b) a site on the DLG5 gene or in the vicinity DNA region of the gene, the 10001 position or the 37583 position in the nucleotide sequence set forth in SEQ ID NO: 22
(23b) a site on the LDLC gene or a nearby DNA region of the gene, and the site at position 18563 in the base sequence set forth in SEQ ID NO: 23
(24b) A site on the AQP8 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 24
(25b) A site on the LARGE gene or a DNA region in the vicinity of the gene, the site at positions 10001, 13103, or 15448 in the nucleotide sequence set forth in SEQ ID NO: 25
(26b) a site on the BLZF1 gene or in the vicinity DNA region of the gene, the site at position 20031 in the base sequence set forth in SEQ ID NO: 26
(27b) A site on the RGS5 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 27
(28b) a site on the FRAP1 gene or in the vicinity DNA region of the gene, the site at position 17020 in the base sequence set forth in SEQ ID NO: 28
(28b-2) a site on the FRAP1 gene or in the vicinity DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 30
(29b) a site on the LEP gene or a DNA region in the vicinity of the gene, the site at position 10503 in the base sequence set forth in SEQ ID NO: 29
[6] When the base type in the polymorphic site according to (1b) to (29b) in [5] is the following (1c) to (29c), respectively, it is type 2 diabetes (type 2 diabetes susceptibility) Or having a predisposition to susceptibility to type 2 diabetes), the method according to [5],
(1c) A site on the MET gene or a nearby DNA region of the gene, wherein the base species at position 14258 in the base sequence described in SEQ ID NO: 1 is T, or the base species at position 19768 is T
(2c) A site on the NPHS1 gene or a nearby DNA region of the gene, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 2 is A, or the base type at position 17848 is A
(3c) The SLC1A1 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 3 is G
(4c) The SERPINB2 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 24578 in the nucleotide sequence set forth in SEQ ID NO: 4 is A
(5c) a site on the FABP2 gene or a DNA region in the vicinity of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 5 is A
(6c) a ZNF263 gene or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 6 is G
(7c) A site on the TNFA gene or a nearby DNA region of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 7 is A
(8c) a THBS3 gene or a site on a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 8 is C
(9c) The ABCA1 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 17283 in the nucleotide sequence set forth in SEQ ID NO: 9 is T
(10c) The SERPINB10 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 10 is G
(11c) A region on the ALDOB gene or a DNA region in the vicinity of the ALDOB gene, wherein the base species at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 11 is T
(12c) A site on the CTSD gene or a nearby DNA region of the gene, wherein the base type at position 11990 in the base sequence described in SEQ ID NO: 12 is A, or the base type at position 16771 is C
(13c) A site on the STE gene or a nearby DNA region of the gene, wherein the base species at position 18958 in the base sequence described in SEQ ID NO: 13 is G, or the base species at position 21213 is C
(14c) the ABCC8 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 14475 in the nucleotide sequence set forth in SEQ ID NO: 14 is A
(15c) The SERPINA3 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 15 is A
(16c) The RCV1 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 16 is T
(17c) a site on the TFRC gene or a nearby DNA region of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 17 is G
(18c) The MMP19 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 18 is A
(19c) a site on the MYL2 gene or a nearby DNA region of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 19 is T
(20c) a VLDLR gene or a site on a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 20 is T
(21c) a P2RX7 gene or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 21 is C
(22c) a site on the DLG5 gene or a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 22 is G, or the base type at position 37583 is C
(23c) a region on the LDLC gene or a nearby DNA region of the gene, wherein the base type at position 18563 in the base sequence set forth in SEQ ID NO: 23 is G
(24c) a site on the AQP8 gene or a nearby DNA region of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 24 is G
(25c) a site on the LARGE gene or a nearby DNA region of the gene, wherein the base type at position 10001 is T, the base type at position 13103 is G, or the base at position 15448 in the base sequence described in SEQ ID NO: 25 The seed is G
(26c) a BLZF1 gene or a site on a DNA region in the vicinity of the gene, wherein the nucleotide type at position 20031 in the nucleotide sequence set forth in SEQ ID NO: 26 is C
(27c) the RGS5 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 27 is G
(28c) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 17020 in the base sequence set forth in SEQ ID NO: 28 is G
(28c-2) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 30 is G
(29c) the LEP gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10503 in the base sequence set forth in SEQ ID NO: 29 is G
[7] A method for examining type 2 diabetes (whether it is type 2 diabetes susceptibility or a predisposition to type 2 diabetes susceptibility), comprising the following steps (a) and (b):
(A) a step of determining a base type for a polymorphic site on the gene according to any one of the following (1) to (14) or a DNA region adjacent to the gene in a subject;
(1) MET, (2) NPHS1, (3) SERPINB2, (4) ABCA1, (5) CTSD, (6) STE, (7) ABCC8, (8) MYL2, (9) DLG5, (10) LDLC, (11) LARGE, (12) BLZF1, (13) FRAP1, (14) LEP
(B) The base species determined in the step (a) is a polymorphic site (haplotype in the gene or the DNA region in the vicinity of the gene showing the haplotype described in any of the following (1 ′) to (14 ′): Step to compare with the base type of the polymorphic site on the block)
(1 ′) Polymorphic sites on the MET gene, and the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the base sequence set forth in SEQ ID NO: 1 are G, T, and T, respectively. Haplotype, which is
A haplotype that is a polymorphic site on the MET gene, wherein the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the nucleotide sequence set forth in SEQ ID NO: 1 are G, C, and C, respectively Or
A haplotype that is a polymorphic site on the MET gene, wherein the base types of the polymorphic sites at positions 19768 and 24209 in the base sequence set forth in SEQ ID NO: 1 are T and T, respectively.
(2 ′) Polymorphic sites on the NPHS1 gene, wherein the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2 are A, C, and A, respectively. Haplotype, which is
A haplotype that is a polymorphic site on the NPHS1 gene, wherein the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2 are G, G, and G, respectively Or
A haplotype that is a polymorphic site on the NPHS1 gene, and whose base types of the polymorphic sites at positions 10001, 16198, and 17848 in the nucleotide sequence set forth in SEQ ID NO: 2 are G, C, and G, respectively
(3 ′) a haplotype that is a polymorphic site on the SERPINB2 gene, wherein the base types of the polymorphic sites at positions 10001 and 24578 in the base sequence set forth in SEQ ID NO: 4 are C and A, or
A haplotype that is a polymorphic site on the SERPINB2 gene, wherein the base types of the polymorphic sites at positions 10001 and 24578 in the base sequence described in SEQ ID NO: 4 are T and T, respectively.
(4 ′) Polymorphic sites on the ABCA1 gene, wherein the base types of the polymorphic sites at positions 10001, 15824, 17283, 20515, and 28564 in the base sequence set forth in SEQ ID NO: 9 are A haplotype that is C, A, C, T, and G, or
A haplotype that is a polymorphic site on the ABCA1 gene, wherein the base types of the polymorphic sites at positions 10001 and 11000 in the nucleotide sequence set forth in SEQ ID NO: 71 are G and C, respectively.
(5 ′) Polymorphic sites on the CTSD gene, wherein the base types of the polymorphic sites at positions 10001, 11990, and 16771 in the base sequence set forth in SEQ ID NO: 12 are C, G, and T, respectively. Is a haplotype
(6 ′) Polymorphic sites on the STE gene, wherein the base types of the polymorphic sites at positions 10001, 18958, 19354, 19567, and 20971 in the base sequence set forth in SEQ ID NO: 13 are A haplotype that is A, G, T, T, and C, or
A haplotype that is a polymorphic site on the STE gene and whose base types of the polymorphic sites at positions 20971 and 21213 in the base sequence set forth in SEQ ID NO: 13 are C and C, respectively.
(7 ′) A haplotype that is a polymorphic site on the ABCC8 gene, and whose base type of the polymorphic site at position 14475 in the base sequence described in SEQ ID NO: 14 is A
(8 ′) Polymorphic sites on the MYL2 gene, and the base types of the polymorphic sites at positions 10001, 18386, 42589, and 83500 in the nucleotide sequence set forth in SEQ ID NO: 19 are C and T, respectively. , T, and C haplotypes
(9 ′) Polymorphic sites on the DLG5 gene, the base types of the polymorphic sites at positions 10001, 24016, 29469, and 37583 in the base sequence set forth in SEQ ID NO: 22 are G and C , G, and C haplotypes, polymorphic sites on the DLG5 gene, and the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence set forth in SEQ ID NO: 22 are C Haplotypes that are G, C, or
A haplotype that is a polymorphic site on the DLG5 gene, wherein the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence set forth in SEQ ID NO: 22 are C, G, and T, respectively
(10 ′) a haplotype that is a polymorphic site on the LDLC gene, wherein the base species of the polymorphic site at positions 10001 and 18563 in the nucleotide sequence set forth in SEQ ID NO: 23 are G and A, or
Haplotypes that are polymorphic sites on the LDLC gene, and whose base types of the polymorphic sites at positions 10001 and 18563 in the nucleotide sequence set forth in SEQ ID NO: 23 are G and G, respectively
(11 ′) Polymorphic sites on the LARGE gene, wherein the base types of the polymorphic sites at positions 10001, 13103, 15448, 36690, and 43240 in the nucleotide sequence set forth in SEQ ID NO: 25 are Haplotypes that are T, G, G, T, and A
(12 ′) A haplotype that is a polymorphic site on the BLZF1 gene, wherein the base types of the polymorphic sites at positions 10001 and 20031 in the base sequence set forth in SEQ ID NO: 26 are G and T, respectively.
(13 ′) A haplotype that is a polymorphic site on the FRAP1 gene and the base type of the polymorphic site at position 17020 in the base sequence set forth in SEQ ID NO: 28 is G
(14 ′) A haplotype that is a polymorphic site on the LEP gene, and whose base types of the polymorphic sites at positions 10001 and 10503 in the nucleotide sequence set forth in SEQ ID NO: 29 are G and A, respectively
In the above method, for example, for a test sample obtained from the test subject, 2 subjects have used the presence or absence of the haplotype (block) described in any one of (1 ′) to (14 ′) as an index. Is a method for testing whether or not there is a predisposition to susceptibility or resistance to type 2 diabetes,
[8] [7] The gene according to (1) to (14) of (a) or a polymorphic site in a DNA region adjacent to the gene is any of the following (1) to (14): The inspection method according to [7], which is a polymorphic site,
(1) The MET gene or a site on the DNA region in the vicinity of the gene, which is the position 765, position 1383, position 1890, position 2099, position 2814, position 3293, position 3752 in the nucleotide sequence set forth in SEQ ID NO: 1. 3774, 3927, 4690, 7419, 8219, 8201, 10001, 10318, 13766, 13789, 14258, 15427, 16275, 16597, 16932, 19768, 19835, 230032 , 20240, 20537, 20885, 21546, 24209, 24271, 24376, 24642, 26028, 26226, or 34209
(2) a site on the NPHS1 gene or a DNA region in the vicinity of the gene, the positions 172, 186, 211, 2554, 2558, 2558, 10001, 10001 in the nucleotide sequence set forth in SEQ ID NO: 2; 10176th, 11840th, 13759th, 14645th, 14967th, 16198th, 16439th, 17635th, 17685th, 17794th, 17805th, 17848th, 17849th, 18491th, 19955th, 20955th, 20613th 21533, 21582, 21811, 21812, 22190, 22965, 22982, 23660, 23780, 24040, 24172, 25334, or 26585 polymorphic sites
(3) SERPINB2 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 4, positions 2853, 2891, 5218, 5317, 7560, 8054, 8771, 10001 , 10329, 11781, 12063, 12705, 13125, 13691, 13913, 13916, 13917, 14123, 14169, 14453, 14456, 14483, 16526, 16556, 17643, 17725, 17824, 18076, 18227, 18500, 18687, 18741, 18744, 18875, 19045, 19141, 19149, 19190, 19429, 19548, 1956 , 19624, 19645, 19814, 20049, 20082, 20097, 20243, 22122, 22495, 22516, 22661, 22816, 23007, 23904, 24005, 24418, Any of the polymorphic sites at positions 24578, 24855, 24855, 25505, 25538, 25564, 25586, 26017, 26234, 26557, 26621, 30155, 30267, or 30607
(4) ABCA1 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 9, positions 993, 1779, 1813, 3450, 4287, 4382, 7270, 7528, 8383, 8783, 8798, 9798, 9254, 10001, 10999, 11122, 11720, 12473, 12560, 12591, 12664, 12916, 13403, 13605, 13691 14457, 14737, 15278, 15345, 15345, 15636, 15636, 15824, 16793, 17022, 17037, 17283, 17576, 18167, 18619, 18632, 18842 Rank, 18843 18926, 19404, 19404, 19809, 20447, 20514, 20515, 20315, 21963, 22882, 23081, 23157, 23579, 23553, 24389, 25814, 26198, 26655 , 26902, 27367, 27535, 27701, 28175, 28467, 28564, 28669, 28774, 28939, 28937, 28944, 29037, 30091, 30613, 30975, Any of the polymorphic sites at positions 32803, 32803, 33435, 34263, 34264, 34812, 35306, 35339, 35473, 36217, 37596, or 37855, or
ABCA1 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 71, positions 1206, 1905, 1979, 2008, 2054, 2286, 2303, 2808, 3176, 3413, 3415, 3548, 3548, 3669, 3756, 3983, 4521, 4544, 4577, 4609, 5226, 5833, 5868, 6422, 6532, 6583 6696th, 7043th, 7198th, 7324th, 7395th, 7484th, 7716th, 7787th, 7787th, 8197th, 8350th, 9541th, 9643th, 9793th, 100001th, 10348th, 10415th, 10741th , 10826, 11000, 11024, 1112 , 11185, 11286, 11452, 11471, 11653, 11746, 11858, 12114, 12225, 12520, 12725, 12896, 12899, 12905, 13118, 13492, 13589th, 13836th, 14226th, 14391th, 14489th, 14536th, 14813th, 15071th, 15215th, 15377th, 15399th, 15644th, 15687th, 15830th, 16103th, 16112th, 16139th 16754, 17303, 18237, 18379, 18618, 18903, 18921, 19014, 19034, 19074, 19149, 19165, 19167, 19365, 1 373 of, 19,376 positions, 19,386 positions, one of the polymorphic sites of the 19487 position or 19659 place
(5) a CTSD gene or a site on a DNA region in the vicinity of the gene, and positions 813, 815, 844, 861, 954, 1026, 1159 in the nucleotide sequence set forth in SEQ ID NO: 12; 1299, 5464, 5600, 7144, 7707, 7726, 7739, 7778, 7803, 7803, 7841, 7842, 7886, 7939, 7963, 8058, 8333, 9948 , 10001, 11990, 12342, 12807, 16266, or 16771
(6) A site on the STE gene or a DNA region in the vicinity of the gene, and in positions 57, 468, 1881, 2955, 3460, 3460, 3976, 4537 in the nucleotide sequence set forth in SEQ ID NO: 13. 4538, 4722, 4867, 4873, 4947, 5025, 7043, 7676, 8581, 9035, 9347, 9686, 9882, 9894, 10001, 10003, 12142 , 13520, 13551, 13797, 14492, 14753, 14933, 15684, 17022, 18264, 18568, 18958, 19194, 19330, 19354, 19355, 19567, 19785 Rank, 20511th, 20612th, 206 7 position, 20,936 positions, 20,961 positions, 20,971 positions, 21213-position, 21336 positions, 21,609 positions, or polymorphic sites 21914 position, or 21,983 of
(7) ABCC8 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence described in SEQ ID NO: 14, positions 6978, 7358, 7359, 7649, 7755, 8068, 8214, 8215, 8308, 9658, 9761, 9861, 9811, 9828, 10001, 10095, 10125, 11658, 11684, 11708, 11767, 11883, 111998, 12109, 12308 13620th, 13664th, 13912th, 14297th, 14309th, 14320th, 14323th, 14367th, 14393th, 14475th, 1459th, 14722th, 15294th, 15601th, 15657th, 16160th, 16230 Rank, 1636 Position, 16417 positions, 17,695 positions, 18213-position, 18815 positions, 19,093 positions, 20082-position, 20211-position, 20461 positions, 21,547 positions, 21995-position, or polymorphic sites 22825 position, or 23,901 of
(8) MYL2 gene or a site on the DNA region in the vicinity of the gene, and the 3147, 3213, 4686, 7343, 7892, 10001, 1001, 10123 positions in the nucleotide sequence set forth in SEQ ID NO: 19; 11264th, 11992th, 15532th, 15681, 15957, 15988, 16641, 18386, 18782, 20354, 21531, 23245, 27740, 30669, 30670, 32077, 32732 , 32907, 32955, 42589, 43029, 47354, 64777, 71869, 71196, 74274, 75551, 76343, 76344, 78270, 78339, 78689, 78743, 7880 , 78807, 79215, 79319, 79357, 79460, 79463, 80034, 80062, 80076, 80401, 80475, 80599, 80715, 80865, 80943, 80952, 80974, 80991, 81130, 81373, 81428, 81543, 81682, 82422, 82545, 82792, 82929, 83265, 83266, 83394, 83463, 83500, 83578 , 83612, 83650, 84790, 8492, 84945, 85214, 85383, 85384, 85550, 85984, 86109, 86539, 86542, 87169, 8169 170, 87353, 87671, 87953, 89554, 88057, 88178, 88240, 88258, 88271, 88272, 88307, 88512, 88695, 88778, 88798, 89124 Or any polymorphic position at position 91081
(9) a DLG5 gene or a site on the DNA region in the vicinity of the gene, and the 2nd, 121st, 1438th, 1669th, 2180th, 2436th, 2543th position in the nucleotide sequence set forth in SEQ ID NO: 22; 2619, 3715, 3727, 5327, 5256, 5335, 5339, 5688, 5752, 6304, 6627, 7195, 7342, 7603, 8273, 8905, 8988, 9013 , 9057, 10001, 10372, 10453, 10468, 10468, 10512, 11038, 11055, 11597, 11681, 11719, 11922, 12034, 12049, 12176, 12554 , 13481, 13864, 14390, 5044, 15598, 16176, 16870, 17475, 17649, 18077, 18585, 18619, 18677, 19222, 19731, 19750, 20758, 20962, 21073, 21680 21867th, 22385th, 22874th, 23191st, 24016th, 24520th, 24993th, 25460th, 26155th, 26804th, 26980th, 27882th, 29234th, 29469th, 30254th, 30742th, 30742 , 30756, 30756, 31671, 31874, 31940, 32626, 33393, 33581, 34240, 35294, 35666, 36587, 36653, 3694 , 37408, 37583, 37612, 38462, 38470, 38485, 39065, 39146, 40612, 40641, 41242, 42217, 42539, 45934, 46033, or 46895 Any polymorphic site of
(10) LDLC gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 23, positions 1580, 1754, 1882, 3062, 3507, 4507, 4800, 4905, 5569, 5608, 5643, 5665, 5593, 5864, 5884, 5981, 6169, 6808, 6858, 7194, 7283, 7728, 7835, 8049, 8049 , 8265, 9209, 9445, 9646, 9647, 9716, 9716, 9775, 10001, 10027, 10054, 10193, 10208, 10826, 10908, 10997, 11336, 11754 Rank, 11895, 12031, 12327, 1 464, 12561, 14051, 15366, 15778, 17723, 17772, 17783, 17811, 17815, 17862, 17900, 17962, 18184, 18189, 18297, 18411 , 18512, 18563, 19125, 19152, 200092, 21227, 21506, 21617, 21855, 23090, 25700, 26468, 26638, 27682, 27755, 27888, 27973 Position, 27995, 28123, 28127, 28144, 28153, or 28271 polymorphic site
(11) LARGE gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 25, position 1816, position 3116, position 3117, position 3118, position 3154, position 3157, position 3191, 3197, 3205, 3291, 3622, 3652, 3652, 3792, 4488, 5076, 5474, 5481, 5492, 5503, 5654, 5672, 6563, 6843, 6908 7119, 7538, 7538, 7721, 8403, 8626, 10001, 12636, 13103, 14794, 15370, 15448, 15599, 15564, 16045, 16261, 16277 Rank, 16861, 17360, 17611 17714, 17809, 18221, 18221, 18221, 18376, 18536, 19063, 22294, 23404, 23612, 25125, 25134, 25662, 25705, 25751, 27407, 28166 28567, 30302, 30316, 30353, 30354, 30391, 33594, 33650, 33754, 36690, 37753, 37895, 37900, 39104, 39745, 41404, 41646 Rank, 41711, 42020, 42080, 42974, 43052, 43240, 43621, 44734, 44958, 46920, 47431, 47573, 48101, 481 7 position, 48,306 positions, 48,807 positions, 49049 positions, 49,848 positions, 49,860 positions, 49,903 positions, 50,114 positions, 50,223 positions, 52,057 positions, 52,543 positions, one of the polymorphic sites of 52,693 positions, or 53,061 of
(12) a BLZF1 gene or a site on a DNA region in the vicinity of the gene, and positions 1283, 4288, 7520, 7599, 7740, 8745, 9050 in the nucleotide sequence set forth in SEQ ID NO: 26; 9862, 10001, 10124, 10168, 10168, 10339, 11017, 13737, 13747, 18354, 20031, 20072, 22094, 24082, 24305, 24478, 24552, 25273 , 25351, 29096, 29100, 2976
(13) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, and positions 3455, 7826, 8715, 8783, 9042, 9390, 9462 in the nucleotide sequence set forth in SEQ ID NO: 28; 9709, 10001, 10635, 10748, 12794, 12849, 12915, 13169, 17020, 18586, 21586, 21123, or 21128 polymorphic sites
(14) a LEP gene or a site on a DNA region in the vicinity of the gene, wherein the positions 244, 575, 675, 1487, 1644, 2343, 2715 in the nucleotide sequence set forth in SEQ ID NO: 29; 2,763, 3098, 3589, 3880, 5787, 5865, 6436, 7392, 7995, 8161, 8544, 9230, 9795, 10001, 10199, 10503, 111975 , 14981, 15681, or 19300
[9] [7] The polymorphic site in the gene according to (1) to (14) of (a) is the polymorphic site according to any of the following (1) to (14), [ 7],
(1) A polymorphic site on the MET gene, the polymorphic site at positions 10001, 14258, 19768, or 24209 in the nucleotide sequence set forth in SEQ ID NO: 1.
(2) A polymorphic site on the NPHS1 gene, the polymorphic site at positions 10001, 16198 or 17848 in the nucleotide sequence set forth in SEQ ID NO: 2.
(3) A polymorphic site on the SERPINB2 gene, which is a polymorphic site at position 10001 or 24578 in the nucleotide sequence set forth in SEQ ID NO: 4.
(4) a polymorphic site on the ABCA1 gene, the polymorphic site at positions 10001, 15824, 17283, 20515, or 28564 in the nucleotide sequence set forth in SEQ ID NO: 9; or
Polymorphic sites on ABCA1 gene, polymorphic sites at positions 10001 and 11000 in the nucleotide sequence set forth in SEQ ID NO: 71
(5) A polymorphic site on the CTSD gene, which is a polymorphic site at positions 10001, 11990, or 16771 in the nucleotide sequence set forth in SEQ ID NO: 12.
(6) A polymorphic site on the STE gene, the polymorphic site at positions 10001, 18958, 19354, 19567, 20971, or 21213 in the nucleotide sequence set forth in SEQ ID NO: 13
(7) A polymorphic site on the ABCC8 gene, which is the polymorphic site at position 10001 or 14475 in the nucleotide sequence set forth in SEQ ID NO: 14
(8) A polymorphic site on the MYL2 gene, the polymorphic site at positions 10001, 18386, 42589, or 83500 in the nucleotide sequence set forth in SEQ ID NO: 19
(9) A polymorphic site on the DLG5 gene, the polymorphic site at positions 10001, 24016, 29469, or 37583 in the nucleotide sequence set forth in SEQ ID NO: 22
(10) A polymorphic site on the LDLC gene, the polymorphic site at position 10001 or 18563 in the nucleotide sequence set forth in SEQ ID NO: 23
(11) A polymorphic site on the LARGE gene, the polymorphic site at positions 10001, 13103, 15448, 36690, or 43240 in the nucleotide sequence set forth in SEQ ID NO: 25
(12) A polymorphic site on the BLZF1 gene, the polymorphic site at position 10001 or 20031 in the nucleotide sequence set forth in SEQ ID NO: 26
(13) A polymorphic site on the FRAP1 gene, the polymorphic site at position 10001 or 17020 in the nucleotide sequence set forth in SEQ ID NO: 28
(14) A polymorphic site on the LEP gene, the polymorphic site at position 10001 or 10503 in the nucleotide sequence set forth in SEQ ID NO: 29
[10] The method according to any one of [3] to [9], further comprising a step of preparing DNA containing a polymorphic site from a biological sample of a subject.
The present invention also provides an oligonucleotide for type 2 diabetes test for use in the test method.
More specifically, for example, an oligonucleotide primer or an oligonucleotide probe used in the above inspection method can be mentioned. More specifically,
[11] A diagnostic agent for type 2 diabetes comprising an oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to the DNA containing the polymorphic site according to any one of (1a) to (29a) in [4] (Test reagent),
[12] Type 2 diabetes test agent (reagent for test) comprising a solid phase to which a nucleotide probe that hybridizes with the DNA containing the polymorphic site according to any one of (1a) to (29a) of [4] is fixed ),
[13] A test agent for type 2 diabetes (test reagent) comprising a primer oligonucleotide for amplifying a DNA containing the polymorphic site according to any one of (1a) to (29a) in [4] It is to provide.
The present inventors have identified a gene (DM-related gene) associated with type 2 diabetes (DM). In patients with DM, mutations are found significantly on the gene, so it is possible to examine whether or not the subject is DM by examining the presence or absence of mutations on the gene. .
Table 1 shows the name of the DM-related gene of the present invention, the position of the gene on the chromosome, and the GenBank accession number of the gene. Information on the base sequences of these genes and the amino acid sequences of the proteins encoded by the genes can be easily obtained from the GenBank accession numbers shown in Table 1. Moreover, those skilled in the art can easily obtain information on the base sequence of a gene and the amino acid sequence of a protein encoded by the gene from a public gene database or literature database based on the gene notation (gene name) shown in Table 1. It is possible to obtain.

The present invention provides a test method for type 2 diabetes characterized by detecting a mutation in a gene according to any one of the following (1) to (29) for a subject.
(1) MET, (2) NPHS1, (3) SLC1A1, (4) SERPINB2, (5) FABP2, (6) ZNF263, (7) TNF, (8) THBS3, (9) ABCA1, (10) SERPINB10, (11) ALDOB, (12) CTSD, (13) STE, (14) ABCC8, (15) SERPINA3, (16) RCV1, (17) TFRC, (18) MMP19, (19) MYL2, (20) VLDLR, (21) P2RX7, (22) DLG5, (23) LDLC, (24) AQP8, (25) LARGE, (26) BLZF1, (27) RGS5, (28) FRAP1, (29) LEP
The “gene according to any one” of the present invention means at least one gene of any one of (1) to (29) above. That is, the present invention includes cases where type 2 diabetes is tested by detecting mutations in the plurality of genes described in (1) to (29) above.
In the present invention, “examination of type 2 diabetes” refers to whether or not a subject has a high or low possibility of suffering from type 2 diabetes, and if the subject is already suffering from DM, the cause and the type of the disease are determined. Includes an inspection to do. In the method of the present invention, when a mutation is detected in each of the genes (1) to (29), it is determined that the subject is likely to suffer from type 2 diabetes. Even a subject who has already suffered from type 2 diabetes can determine the cause and type of onset of type 2 diabetes, and can be used to determine a treatment policy.
Although it is difficult to predetermine the position of the “mutation” in the method of the present invention, it is usually in the ORF of the gene or a region that controls the expression of the gene (eg, promoter region, enhancer region, etc.) It is in. In addition, the “mutation” is a mutation that changes the expression level of the gene, changes properties such as the stability of mRNA, or changes the activity of the protein encoded by the gene. There is no particular limitation, and examples include base addition, deletion, substitution, and insertion mutation.
The present inventors have succeeded in finding a polymorphic mutation significantly related to DM in each of the above-mentioned genes (1) to (29) or a DNA region near the gene in DM patients. Therefore, the DM can be examined by using the presence or absence of mutation as an index (determining the base type) for each of the genes (1) to (29) or the polymorphic site in the vicinity of the gene. Is possible. The above-mentioned “near DNA region of the gene” usually refers to a region on the chromosome in the vicinity of the gene. The vicinity is not particularly limited, but is usually a DNA region containing the polymorphic site of the present invention, and preferably refers to a region within 10 kb from the end portion of the gene.
Polymorphisms in the range of 10 kb before and after, that is, 20 kb, are highly likely to be linked as reported by Gabriel et al. (Gabriel SB, Schaffner SF, Nguyen H et al. The structure of haplotypes 2 blocks in the block 96. , 2225-9.2002).
The genes (1) to (29) above and the DNA sequences in the vicinity of the genes are shown in SEQ ID NOs: 1 to 29, respectively. (That is, the nucleotide sequence described in SEQ ID NO: 1 represents the MET gene and the DNA sequence in the vicinity of the gene.) Further, SEQ ID NO: 30 includes the FRAP gene represented by SEQ ID NO: 28 and the gene. The FRAP gene and the DNA sequence in the vicinity of the gene other than the nearby DNA region are shown.
In a preferred embodiment of the present invention, there is provided a method for testing type 2 diabetes, which comprises detecting a single nucleotide polymorphism mutation in the gene according to any one of (1) to (29) above.
A polymorphism is generally defined genetically as a change in a base in one gene that is present at a frequency of 1% or more in the population, but the “polymorphism” in the present invention is this definition. Not limited to. Examples of the polymorphism in the present invention include, but are not limited to, a single nucleotide polymorphism and a polymorphism in which one to several tens of bases (sometimes several thousand bases) are deleted or inserted. Furthermore, the number of polymorphic sites is not limited to one, and may have a plurality of polymorphisms.
In another aspect of the present invention, the base type of the polymorphic site in the gene according to any one of the above (1) to (29) or a DNA region adjacent to the gene is determined. Diabetes testing method.
The “polymorphic site” in the above-described method of the present invention is not particularly limited as long as it is a polymorphism present on each of the genes (1) to (29) or a DNA region in the vicinity of the gene. Specifically, as polymorphic sites that can be used in the test method of the present invention, the following (1a) to (29a) that are present on each of the genes (1) to (29) or on a DNA region near the gene: Mention may be made of polymorphic sites. (In the present specification, these polymorphic sites are sometimes simply referred to as “polymorphic sites of the present invention”.)
(1a) a MET gene or a site on a DNA region in the vicinity of the gene, the positions 765, 1383, 1890, 2099, 2814, 3293, 3752 in the nucleotide sequence set forth in SEQ ID NO: 1; 3774, 3927, 4690, 7419, 8219, 8201, 10001, 10318, 13766, 13789, 14258, 15427, 16275, 16597, 16932, 19768, 19835, 230032 , 20240, 20537, 20885, 21546, 24209, 24271, 24376, 24642, 26028, 26226, or 34209
(2a) the NPHS1 gene or a site on the DNA region in the vicinity of the gene, the positions 172, 186, 211, 2554, 2558, 2558, 10001, 10001 in the nucleotide sequence set forth in SEQ ID NO: 2; 10176th, 11840th, 13759th, 14645th, 14967th, 16198th, 16439th, 17635th, 17685th, 17794th, 17805th, 17848th, 17849th, 18491th, 19955th, 20955th, 20613th 21533, 21582, 21811, 21812, 22190, 22965, 22982, 23660, 23780, 24040, 24172, 25334, or 26585 polymorphic sites
(3a) SLC1A1 gene or a site on the DNA region in the vicinity of the gene, and positions 1142, 2732, 2734, 7220, 7001, 12001, 12276, 12922 in the nucleotide sequence set forth in SEQ ID NO: 3. Or any polymorphic site at position 18049
(4a) SERPINB2 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence of SEQ ID NO: 4, positions 2853, 2891, 5218, 5317, 7560, 8054, 8771, 10001 , 10329, 11781, 12063, 12705, 13125, 13691, 13913, 13916, 13917, 14123, 14169, 14453, 14456, 14483, 16526, 16556, 17643, 17725, 17824, 18076, 18227, 18500, 18687, 18741, 18744, 18875, 19045, 19141, 19149, 19190, 19429, 19548, 195 7th, 19624th, 19645th, 19814th, 200049th, 20082th, 20097th, 20243th, 22243th, 22122th, 22516th, 22561th, 22816th, 23007th, 23904th, 24005th, 24418th Any of the polymorphic sites at positions 24578, 24855, 24855, 25505, 25538, 25564, 25586, 26017, 26234, 26557, 26621, 30155, 30267, or 30607
(5a) the FABP2 gene or a site on the DNA region in the vicinity of the gene, and the positions 2970, 3174, 3175, 4525, 4556, 4556, 4558, 4636 in the nucleotide sequence set forth in SEQ ID NO: 5; 4646, 4774, 5726, 5736, 5852, 7203, 7253, 7310, 8239, 8251, 8296, 8312, 8974, 10001, 10107, 10424, 10464 , 10491, 10794, 11922, 11957, 12820, 16006, 16256, 16309, 1631, 16360, 16923, 17877, 18408, 18649, 18715, 18783, or Any polymorphic part in 19878
(6a) a ZNF263 gene or a site on the DNA region in the vicinity of the gene, and positions 876, 1552, 3213, 3269, 6401, 6425, 6602 in the nucleotide sequence set forth in SEQ ID NO: 6; 9595th, 10001th, 10252th, 10269th, 10294th, 11377th, 12469th, 13642th, 13643th, 13879th, 14367th, 16133th, 17084th, 1765th, or 19263 Mold part
(7a) a TNFA gene or a site on the DNA region in the vicinity of the gene, the positions 175, 2401, 2773, 4452, 4671, 4672, 5184 in the nucleotide sequence set forth in SEQ ID NO: 7; 5609, 5820, 5941, 5942, 5942, 6245, 6315, 6316, 6487, 6603, 6730, 6867, 6931, 6935, 6958, 7953, 8022 , 8133, 8482, 8628, 8650, 8656, 8827, 8829, 8865, 8938, 8945, 8994, 9205, 9269, 9275, 10001, 10002, 1023, 10363 , 10399, 10680, 10765, 10816, 110 2nd, 11565, 11194, 12669, 12936, 12963, 13024, 13128, 13154, 13289, 13538, 13539, 13594, 13632, 13646, 13688, 13822 13855, 13863, 13966, 13993, 14370, 14542, 14625, 15030, 15064, 15581, 15730, 15975, 16626, 16713, 16734, 17240, 17369 Position, polymorphic site at positions 17698, 17711, or 19348
(8a) THBS3 gene or a site on the DNA region in the vicinity of the gene, which is 3098, 3103, 3143, 3178, 4543, 6977, 7158 in the nucleotide sequence set forth in SEQ ID NO: 8; Polymorphic site at any of the positions 10001, 10445, 11728, 13012, 13514, 16404, 18088, 18708, 18776, 18989, 19465, 19769, or 19786
(9a) ABCA1 gene or a site on the DNA region in the vicinity of the gene, wherein the positions 993, 1779, 1813, 3450, 4287, 4287, 4382, 7270 in the nucleotide sequence set forth in SEQ ID NO: 9; 7528, 8383, 8783, 8798, 9798, 9254, 10001, 10999, 11122, 11720, 12473, 12560, 12591, 12664, 12916, 13403, 13605, 13691 14457, 14737, 15278, 15345, 15345, 15636, 15636, 15824, 16793, 17022, 17037, 17283, 17576, 18167, 18619, 18632, 18842 Rank, 1884 18926, 19404, 19404, 19809, 20447, 20514, 20514, 20515, 21963, 22888, 23081, 23157, 23777, 23533, 24389, 25814, 26198, 26655, 26902, 27367, 27535, 27701, 28175, 28467, 28564, 28669, 28774, 28839, 28937, 28944, 29037, 30091, 30613, 30975 , 32803, 32803, 33435, 34263, 34264, 34812, 35306, 35339, 35473, 36217, 37596, or 37855
(10a) SERPINB10 gene or a site on the DNA region in the vicinity of the gene, and positions 145, 4845, 6757, 6859, 7151, 7827, 8066 in the nucleotide sequence set forth in SEQ ID NO: 10; Any polymorphic site at positions 10001, 12885, 13227, 13432, 13563, 15439, 15447, 15729, 15759, 15585, 16094, 16330, or 18565
(11a) ALDOB gene or a site on a DNA region in the vicinity of the gene, wherein the nucleotide position is 99, 380, 396, 399, 712, 934, 1534 in the nucleotide sequence of SEQ ID NO: 11. 1612, 1948, 2079, 2101, 4161, 4318, 4318, 4318, 5091, 6623, 6718, 7796, 7859, 8190, 8203, 9104, 9621, 9642 , 10001, 10489, 11443, 11592, 11593, 11616, 11616, 11617, 12281, 12457, 12802, 13993, 14026, 14800, 16654, 16822, 17133, 17239 , 18216, 18709, 19610 One of the polymorphic sites, or 19,613 positions
(12a) a site on the CTSD gene or a nearby DNA region of the gene, and positions 813, 815, 844, 861, 954, 1026, 1159 in the nucleotide sequence set forth in SEQ ID NO: 12; 1299, 5464, 5600, 7144, 7707, 7726, 7739, 7778, 7803, 7803, 7841, 7842, 7886, 7939, 7963, 8058, 8333, 9948 , 10001, 11990, 12342, 12807, 16266, or 16771
(13a) A site on the STE gene or a DNA region in the vicinity of the gene, and in positions 57, 468, 1881, 2955, 3460, 3460, 3976, 4537 in the nucleotide sequence set forth in SEQ ID NO: 13. 4538, 4722, 4867, 4873, 4947, 5025, 7043, 7676, 8581, 9035, 9347, 9686, 9882, 9894, 10001, 10003, 12142 , 13520, 13551, 13797, 14492, 14753, 14933, 15684, 17022, 18264, 18568, 18958, 19194, 19330, 19354, 19355, 19567, 19785 Rank, 20511th, 20612th, 2 627 of, 20,936 positions, 20,961 positions, 20,971 positions, 21213-position, 21336 positions, 21,609 positions, or polymorphic sites 21914 position, or 21,983 of
(14a) ABCC8 gene or a site on the DNA region in the vicinity of the gene, and positions 6978, 7358, 7359, 7649, 7755, 7755, 8068, 8214 in the nucleotide sequence set forth in SEQ ID NO: 14. 8215, 8308, 9658, 9761, 9861, 9811, 9828, 10001, 10095, 10125, 11658, 11684, 11708, 11767, 11883, 111998, 12109, 12308 13620th, 13664th, 13912th, 14297th, 14309th, 14320th, 14323th, 14367th, 14393th, 14475th, 1459th, 14722th, 15294th, 15601th, 15657th, 16160th, 16230 Rank, 16 67 position, 16417 positions, 17,695 positions, 18213-position, 18815 positions, 19,093 positions, 20082-position, 20211-position, 20461 positions, 21,547 positions, 21995-position, or polymorphic sites 22825 position, or 23,901 of
(15a) SERPINA3 gene or a site on the DNA region in the vicinity of the gene, which is a position 937, 1145, 1878, 2345, 2559, 3559, 3250, 3513 in the nucleotide sequence set forth in SEQ ID NO: 15. 4402, 5776, 6659, 7453, 7934, 7934, 8000, 8049, 8204, 8543, 8554, 8935, 9928, 10001, 10233, 10472, 10479, 10525 , 10544, 10629, 13339, 13348, 16287, 16389, 16626, or 17880
(16a) the RCV1 gene or a site on the DNA region in the vicinity of the gene, and the 25th position, 49th position, 469th position, 557th position, 592th position, 2730th position, 2754th position in the nucleotide sequence set forth in SEQ ID NO: 16; 5761, 6154, 6183, 8510, 8529, 8542, 8590, 9838, 10001, 10132, 10133, 10649, 10702, 10866, 12560, 15632, 15527 , 15853, 15994, 16617, 16790, 16841, 17010, 17348, 17656, 17656, 17816, 17820, 17882, 17882, 18061, 18101, 18304, Any polymorphic site at position 19485
(17a) a site on a TFRC gene or a DNA region in the vicinity of the gene, and positions 836, 1019, 2511, 4016, 4068, 4169, 4169, 4269 in the nucleotide sequence set forth in SEQ ID NO: 17; 4481, 5398, 5436, 5450, 5730, 5937, 6044, 6313, 6713, 6759, 6794, 6833, 7528, 8047, 8048, 8080, 9248 , 9361, 9417, 9734, 10001, 11461, 11845, 11852, 12086, 12596, 12864, 12930, 13969, 13969, 14119, 14280, 16833, or Any polymorphic site at position 17047
(18a) a position on the MMP19 gene or a DNA region in the vicinity of the gene, the positions 6329, 7329, 7715, 8138, 8507, 8551, 8551, 8574 in the nucleotide sequence set forth in SEQ ID NO: 18. Any polymorphic site at positions 10001, 11138, 11343, 13151, 13868, or 18923
(19a) MYL2 gene or a site on the DNA region in the vicinity of the gene, and the nucleotide sequence described in SEQ ID NO: 19 is position 3147, position 3213, position 4686, position 7343, position 7789, position 7801, position 10001, position 10123, 11264th, 11992th, 15532th, 15681, 15957, 15988, 16641, 18386, 18782, 20354, 21531, 23245, 27740, 30669, 30670, 32077, 32732 32907, 32955, 42589, 43029, 47354, 64777, 71869, 71196, 74274, 75551, 76343, 76344, 78270, 78339, 78689, 78743, 78 06, 78807, 79215, 79319, 79357, 79460, 79463, 790034, 80034, 80062, 800076, 80401, 80475, 80599, 80715, 80865, 80943, 80952 80974, 80991, 81130, 81373, 81428, 81543, 81682, 82422, 82545, 82792, 82929, 83265, 83266, 83394, 83463, 83500, 83578 , 83612, 83650, 84790, 8492, 84945, 85214, 85383, 85384, 85550, 85984, 86109, 86539, 86542, 87169 87170, 87353, 87671, 87953, 89554, 88057, 88178, 88240, 88258, 88271, 88272, 88307, 88512, 88695, 88778, 88798, 89124 Or any polymorphic position at position 91081
(20a) a VLDLR gene or a site on a DNA region in the vicinity of the gene, and positions 6297, 6733, 7994, 9242, 9952, 9901, 11801, 11838 in the nucleotide sequence set forth in SEQ ID NO: 20; Any polymorphic site at positions 12995, 13224, 13277, 18702, 18896, or 19214
(21a) the P2RX7 gene or a site on the DNA region in the vicinity of the gene, and the 99th position, the 694th position, the 2765th position, the 2765th position, the 3101st position, the 3117th position, the 3127th position, 3309th, 3346th, 3802th, 3916th, 4206th, 4430th, 5937th, 6705th, 7378th, 8312th, 8399th, 9928th, 10001, 100th, 10042th, 10277th, 10440th, 10461th , 10479, 10527, 10534, 10550, 10701, 10909, 11066, 11198, 11575, 11631, 11765, 11976, 12119, 12158, 12207, 12272, 12272 Rank, 12498, 1256 , 12791, 12856, 12860, 13207, 13383, 13590, 13860, 13869, 13880, 13881, 13984, 14048, 14059, 14210, 14806, 14921, 15331, 15488, 15558, 15651, 15651, 15686, 15791, 16051, 16082, 16137, 16492, 16649, 16784, 16788, 17072, 17076, 17379, 17527 17593, 17623, 17719, 17998, 18005, 18048, 18288, 18412, 18588, 18640, 19340, 19545, 19676, or 19685 One of the polymorphic sites of
(22a) a DLG5 gene or a site on the DNA region in the vicinity of the gene, and the 2nd position, 121st position, 1438th position, 1669th position, 2180th position, 2436th position, 2543th position in the nucleotide sequence set forth in SEQ ID NO: 22; 2619, 3715, 3727, 5327, 5256, 5335, 5339, 5688, 5752, 6304, 6627, 7195, 7342, 7603, 8273, 8905, 8988, 9013 , 9057, 10001, 10372, 10453, 10468, 10468, 10512, 11038, 11055, 11597, 11681, 11719, 11922, 12034, 12049, 12176, 12554 , 13481, 13864, 14390 15044, 15598, 16176, 16870, 17475, 17649, 18077, 18585, 18619, 18677, 19222, 19731, 19750, 20758, 20962, 21073, 21680 , 21867, 22385, 22874, 23191, 24016, 24520, 24993, 25460, 26155, 26804, 26980, 27882, 29234, 29469, 30254, 30742, 30742, 30756, 30756, 31671, 31874, 31940, 32626, 33393, 33581, 34240, 35294, 35666, 36587, 36653, 36 41st, 37408th, 37583, 37612, 38462, 38470, 38470, 38485, 39146, 39146, 40612, 40641, 41242, 42217, 42539, 45934, 46033, or 46895 Any polymorphic site
(23a) a site on the LDLC gene or a nearby DNA region of the gene, and in the base sequence described in SEQ ID NO: 23, positions 1580, 1754, 1882, 3062, 3507, 4507, 4800, 4905, 5569, 5608, 5643, 5665, 5593, 5864, 5884, 5981, 6169, 6808, 6858, 7194, 7283, 7728, 7835, 8049, 8049 , 8265, 9209, 9445, 9646, 9647, 9716, 9716, 9775, 10001, 10027, 10054, 10193, 10208, 10826, 10908, 10997, 11336, 11754 , 11895, 12031, 12327, 2464, 12561, 14051, 15366, 15778, 17723, 17772, 17783, 17811, 17815, 17862, 17900, 17962, 18184, 18189, 18297, 18411 , 18512, 18563, 19125, 19152, 200092, 21227, 21506, 21617, 21855, 23090, 25700, 26468, 26638, 27682, 27755, 27888, 27973 Position, 27995, 28123, 28127, 28144, 28153, or 28271 polymorphic site
(24a) AQP8 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence set forth in SEQ ID NO: 24, positions 6722, 9643, 9934, 10001, 10002, 13682, 14629, 15376, Polymorphic site at either position 15808 or 16082
(25a) a LARGE gene or a site on a DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 25, positions 1816, 3116, 3117, 3118, 3154, 3157, 3191, 3197, 3205, 3291, 3622, 3652, 3652, 3792, 4488, 5076, 5474, 5481, 5492, 5503, 5654, 5672, 6563, 6843, 6908 7119, 7538, 7538, 7721, 8403, 8626, 10001, 12636, 13103, 14794, 15370, 15448, 15599, 15564, 16045, 16261, 16277 Rank, 16861, 17360, 17611 17714, 17809, 18221, 18221, 18376, 18536, 19063, 22294, 23404, 23612, 25125, 25134, 25662, 25705, 25751, 27407, 28166 , 28567, 30302, 30316, 30353, 30354, 30391, 33594, 33650, 33754, 36690, 37753, 37895, 37900, 39104, 39745, 41404, 41646, 41711, 42020, 42080, 42974, 43052, 43240, 43621, 44734, 44958, 46920, 47431, 47573, 48101, 48 Position 27, 48306-position, 48,807 positions, 49049 positions, 49,848 positions, 49,860 positions, 49,903 positions, 50,114 positions, 50,223 positions, 52,057 positions, 52,543 positions, one of the polymorphic sites of 52,693 positions, or 53,061 of
(26a) a BLZF1 gene or a site on the DNA region in the vicinity of the gene, and positions 1283, 4288, 7520, 7599, 7740, 7745, 8745 and 9050 in the nucleotide sequence set forth in SEQ ID NO: 26; 9862, 10001, 10124, 10168, 10168, 10339, 11017, 13737, 13747, 18354, 20031, 20072, 22094, 24082, 24305, 24478, 24552, 25273 , 25351, 29096, 29100, 2976
(27a) RGS5 gene or a site on the DNA region in the vicinity of the gene, which is a position of 733, 848, 1039, 1351, 1569, 1831, 2078 in the nucleotide sequence set forth in SEQ ID NO: 27; 2707, 2794, 2904, 2995, 2995, 3122, 3520, 4351, 4824, 5189, 5205, 5206, 5525, 5779, 6487, 7712, 10001, 10208 , 10235, 10868, 11092, 11617, 11666, 12216, 12326, 12741, 13396, 13890, 14702, 15823, 16705, or 19086
(28a) the FRAP1 gene or a site on the DNA region in the vicinity of the gene, and positions 3455, 7826, 8715, 8783, 9042, 9390, 9462 in the nucleotide sequence set forth in SEQ ID NO: 28; 9709, 10001, 10635, 10748, 12,794, 12849, 12,915, 13,169, 17020, 18586, 21586, 21123, or 21128 polymorphic sites,
Or
(28a-2) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence described in SEQ ID NO: 30, positions 9598, 10001, 10306, 11181, 11480, 13593, 13721 Position, 13792, 15314, 15623, 15782, 16708, 16748, or 19630 polymorphic sites
(29a) a LEP gene or a site on a DNA region in the vicinity of the gene, the positions 244, 575, 675, 1487, 1644, 2343, 2715 in the nucleotide sequence set forth in SEQ ID NO: 29; 2,763, 3098, 3589, 3880, 5787, 5865, 6436, 7392, 7995, 8161, 8544, 9230, 9795, 10001, 10199, 10503, 111975 , 14981, 15681, or 19300
In more detail, it shows in the following Tables 2-31.
The DNA in the genome usually has a double-stranded DNA structure complementary to each other. Therefore, in the present specification, even when a DNA sequence in one strand is shown for convenience, it is understood that a sequence complementary to the sequence (base) is also disclosed as a matter of course. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious.
[Met gene or polymorphic site on the DNA region near the gene]

Some base types listed in the columns of “Base 1” and “Base 2” in the table indicate the complementary strand side of the site, but those skilled in the art will be able to access the listed polymorphic banks. Based on the session number and the like, it is possible to appropriately acquire information on the base type for the site.
[NPHS1 gene or a polymorphic site on the DNA region near the gene]

[Polymorphic site on SLC1A1 gene or DNA region near the gene]

[Polymorphic site on the SERPINB2 gene or a nearby DNA region of the gene]

[Polymorphic site on FABP2 gene or DNA region near the gene]

[Polymorphic site on the ZNF263 gene or a nearby DNA region of the gene]

[TNFA gene or polymorphic site on the DNA region near the gene]

[THBS3 gene or polymorphic site on the DNA region near the gene]

[A polymorphic site on the ABCA1 gene or a nearby DNA region of the gene]

In Table 10-2 above, * indicates an assayed SNP, ** indicates a significant single SNP, double shaded range indicates linkage disequilibrium block, and dot shaded range indicates linkage disequilibrium. Represents the area to be processed.
[Polymorphic site on the SERPINB10 gene or a nearby DNA region of the gene]

[ALDOB gene or polymorphic site on the DNA region near the gene]

[CTSD gene or polymorphic site on the DNA region near the gene]

[STE gene or polymorphic site on DNA region near the gene]

[A polymorphic site on the ABCC8 gene or a nearby DNA region of the gene]

[Polymorphic site on the SERPINA3 gene or a DNA region near the gene]

[RCV1 gene or a polymorphic site on the DNA region near the gene]

[TFRC gene or polymorphic site on the DNA region near the gene]

[Polymorphic site on MMP19 gene or DNA region near the gene]

[The polymorphic site on the MYL2 gene or a nearby DNA region of the gene]

[VLDLR gene or polymorphic site in the DNA region near the gene]

[Polymorphic site on P2RX7 gene or DNA region near the gene]

[Polymorphic site on DLG5 gene or DNA region near the gene]

[LDLC gene or polymorphic site on the DNA region in the vicinity of the gene]

[Polymorphic site on AQP8 gene or DNA region near the gene]

[LARGE gene or polymorphic site in the DNA region near the gene]

[Polymorphic site on BLZF1 gene or DNA region near the gene]

[Polymorphic site on RGS5 gene or DNA region near the gene]

[Polymorphic site on FRAP1 gene or DNA region near the gene]

“Serial No. in SEQ ID NO.” Described in the above table corresponds to the serial number of SEQ ID NO: 28.
[Polymorphic site on LEP gene or near DNA region of the gene]

[Polymorphic site on FRAP1 gene or DNA region near the gene]

“Serial No. in SEQ ID NO.” Described in the above table corresponds to the serial number of SEQ ID NO: 30.
In the table above, the description content in the SNPID * column is the registration ID of the dbSNP database if rs is prefixed, and the registration ID of the JSNP database if IMS-JST is added. The dbSNP database is available on the website (http://www.ncbi.nlm.nih.gov/SNP/index.html), and the JSNP database is available on the website (http://snp.ims.u-tokyo.ac. jp), and by searching on the website using the registered ID number described in the SNPID * column, detailed information of SNPs in each base sequence (for example, position on the chromosome, polymorphic site) Base types, sequences before and after, etc.). When such information is used, those skilled in the art can easily perform the inspection described in the present invention. The base type of the polymorphic site shown in the above table may indicate the base type on the complementary strand side with respect to the sequence shown in the sequence table, but the sequence before and after published in the dbSNP and JSNP databases If it is used, it is easy for those skilled in the art to confirm the difference, and in conducting the test, the other result can be determined inevitably by examining either the plus strand or the minus strand.
Moreover, those skilled in the art usually use the registration ID number assigned to the polymorphism disclosed in the present specification, for example, the rs number in the dbSNP database, the IMS-JST number in the JSNP database, and the ssj number to determine the polymorphism of the present invention. The actual position on the genome and the sequence before and after the site can be easily known. Thus, even if it is impossible to know, the person skilled in the art will appropriately determine the actual genome corresponding to the polymorphic site from the information on the base sequence and the polymorphic site shown in SEQ ID NOs: 1 to 30. Knowing the top position is easy. For example, the position of the polymorphic site of the present invention on the genome can be known by making an inquiry with a publicly available genome database or the like. That is, even if there is a slight difference in base sequence between the base sequence listed in the sequence listing and the actual base sequence on the genome, By performing a homology search or the like, it is possible to accurately know the actual genomic position of the polymorphic site of the present invention. Even when the position on the genome cannot be specified, it is easy to perform the test described in the present invention from the sequence listing and polymorphic site information described in this specification.
As an example of the sequences before and after the polymorphic site of the present invention (on the 5 ′ side and 3 ′ side), the sequences before and after the partial polymorphic site are exemplified in Table 32 and Table 33 below.

In the inspection method of the present invention, it is preferable to determine the base type for the polymorphic site described in any of (1b) to (29b) below.
(1b) A site on the MET gene or a nearby DNA region of the gene, and the site at position 14258 or 19768 in the nucleotide sequence set forth in SEQ ID NO: 1.
(2b) a site on the NPHS1 gene or a DNA region in the vicinity of the gene, the 10001 position or the 17848 position in the nucleotide sequence set forth in SEQ ID NO: 2
(3b) A site on the SLC1A1 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 3
(4b) A site on the SERPINB2 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 4
(5b) a site on the FABP2 gene or a nearby DNA region of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 5
(6b) A site on the ZNF263 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 6
(7b) A site on the TNFA gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 7
(8b) a site on the THBS3 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 8
(9b) A site on the ABCA1 gene or a nearby DNA region of the gene, and the site at position 17283 in the base sequence set forth in SEQ ID NO: 9
(10b) a site on the SERPINB10 gene or a nearby DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 10
(11b) A site on the ALDOB gene or a nearby DNA region of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 11
(12b) a site on the CTSD gene or a DNA region in the vicinity of the gene, which is located at position 11990 or position 16771 in the nucleotide sequence set forth in SEQ ID NO: 12
(13b) a site on the STE gene or a DNA region in the vicinity of the STE gene, and a site at position 18958 or 21213 in the nucleotide sequence set forth in SEQ ID NO: 13
(14b) a site on the ABCC8 gene or a DNA region in the vicinity of the gene, which is located at position 14475 in the base sequence set forth in SEQ ID NO: 14
(15b) a site on the SERPINA3 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 15
(16b) A site on the RCV1 gene or a DNA region in the vicinity of the gene, the 10001 position in the nucleotide sequence set forth in SEQ ID NO: 16
(17b) A site on the TFRC gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 17
(18b) a site on the MMP19 gene or in the vicinity DNA region of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 18
(19b) A site on the MYL2 gene or a nearby DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 19
(20b) a site on the VLDLR gene or a DNA region in the vicinity of the gene, the 10001 position in the nucleotide sequence set forth in SEQ ID NO: 20
(21b) a site on the P2RX7 gene or a nearby DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 21
(22b) a site on the DLG5 gene or in the vicinity DNA region of the gene, the 10001 position or the 37583 position in the nucleotide sequence set forth in SEQ ID NO: 22
(23b) a site on the LDLC gene or a nearby DNA region of the gene, and the site at position 18563 in the base sequence set forth in SEQ ID NO: 23
(24b) A site on the AQP8 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the base sequence set forth in SEQ ID NO: 24
(25b) A site on the LARGE gene or a DNA region in the vicinity of the gene, the site at positions 10001, 13103, or 15448 in the nucleotide sequence set forth in SEQ ID NO: 25
(26b) a site on the BLZF1 gene or in the vicinity DNA region of the gene, the site at position 20031 in the base sequence set forth in SEQ ID NO: 26
(27b) A site on the RGS5 gene or a DNA region in the vicinity of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 27
(28b) a site on the FRAP1 gene or in the vicinity DNA region of the gene, the site at position 17020 in the base sequence set forth in SEQ ID NO: 28
(28b-2) a site on the FRAP1 gene or in the vicinity DNA region of the gene, the site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 30
(29b) a site on the LEP gene or a DNA region in the vicinity of the gene, the site at position 10503 in the base sequence set forth in SEQ ID NO: 29
Further, in a preferred embodiment of the present invention, type 2 diabetes is suffered when the base types at the polymorphic sites described in (1b) to (29b) are the following (1c) to (29c), respectively. It is determined to be a thing. It is determined that even a subject who does not suffer from type 2 diabetes is suspected of developing type 2 diabetes or is susceptible to type 2 diabetes.
(1c) A site on the MET gene or a nearby DNA region of the gene, wherein the base species at position 14258 in the base sequence described in SEQ ID NO: 1 is T, or the base species at position 19768 is T
(2c) A site on the NPHS1 gene or a nearby DNA region of the gene, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 2 is A, or the base type at position 17848 is A
(3c) The SLC1A1 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 3 is G
(4c) The SERPINB2 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 24578 in the nucleotide sequence set forth in SEQ ID NO: 4 is A
(5c) a site on the FABP2 gene or a DNA region in the vicinity of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 5 is A
(6c) a ZNF263 gene or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 6 is G
(7c) A site on the TNFA gene or a nearby DNA region of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 7 is A
(8c) a THBS3 gene or a site on a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 8 is C
(9c) The ABCA1 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 17283 in the nucleotide sequence set forth in SEQ ID NO: 9 is T
(10c) The SERPINB10 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 10 is G
(11c) A region on the ALDOB gene or a DNA region in the vicinity of the ALDOB gene, wherein the base species at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 11 is T
(12c) A site on the CTSD gene or a nearby DNA region of the gene, wherein the base type at position 11990 in the base sequence described in SEQ ID NO: 12 is A, or the base type at position 16771 is C
(13c) A site on the STE gene or a nearby DNA region of the gene, wherein the base species at position 18958 in the base sequence described in SEQ ID NO: 13 is G, or the base species at position 21213 is C
(14c) the ABCC8 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 14475 in the nucleotide sequence set forth in SEQ ID NO: 14 is A
(15c) The SERPINA3 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 15 is A
(16c) The RCV1 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 16 is T
(17c) a site on the TFRC gene or a nearby DNA region of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 17 is G
(18c) The MMP19 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 18 is A
(19c) a site on the MYL2 gene or a nearby DNA region of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 19 is T
(20c) a VLDLR gene or a site on a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 20 is T
(21c) a P2RX7 gene or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 21 is C
(22c) a site on the DLG5 gene or a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 22 is G, or the base type at position 37583 is C
(23c) a region on the LDLC gene or a nearby DNA region of the gene, wherein the base type at position 18563 in the base sequence set forth in SEQ ID NO: 23 is G
(24c) a site on the AQP8 gene or a nearby DNA region of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 24 is G
(25c) a site on the LARGE gene or a nearby DNA region of the gene, wherein the base type at position 10001 is T, the base type at position 13103 is G, or the base at position 15448 in the base sequence described in SEQ ID NO: 25 The seed is G
(26c) a BLZF1 gene or a site on a DNA region in the vicinity of the gene, wherein the nucleotide type at position 20031 in the nucleotide sequence set forth in SEQ ID NO: 26 is C
(27c) the RGS5 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 27 is G
(28c) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 17020 in the base sequence set forth in SEQ ID NO: 28 is G
(28c-2) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 30 is G
(29c) the LEP gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10503 in the base sequence set forth in SEQ ID NO: 29 is G
In the present invention, it is considered that the polymorphic site and its surrounding DNA region are strongly linked even if other than the polymorphic site. DM can also be examined by determining the species. That is, this “neighboring polymorphic site” (for example, Table 2 to Table 2) is used for a small population of humans including DM patients whose base species of the polymorphic site are those described in the above (1c) to (29c). 31) is determined in advance. Next, a base type in the subject is determined for this “neighboring polymorphic site” and compared with the previously determined base type, thereby enabling a test for type 2 diabetes. When the base type is the same as the predetermined base type, it is determined that the subject is likely / not likely to suffer from type 2 diabetes. In the case of a subject who already suffers from type 2 diabetes, the cause and type of the onset of type 2 diabetes can be determined, which can be used to determine a treatment policy.
Hereinafter, the case of the MET gene will be described as an example. First, for example, for a small population of humans including a DM patient whose base type of the polymorphic site at position 10001 in the base sequence described in SEQ ID NO: 1 on the MET gene is G, a nearby polymorphic site, for example, position 8214 The base type of the polymorphic site is determined. If the base type of this site is T in the DM patient group, the base type of the polymorphic site at position 8214 is examined for the subject, and if the base type of this site is also T, The person is determined to be susceptible to type 2 diabetes.
As described above, after the region on the DM-related gene is clarified by the present invention, a test on DM can be performed without imposing an excessive burden on those skilled in the art.
In addition, the analysis of the human genome has progressed, and polymorphism information such as the entire nucleotide sequence, SNP, microsatellite, VNTR, and RFLPs has been enhanced. Now that the details of the genome sequence are becoming clear, the biggest concern is to analyze the relationship between genes or specific sequences and functions (phenotypes such as disease and drug responsiveness). In particular, lifestyle-related diseases such as diabetes and hypertension are very difficult to analyze because they develop due to multiple disease susceptibility genes and environmental factors. One of the promising methods for solving this is genetic statistical analysis using linkage disequilibrium.
Human chromosomes exist in pairs, each from a father and mother. A haplotype refers to a combination of individual genotypes related to one of them, and indicates how loci are arranged on one chromosome derived from each parent. Since the chromosomes are inherited from the parents one by one, if recombination does not occur during gametogenesis, the genes on one chromosome are always transmitted to the child together, that is, linked. However, since recombination actually occurs during meiosis, even a gene on a single chromosome is not necessarily linked. Conversely, even when genetic recombination occurs, loci that are close to each other on the same chromosome are strongly linked.
When such a phenomenon is observed in a group and non-independence of allyl is recognized, it is called linkage disequilibrium. For example, when 3 loci are observed, there are 2 haplotypes present if there is no linkage disequilibrium between them. ³ Each frequency is predicted from the frequency of each locus, but if there is linkage disequilibrium, 2 ³ There are fewer haplotypes than streets, and the frequency is different from the predicted value.
In recent years, it has been shown that haplotypes are useful for linkage disequilibrium analysis (Genetic Epidemiology 23: 221-233). Yes, it has been clarified that a part (haplotype) transmitted from an ancestor to a descendant as one region is common across races (Science 226, 5576: 2225-2229).
That is, if a haplotype associated with type 2 diabetes is found, the type 2 diabetes can be examined by detecting the haplotype. The present inventors have succeeded in finding a haplotype associated with type 2 diabetes through intensive studies.
Therefore, the present invention is characterized by detecting the haplotype associated with type 2 diabetes present in the gene according to any one of the following (1) to (14) and / or a DNA region in the vicinity of the gene. A method for testing type 2 diabetes is provided.
(1) MET, (2) NPHS1, (3) SERPINB2, (4) ABCA1, (5) CTSD, (6) STE, (7) ABCC8, (8) MYL2, (9) DLG5, (10) LDLC, (11) LARGE, (12) BLZF1, (13) FRAP1, (14) LEP
In this method, when “a haplotype associated with type 2 diabetes” is detected for a subject, it is determined that the subject is likely to suffer from type 2 diabetes. In the case of a subject who already suffers from type 2 diabetes, the cause and type of the onset can be determined, which can be used to determine a treatment policy.
The above-mentioned “haplotype associated with type 2 diabetes” can specifically indicate the following haplotype. In addition, “susceptible” refers to being susceptible to type 2 diabetes, and “protective” refers to being less susceptible to type 2 diabetes.
(1 ′) Polymorphic sites on the MET gene, and the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the base sequence set forth in SEQ ID NO: 1 are G, T, and T, respectively. Haplotype, which is
A haplotype that is a polymorphic site on the MET gene, wherein the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the nucleotide sequence set forth in SEQ ID NO: 1 are G, C, and C, respectively Or
A haplotype that is a polymorphic site on the MET gene, wherein the base types of the polymorphic sites at positions 19768 and 24209 in the base sequence set forth in SEQ ID NO: 1 are T and T, respectively.
More specifically, the polymorphic sites on the MET gene, the base species of the polymorphic sites at positions 10001, 14258, and 19768 in the base sequence set forth in SEQ ID NO: 1, are G, T, and T, respectively. Haplotypes that are sensitive to type 2 diabetes, and haplotypes whose base species are G, C, and C, respectively, are resistant to type 2 diabetes. In addition, haplotypes that are polymorphic sites on the MET gene and whose base types of the polymorphic sites at positions 19768 and 24209 in the nucleotide sequence set forth in SEQ ID NO: 1 are T and T, respectively, are susceptible to type 2 diabetes. is there.
(2 ′) Polymorphic sites on the NPHS1 gene, wherein the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2 are A, C, and A, respectively. Haplotype, which is
A haplotype that is a polymorphic site on the NPHS1 gene, wherein the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2 are G, G, and G, respectively Or
A haplotype that is a polymorphic site on the NPHS1 gene, and whose base types of the polymorphic sites at positions 10001, 16198, and 17848 in the nucleotide sequence set forth in SEQ ID NO: 2 are G, C, and G, respectively
More specifically, the polymorphic sites on the NPHS1 gene, the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2, are G, G, and G, respectively. A haplotype that is susceptible to type 2 diabetes, a haplotype whose base species is G, C, and G is susceptible to type 2 diabetes, and a haplotype whose base species is A, C, and A is resistant to type 2 diabetes It is sex.
(3 ′) a haplotype that is a polymorphic site on the SERPINB2 gene, wherein the base types of the polymorphic sites at positions 10001 and 24578 in the base sequence set forth in SEQ ID NO: 4 are C and A, or
A haplotype that is a polymorphic site on the SERPINB2 gene, wherein the base types of the polymorphic sites at positions 10001 and 24578 in the base sequence described in SEQ ID NO: 4 are T and T, respectively.
More specifically, the haplotypes that are polymorphic sites on the SERPINB2 gene and whose base types of the polymorphic sites at positions 10001 and 24578 in the base sequence described in SEQ ID NO: 4 are C and A, respectively, are type 2. Haplotypes that are susceptible to diabetes and whose base species are T and T are type 2 diabetes resistant.
(4 ′) Polymorphic sites on the ABCA1 gene, wherein the base types of the polymorphic sites at positions 10001, 15824, 17283, 20515, and 28564 in the base sequence set forth in SEQ ID NO: 9 are A haplotype that is C, A, C, T, and G, or
A haplotype that is a polymorphic site on the ABCA1 gene, wherein the base types of the polymorphic sites at positions 10001 and 11000 in the nucleotide sequence set forth in SEQ ID NO: 71 are G and C, respectively.
More specifically, each of the polymorphic sites on the ABCA1 gene, the base types of the polymorphic sites at positions 10001, 15824, 17283, 20515, and 28564 in the base sequence set forth in SEQ ID NO: 9, The haplotypes C, A, C, T, and G are type 2 diabetes resistant, polymorphic sites on the ABCA1 gene, and the polymorphic sites at positions 10001 and 11000 in the nucleotide sequence set forth in SEQ ID NO: 71 Haplotypes whose base species at the type site are G and C, respectively, are susceptible to type 2 diabetes.
(5 ′) Polymorphic sites on the CTSD gene, wherein the base types of the polymorphic sites at positions 10001, 11990, and 16771 in the base sequence set forth in SEQ ID NO: 12 are C, G, and T, respectively. Is a haplotype
More specifically, the polymorphic sites on the CTSD gene, the base types of the polymorphic sites at positions 10001, 11990, and 16771 in the base sequence set forth in SEQ ID NO: 12, are C, G, and T, respectively. A haplotype that is resistant to type 2 diabetes.
(6 ′) Polymorphic sites on the STE gene, wherein the base types of the polymorphic sites at positions 10001, 18958, 19354, 19567, and 20971 in the base sequence set forth in SEQ ID NO: 13 are A haplotype that is A, G, T, T, and C, or
A haplotype that is a polymorphic site on the STE gene and whose base types of the polymorphic sites at positions 20971 and 21213 in the base sequence set forth in SEQ ID NO: 13 are C and C, respectively.
More specifically, the polymorphic sites on the STE gene, the base types of the polymorphic sites at positions 10001, 18958, 19354, 19567, and 20971 in the base sequence set forth in SEQ ID NO: 13, respectively, Haplotypes that are A, G, T, T, and C are susceptible to type 2 diabetes. In addition, haplotypes that are polymorphic sites on the STE gene and whose base types of the polymorphic sites at positions 20971 and 21213 in the nucleotide sequence set forth in SEQ ID NO: 13 are C and C, respectively, are susceptible to type 2 diabetes. It is.
(7 ′) A haplotype that is a polymorphic site on the ABCC8 gene, and whose base type of the polymorphic site at position 14475 in the base sequence described in SEQ ID NO: 14 is A
More specifically, a haplotype that is a polymorphic site on the ABCC8 gene and whose base type of the polymorphic site at position 14475 in the base sequence described in SEQ ID NO: 14 is A is susceptible to type 2 diabetes.
(8 ′) Polymorphic sites on the MYL2 gene, and the base types of the polymorphic sites at positions 10001, 18386, 42589, and 83500 in the nucleotide sequence set forth in SEQ ID NO: 19 are C and T, respectively. , T, and C haplotypes
More specifically, it is a polymorphic site on the MYL2 gene, and the base types of the polymorphic sites at positions 10001, 18386, 42589, and 83500 in the base sequence described in SEQ ID NO: 19 are C, T The haplotypes that are T, C, and C are susceptible to type 2 diabetes.
(9 ′) Polymorphic sites on the DLG5 gene, the base types of the polymorphic sites at positions 10001, 24016, 29469, and 37583 in the base sequence set forth in SEQ ID NO: 22 are G and C , G, and C haplotypes
A haplotype that is a polymorphic site on the DLG5 gene, wherein the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence set forth in SEQ ID NO: 22 are C, G, and C, respectively Or
A haplotype that is a polymorphic site on the DLG5 gene, wherein the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence set forth in SEQ ID NO: 22 are C, G, and T, respectively
More specifically, the polymorphic sites on the DLG5 gene, the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the base sequence set forth in SEQ ID NO: 22, are C, G, and C, respectively. A haplotype that is susceptible to type 2 diabetes and a haplotype whose base species are C, G, and T, respectively, is resistant to type 2 diabetes.
(10 ′) a haplotype that is a polymorphic site on the LDLC gene, wherein the base species of the polymorphic site at positions 10001 and 18563 in the nucleotide sequence set forth in SEQ ID NO: 23 are G and A, or
Haplotypes that are polymorphic sites on the LDLC gene, and whose base types of the polymorphic sites at positions 10001 and 18563 in the nucleotide sequence set forth in SEQ ID NO: 23 are G and G, respectively
More specifically, haplotypes that are polymorphic sites on the LDLC gene and whose base types of the polymorphic sites at positions 10001 and 18563 in the base sequence described in SEQ ID NO: 23 are G and G, respectively, are type 2. Haplotypes that are susceptible to diabetes and whose base species are G and A, respectively, are resistant to type 2 diabetes.
(11 ′) Polymorphic sites on the LARGE gene, wherein the base types of the polymorphic sites at positions 10001, 13103, 15448, 36690, and 43240 in the nucleotide sequence set forth in SEQ ID NO: 25 are Haplotypes that are T, G, G, T, and A
More specifically, each of the polymorphic sites on the LARGE gene, the base types of the polymorphic sites at positions 10001, 13103, 15448, 36690, and 43240 in the base sequence set forth in SEQ ID NO: 25, Haplotypes that are T, G, G, T, and A are susceptible to type 2 diabetes.
(12 ′) A haplotype that is a polymorphic site on the BLZF1 gene, wherein the base types of the polymorphic sites at positions 10001 and 20031 in the base sequence set forth in SEQ ID NO: 26 are G and T, respectively.
More specifically, a haplotype that is a polymorphic site on the BLZF1 gene and whose base types of the polymorphic sites at positions 10001 and 20031 in the base sequence described in SEQ ID NO: 26 are G and T, respectively, is type 2 diabetes. Resistant.
(13 ′) A haplotype that is a polymorphic site on the FRAP1 gene and the base type of the polymorphic site at position 17020 in the base sequence set forth in SEQ ID NO: 28 is G
More specifically, a haplotype that is a polymorphic site on the FRAP1 gene and in which the base type of the polymorphic site at position 17020 in the base sequence described in SEQ ID NO: 28 is G is susceptible to type 2 diabetes.
(14 ′) A haplotype that is a polymorphic site on the LEP gene, and whose base types of the polymorphic sites at positions 10001 and 10503 in the nucleotide sequence set forth in SEQ ID NO: 29 are G and A, respectively
More specifically, haplotypes that are polymorphic sites on the LEP gene and whose base types of the polymorphic sites at positions 10001 and 10503 in the nucleotide sequence set forth in SEQ ID NO: 29 are G and A, respectively, are type 2 diabetes. Sensitive.
In a preferred embodiment of the above method, it is a method for examining type 2 diabetes, comprising the following steps (a) and (b).
(A) a step of determining a base type for the gene according to any one of (1) to (14) or a polymorphic site on a DNA region in the vicinity of the gene in a subject;
(B) The base type determined in step (a) is the base type of the polymorphic site in the gene showing the haplotype according to any one of the above (1 ′) to (14 ′) or a nearby DNA region of the gene And the process to compare
As the polymorphic site in the step (a), preferably, the following polymorphic sites can be shown.
(1) The MET gene or a site on the DNA region in the vicinity of the gene, which is the position 765, position 1383, position 1890, position 2099, position 2814, position 3293, position 3752 in the nucleotide sequence set forth in SEQ ID NO: 1. 3774, 3927, 4690, 7419, 8219, 8201, 10001, 10318, 13766, 13789, 14258, 15427, 16275, 16597, 16932, 19768, 19835, 230032 , 20240, 20537, 20885, 21546, 24209, 24271, 24376, 24642, 26028, 26226, or 34209
(2) a site on the NPHS1 gene or a DNA region in the vicinity of the gene, the positions 172, 186, 211, 2554, 2558, 2558, 10001, 10001 in the nucleotide sequence set forth in SEQ ID NO: 2; 10176th, 11840th, 13759th, 14645th, 14967th, 16198th, 16439th, 17635th, 17685th, 17794th, 17805th, 17848th, 17849th, 18491th, 19955th, 20955th, 20613th 21533, 21582, 21811, 21812, 22190, 22965, 22982, 23660, 23780, 24040, 24172, 25334, or 26585 polymorphic sites
(3) SERPINB2 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 4, positions 2853, 2891, 5218, 5317, 7560, 8054, 8771, 10001 , 10329, 11781, 12063, 12705, 13125, 13691, 13913, 13916, 13917, 14123, 14169, 14453, 14456, 14483, 16526, 16556, 17643, 17725, 17824, 18076, 18227, 18500, 18687, 18741, 18744, 18875, 19045, 19141, 19149, 19190, 19429, 19548, 1956 , 19624, 19645, 19814, 20049, 20082, 20097, 20243, 22122, 22495, 22516, 22661, 22816, 23007, 23904, 24005, 24418, Any of the polymorphic sites at positions 24578, 24855, 24855, 25505, 25538, 25564, 25586, 26017, 26234, 26557, 26621, 30155, 30267, or 30607
(4) ABCA1 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 9, positions 993, 1779, 1813, 3450, 4287, 4382, 7270, 7528, 8383, 8783, 8798, 9798, 9254, 10001, 10999, 11122, 11720, 12473, 12560, 12591, 12664, 12916, 13403, 13605, 13691 14457, 14737, 15278, 15345, 15345, 15636, 15636, 15824, 16793, 17022, 17037, 17283, 17576, 18167, 18619, 18632, 18842 Rank, 18843 18926, 19404, 19404, 19809, 20447, 20514, 20515, 21963, 22888, 23081, 23157, 23579, 23553, 24389, 25814, 26198, 26655 , 26902, 27367, 27535, 27701, 28175, 28467, 28564, 28669, 28774, 28939, 28937, 28944, 29037, 30091, 30613, 30975, Any of the polymorphic sites at positions 32803, 32803, 33435, 34263, 34264, 34812, 35306, 35339, 35473, 36217, 37596, or 37855, or BCA1 gene or a site on the DNA region in the vicinity of the gene, which is 1206, 1905, 1979, 2008, 2054, 2286, 2303, 2808 in the nucleotide sequence set forth in SEQ ID NO: 71, 3176, 3413, 3415, 3548, 3548, 3669, 3756, 3983, 4521, 4544, 4577, 4609, 5226, 5833, 5868, 6422, 6532, 6583 6696th, 7043th, 7198th, 7324th, 7395th, 7484th, 7716th, 7787th, 7787th, 8197th, 8350th, 9541th, 9643th, 9793th, 100001th, 10348th, 10415th, 10741 , 10826, 11000, 11024, 11129 , 11185, 11286, 11452, 11471, 11653, 11746, 11858, 12114, 12225, 12520, 12725, 12896, 12899, 12905, 13118, 13492, 13589th, 13836th, 14226th, 14391th, 14489th, 14536th, 14813th, 15071th, 15215th, 15377th, 15399th, 15644th, 15687th, 15830th, 16103th, 16112th, 16139th , 16754, 17303, 18237, 18379, 18618, 18903, 18921, 19014, 19034, 19074, 19149, 19165, 19167, 19365, 19 Position 73, 19376-position, 19,386 positions, or polymorphic sites 19487 position or 19659-position
(5) a CTSD gene or a site on a DNA region in the vicinity of the gene, and positions 813, 815, 844, 861, 954, 1026, 1159 in the nucleotide sequence set forth in SEQ ID NO: 12; 1299, 5464, 5600, 7144, 7707, 7726, 7739, 7778, 7803, 7803, 7841, 7842, 7886, 7939, 7963, 8058, 8333, 9948 , 10001, 11990, 12342, 12807, 16266, or 16771
(6) A site on the STE gene or a DNA region in the vicinity of the gene, and in positions 57, 468, 1881, 2955, 3460, 3460, 3976, 4537 in the nucleotide sequence set forth in SEQ ID NO: 13. 4538, 4722, 4867, 4873, 4947, 5025, 7043, 7676, 8581, 9035, 9347, 9686, 9882, 9894, 10001, 10003, 12142 , 13520, 13551, 13797, 14492, 14753, 14933, 15684, 17022, 18264, 18568, 18958, 19194, 19330, 19354, 19355, 19567, 19785 Rank, 20511th, 20612th, 206 7 position, 20,936 positions, 20,961 positions, 20,971 positions, 21213-position, 21336 positions, 21,609 positions, or polymorphic sites 21914 position, or 21,983 of
(7) ABCC8 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence described in SEQ ID NO: 14, positions 6978, 7358, 7359, 7649, 7755, 8068, 8214, 8215, 8308, 9658, 9761, 9861, 9811, 9828, 10001, 10095, 10125, 11658, 11684, 11708, 11767, 11883, 111998, 12109, 12308 13620th, 13664th, 13912th, 14297th, 14309th, 14320th, 14323th, 14367th, 14393th, 14475th, 1459th, 14722th, 15294th, 15601th, 15657th, 16160th, 16230 Rank, 1636 Position, 16417 positions, 17,695 positions, 18213-position, 18815 positions, 19,093 positions, 20082-position, 20211-position, 20461 positions, 21,547 positions, 21995-position, or polymorphic sites 22825 position, or 23,901 of
(8) MYL2 gene or a site on the DNA region in the vicinity of the gene, and the 3147, 3213, 4686, 7343, 7892, 10001, 1001, 10123 positions in the nucleotide sequence set forth in SEQ ID NO: 19; 11264th, 11992th, 15532th, 15681, 15957, 15988, 16641, 18386, 18782, 20354, 21531, 23245, 27740, 30669, 30670, 32077, 32732 , 32907, 32955, 42589, 43029, 47354, 64777, 71869, 71196, 74274, 75551, 76343, 76344, 78270, 78339, 78689, 78743, 7880 , 78807, 79215, 79319, 79357, 79460, 79463, 80034, 80062, 80076, 80401, 80475, 80599, 80715, 80865, 80943, 80952, 80974, 80991, 81130, 81373, 81428, 81543, 81682, 82422, 82545, 82792, 82929, 83265, 83266, 83394, 83463, 83500, 83578 , 83612, 83650, 84790, 8492, 84945, 85214, 85383, 85384, 85550, 85984, 86109, 86539, 86542, 87169, 8169 170, 87353, 87671, 87953, 89554, 88057, 88178, 88240, 88258, 88271, 88272, 88307, 88512, 88695, 88778, 88798, 89124 Or any polymorphic position at position 91081
(9) a DLG5 gene or a site on the DNA region in the vicinity of the gene, and the 2nd, 121st, 1438th, 1669th, 2180th, 2436th, 2543th position in the nucleotide sequence set forth in SEQ ID NO: 22; 2619, 3715, 3727, 5327, 5256, 5335, 5339, 5688, 5752, 6304, 6627, 7195, 7342, 7603, 8273, 8905, 8988, 9013 , 9057, 10001, 10372, 10453, 10468, 10468, 10512, 11038, 11055, 11597, 11681, 11719, 11922, 12034, 12049, 12176, 12554 , 13481, 13864, 14390, 5044, 15598, 16176, 16870, 17475, 17649, 18077, 18585, 18619, 18677, 19222, 19731, 19750, 20758, 20962, 21073, 21680 21867th, 22385th, 22874th, 23191st, 24016th, 24520th, 24993th, 25460th, 26155th, 26804th, 26980th, 27882th, 29234th, 29469th, 30254th, 30742th, 30742 , 30756, 30756, 31671, 31874, 31940, 32626, 33393, 33581, 34240, 35294, 35666, 36587, 36653, 3694 , 37408, 37583, 37612, 38462, 38470, 38485, 39065, 39146, 40612, 40641, 41242, 42217, 42539, 45934, 46033, or 46895 Any polymorphic site of
(10) LDLC gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 23, positions 1580, 1754, 1882, 3062, 3507, 4507, 4800, 4905, 5569, 5608, 5643, 5665, 5593, 5864, 5884, 5981, 6169, 6808, 6858, 7194, 7283, 7728, 7835, 8049, 8049 , 8265, 9209, 9445, 9646, 9647, 9716, 9716, 9775, 10001, 10027, 10054, 10193, 10208, 10826, 10908, 10997, 11336, 11754 Rank, 11895, 12031, 12327, 1 464, 12561, 14051, 15366, 15778, 17723, 17772, 17783, 17811, 17815, 17862, 17900, 17962, 18184, 18189, 18297, 18411 , 18512, 18563, 19125, 19152, 200092, 21227, 21506, 21617, 21855, 23090, 25700, 26468, 26638, 27682, 27755, 27888, 27973 Position, 27995, 28123, 28127, 28144, 28153, or 28271 polymorphic site
(11) LARGE gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 25, position 1816, position 3116, position 3117, position 3118, position 3154, position 3157, position 3191, 3197, 3205, 3291, 3622, 3652, 3652, 3792, 4488, 5076, 5474, 5481, 5492, 5503, 5654, 5672, 6563, 6843, 6908 7119, 7538, 7538, 7721, 8403, 8626, 10001, 12636, 13103, 14794, 15370, 15448, 15599, 15564, 16045, 16261, 16277 Rank, 16861, 17360, 17611 17714, 17809, 18221, 18221, 18221, 18376, 18536, 19063, 22294, 23404, 23612, 25125, 25134, 25662, 25705, 25751, 27407, 28166 28567, 30302, 30316, 30353, 30354, 30391, 33594, 33650, 33754, 36690, 37753, 37895, 37900, 39104, 39745, 41404, 41646 Rank, 41711, 42020, 42080, 42974, 43052, 43240, 43621, 44734, 44958, 46920, 47431, 47573, 48101, 481 7 position, 48,306 positions, 48,807 positions, 49049 positions, 49,848 positions, 49,860 positions, 49,903 positions, 50,114 positions, 50,223 positions, 52,057 positions, 52,543 positions, one of the polymorphic sites of 52,693 positions, or 53,061 of
(12) a BLZF1 gene or a site on a DNA region in the vicinity of the gene, and positions 1283, 4288, 7520, 7599, 7740, 8745, 9050 in the nucleotide sequence set forth in SEQ ID NO: 26; 9862, 10001, 10124, 10168, 10168, 10339, 11017, 13737, 13747, 18354, 20031, 20072, 22094, 24082, 24305, 24478, 24552, 25273 , 25351, 29096, 29100, 2976
(13) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, and positions 3455, 7826, 8715, 8783, 9042, 9390, 9462 in the nucleotide sequence set forth in SEQ ID NO: 28; 9709, 10001, 10635, 10748, 12794, 12849, 12915, 13169, 17020, 18586, 21586, 21123, or 21128 polymorphic sites
(14) a LEP gene or a site on a DNA region in the vicinity of the gene, wherein the positions 244, 575, 675, 1487, 1644, 2343, 2715 in the nucleotide sequence set forth in SEQ ID NO: 29; 2,763, 3098, 3589, 3880, 5787, 5865, 6436, 7392, 7995, 8161, 8544, 9230, 9795, 10001, 10199, 10503, 111975 , 14981, 15681, or 19300
More preferably, as the polymorphic site in the step (a), the following polymorphic sites can be shown.
(1) A polymorphic site on the MET gene, the polymorphic site at positions 10001, 14258, 19768, or 24209 in the nucleotide sequence set forth in SEQ ID NO: 1.
(2) A polymorphic site on the NPHS1 gene, the polymorphic site at positions 10001, 16198 or 17848 in the nucleotide sequence set forth in SEQ ID NO: 2.
(3) A polymorphic site on the SERPINB2 gene, which is a polymorphic site at position 10001 or 24578 in the nucleotide sequence set forth in SEQ ID NO: 4.
(4) a polymorphic site on the ABCA1 gene, the polymorphic site at positions 10001, 15824, 17283, 20515, or 28564 in the nucleotide sequence set forth in SEQ ID NO: 9; or
Polymorphic sites on ABCA1 gene, polymorphic sites at positions 10001 and 11000 in the nucleotide sequence set forth in SEQ ID NO: 71
(5) A polymorphic site on the CTSD gene, which is a polymorphic site at positions 10001, 11990, or 16771 in the nucleotide sequence set forth in SEQ ID NO: 12.
(6) A polymorphic site on the STE gene, the polymorphic site at positions 10001, 18958, 19354, 19567, 20971, or 21213 in the nucleotide sequence set forth in SEQ ID NO: 13
(7) A polymorphic site on the ABCC8 gene, which is the polymorphic site at position 10001 or 14475 in the nucleotide sequence set forth in SEQ ID NO: 14
(8) A polymorphic site on the MYL2 gene, the polymorphic site at positions 10001, 18386, 42589, or 83500 in the nucleotide sequence set forth in SEQ ID NO: 19
(9) A polymorphic site on the DLG5 gene, the polymorphic site at positions 10001, 24016, 29469, or 37583 in the nucleotide sequence set forth in SEQ ID NO: 22
(10) A polymorphic site on the LDLC gene, the polymorphic site at position 10001 or 18563 in the nucleotide sequence set forth in SEQ ID NO: 23
(11) A polymorphic site on the LARGE gene, the polymorphic site at positions 10001, 13103, 15448, 36690, or 43240 in the nucleotide sequence set forth in SEQ ID NO: 25
(12) A polymorphic site on the BLZF1 gene, the polymorphic site at position 10001 or 20031 in the nucleotide sequence set forth in SEQ ID NO: 26
(13) A polymorphic site on the FRAP1 gene, the polymorphic site at position 10001 or 17020 in the nucleotide sequence set forth in SEQ ID NO: 28
(14) A polymorphic site on the LEP gene, the polymorphic site at position 10001 or 10503 in the nucleotide sequence set forth in SEQ ID NO: 29
A person skilled in the art can determine the base species in the polymorphic site of the present invention by various methods. For example, it can be carried out by directly determining the base sequence of DNA containing the polymorphic site of the present invention. In this method, first, a DNA sample is prepared from a subject. In the present invention, a DNA sample is based on, for example, a subject's blood, skin, oral mucosa, tissue or cells collected or excised by surgery, chromosomal DNA extracted from body fluids collected for the purpose of examination, or RNA. Can be prepared.
In this method, DNA containing the polymorphic site of the present invention is then isolated. Isolation of the DNA can also be performed by PCR or the like using a primer that hybridizes to the DNA containing the polymorphic site of the present invention and using chromosomal DNA or RNA as a template.
In this method, the base sequence of the isolated DNA is then determined. A person skilled in the art can easily determine the base sequence of the isolated DNA using a DNA sequencer or the like.
In the polymorphic site of the present invention, usually the variation of the base type of the site has already been clarified. “Determining the base type” in the present invention does not necessarily mean that the polymorphic site is a base type of A, G, T, or C. For example, if it is known that the variation of the base type is A or G for a certain polymorphic site, it is sufficient that the base type of the site is found to be “not A” or “not G”. .
Various methods for determining the base type of a polymorphic site whose base variation has been clarified in advance are known. The method for determining the base species of the present invention is not particularly limited. For example, TaqMan PCR method, Acyclo Prime method, MALDI-TOF / MS method and the like have been put to practical use as analysis methods applying the PCR method. In addition, the Invader method and the RCA method are known as methods for determining the base species independent of PCR. Furthermore, the base species can be determined using a DNA array. These methods are briefly described below. Any of the methods described herein can be applied to the determination of the base type of the polymorphic site in the present invention.
[TaqMan PCR method]
The principle of the TaqMan PCR method is as follows. The TaqMan PCR method is an analysis method using a primer set that can amplify a region containing an allele and a TaqMan probe. The TaqMan probe is designed to hybridize to the region containing the allele amplified by this primer set.
If the target base sequence is hybridized under conditions close to the Tm of the TaqMan probe, the hybridization efficiency of the TaqMan probe is significantly reduced due to the difference of one base. When the PCR method is performed in the presence of the TaqMan probe, the extension reaction from the primer eventually reaches the hybridized TaqMan probe. At this time, the TaqMan probe is degraded from its 5 ′ end by the 5′-3 ′ exonuclease activity of DNA polymerase. If the TaqMan probe is labeled with a reporter dye and a quencher, the degradation of the TaqMan probe can be followed as a change in fluorescence signal. That is, when the TaqMan probe is decomposed, the reporter dye is released and the distance from the quencher is increased, thereby generating a fluorescent signal. If the hybridization of the TaqMan probe decreases due to a difference in one base, the TaqMan probe is not decomposed and a fluorescent signal is not generated.
If a TaqMan probe corresponding to a polymorphism is designed and a different signal is generated by the decomposition of each probe, the base species can be simultaneously determined. For example, as a reporter dye, 6-carboxy-fluorescein (FAM) is used for the TaqMan probe of allele A of an allele, and VIC is used for the probe of allele B. In a state where the probe is not decomposed, generation of a fluorescent signal of the reporter dye is suppressed by the quencher. If each probe hybridizes to the corresponding allele, a fluorescence signal corresponding to the hybridization is observed. That is, if either FAM or VIC signal is stronger than the other, it turns out that it is homologous to allele A or allele B. On the other hand, when the allele is hetero, both signals are detected at substantially the same level. By using the TaqMan PCR method, PCR and base type determination can be performed simultaneously on a genome as an analysis target without a time-consuming step such as separation on a gel. Therefore, the TaqMan PCR method is useful as a method that can determine the base species for many subjects.
[Acyclo Prime method]
As a method for determining the base species using the PCR method, the Acyclo Prime method has also been put into practical use. In the Acyclo Prime method, one set of primers for genome amplification and one set of primers for polymorphism detection are used. First, a region containing a polymorphic site in the genome is amplified by PCR. This process is the same as normal genomic PCR. Next, the obtained PCR product is annealed with a polymorphism detection primer to perform an extension reaction. The polymorphism detection primer is designed to anneal to a region adjacent to the polymorphic site to be detected.
At this time, a nucleotide derivative (terminator) labeled with a fluorescent polarizing dye and blocked with 3′-OH is used as a nucleotide substrate for the extension reaction. As a result, only one base complementary to the base at the position corresponding to the polymorphic site is incorporated, and the extension reaction stops. Incorporation of nucleotide derivatives into the primer can be detected by an increase in fluorescence polarization (FP) due to an increase in molecular weight. If two types of labels having different wavelengths are used for the fluorescent polarizing dye, it is possible to specify which of the two types of bases the specific SNPs are. Since the level of fluorescence polarization can be quantified, it is also possible to determine whether an allele is homo or hetero in one analysis.
[MALDI-TOF / MS method]
Base species can also be determined by analyzing PCR products with MALDI-TOF / MS. MALDI-TOF / MS can be used in various fields as an analytical method that can clearly identify slight differences in protein amino acid sequences and DNA base sequences because it can know the molecular weight with great accuracy. Yes. In order to determine the base species by MALDI-TOF / MS, first, a region containing the allele to be analyzed is amplified by PCR. The amplification product is then isolated and its molecular weight is measured by MALDI-TOF / MS. Since the base sequence of the allele is known in advance, the base sequence of the amplification product is uniquely determined based on the molecular weight.
The determination of the base species using MALDI-TOF / MS requires a PCR product separation step and the like. However, accurate base species determination can be expected without using labeled primers or labeled probes. It can also be applied to simultaneous detection of polymorphisms at multiple locations.
[SNPs-specific labeling method using type IIs restriction enzyme]
A method capable of determining a base species at a higher speed using the PCR method has also been reported. For example, the base type of the polymorphic site is determined using a type IIs restriction enzyme. In this method, a primer having a recognition sequence of type IIs restriction enzyme is used for PCR. A general restriction enzyme (type II) used for gene recombination recognizes a specific base sequence and cleaves a specific site in the base sequence. In contrast, type IIs restriction enzymes recognize specific base sequences and cleave sites away from the recognized base sequences. The number of bases between the recognition sequence and the cleavage site is determined by the enzyme. Therefore, if the primer containing the recognition sequence of the type IIs restriction enzyme is annealed at a position separated by the number of bases, the amplified product can be cleaved at the polymorphic site by the type IIs restriction enzyme.
At the end of the amplification product cleaved with the type IIs restriction enzyme, a cohesive end containing the base of SNPs is formed. Here, an adapter having a base sequence corresponding to the sticky end of the amplification product is ligated. The adapter has different base sequences including bases corresponding to polymorphic mutations, and can be labeled with different fluorescent dyes. Finally, the amplification product is labeled with a fluorescent dye corresponding to the base at the polymorphic site.
When a PCR method is performed by combining a primer containing the type IIs restriction enzyme recognition sequence with a capture primer, the amplification product is fluorescently labeled and can be solid-phased using the capture primer. For example, if a biotin-labeled primer is used as a capture primer, the amplification product can be captured on avidin-bound beads. By tracking the fluorescent dye of the amplification product thus captured, the base species can be determined.
[Determination of base species at polymorphic sites using magnetic fluorescent beads]
A technique capable of analyzing a plurality of alleles in parallel in a single reaction system is also known. Analyzing a plurality of alleles in parallel is called multiplexing. In general, a typing method using a fluorescent signal requires fluorescent components having different fluorescent wavelengths for multiplexing. However, there are not so many fluorescent components that can be used for actual analysis. On the other hand, when a plurality of types of fluorescent components are mixed in a resin or the like, a variety of fluorescent signals that can be distinguished from each other can be obtained even with limited types of fluorescent components. Furthermore, if a component that is magnetically adsorbed in the resin is added, it is possible to obtain beads that emit fluorescence and can be separated magnetically. Multiplex polymorphic typing using such magnetic fluorescent beads has been devised (Bioscience and Bioindustry, Vol. 60 No. 12, 821-824).
In multiplexed polymorphic typing using magnetic fluorescent beads, a probe having a terminal complementary to the polymorphic site of each allele is immobilized on the magnetic fluorescent beads. Both are combined so that each allele corresponds to a magnetic fluorescent bead having a unique fluorescent signal. On the other hand, when a probe fixed to a magnetic fluorescent bead is hybridized to a complementary sequence, a fluorescently labeled oligo DNA having a base sequence complementary to an adjacent region on the allele is prepared.
The region containing the allele is amplified by asymmetric PCR, the above-mentioned magnetic fluorescent bead-immobilized probe and fluorescently labeled oligo DNA are hybridized, and both are further ligated. When the end of the magnetic fluorescent bead-immobilized probe has a base sequence complementary to the base of the polymorphic site, ligation is efficiently performed. On the other hand, if the terminal bases are different due to polymorphism, the ligation efficiency of the two decreases. As a result, the fluorescent labeled oligo DNA is bound to each magnetic fluorescent bead only when the sample is a base species complementary to the magnetic fluorescent bead.
By collecting magnetic fluorescent beads by magnetism and detecting the presence of fluorescently labeled oligo DNA on each magnetic fluorescent bead, the base species is determined. Since magnetic fluorescent beads can analyze the fluorescence signal for each bead using a flow cytometer, the signal can be easily separated even if various types of magnetic fluorescent beads are mixed. That is, “multiplexing” in which multiple types of polymorphic sites are analyzed in parallel in a single reaction vessel is achieved.
[Invader method]
A method for genotyping independent of the PCR method has also been put into practical use. For example, in the Invader method, the base type is determined only by three types of oligonucleotides, an allele probe, an invader probe, and a FRET probe, and a special nuclease called cleavase. Of these probes, only the FRET probe requires labeling.
Allele probes are designed to hybridize to regions adjacent to the allele to be detected. On the 5 ′ side of the allele probe, a flap composed of a base sequence unrelated to hybridization is linked. The allele probe has a structure that hybridizes to the 3 ′ side of the polymorphic site and is linked to a flap on the polymorphic site.
On the other hand, the invader probe has a base sequence that hybridizes to the 5 ′ side of the polymorphic site. The base sequence of the invader probe is designed so that the 3 ′ end corresponds to the polymorphic site by hybridization. The base at the position corresponding to the polymorphic site in the invader probe may be arbitrary. That is, both base sequences are designed so that the invader probe and the allele probe hybridize adjacently across the polymorphic site.
When the polymorphic site is a base complementary to the base sequence of the allele probe, when both the invader probe and the allele probe hybridize to the allele, the invader probe enters the base corresponding to the polymorphic site of the allele probe. An (invasion) structure is formed. Cleavease cleaves the strand of the invading side of the oligonucleotide that has formed the invading structure thus formed. Since the cutting occurs on the intrusion structure, the result is that the allele probe flap is cut off. On the other hand, if the base at the polymorphic site is not complementary to the base of the allele probe, there is no competition between the invader probe and the allele probe at the polymorphic site, and no invasion structure is formed. Therefore, the flap is not cut by cleavase.
The FRET probe is a probe for detecting the flap thus separated. The FRET probe constitutes a hairpin loop having a self-complementary sequence on the 5 ′ end side and a single-stranded portion arranged on the 3 ′ end side. The single-stranded portion arranged on the 3 ′ end side of the FRET probe has a base sequence complementary to the flap, and the flap can hybridize there. When the flap hybridizes to the FRET probe, both base sequences are designed so that a structure in which the 3 ′ end of the flap enters the 5 ′ end of the self-complementary sequence of the FRET probe is formed. The cleavase recognizes the invasion structure and cuts it. If the portion of the FRET probe that is cleaved by cleavase is sandwiched and labeled with a reporter dye and quencher similar to TaqMan PCR, the cleavage of the FRET probe can be detected as a change in the fluorescence signal.
Theoretically, the flap should hybridize to the FRET probe even if it is not cut. However, in reality, there is a large difference in the binding efficiency to FRET between the cut flap and the flap present in the state of the allele probe. Therefore, it is possible to specifically detect the cut flap by using the FRET probe.
In order to determine the base type based on the Invader method, two types of allele probes including base sequences complementary to allele A and allele B may be prepared. At this time, the base sequences of the flaps are different base sequences. If two types of FRET probes for detecting the flaps are prepared and each reporter dye can be identified, the base type can be determined based on the same concept as the TacMan PCR method.
The advantage of the Invader method is that the FRET probe is the only oligonucleotide that needs to be labeled. The FRET probe can use the same oligonucleotide regardless of the base sequence to be detected. Therefore, mass production is possible. On the other hand, since the allele probe and the invader probe do not need to be labeled, a genotyping reagent can be manufactured at low cost.
[RCA method]
An example of a method for determining a base species that does not depend on the PCR method is the RCA method. A DNA amplification method based on a reaction in which a DNA polymerase having a strand displacement action synthesizes a long complementary strand using a circular single-stranded DNA as a template is the Rolling Circle Amplification (RCA) method (Lizardri PM et al.,). Nature Genetics 19, 225, 1998). In the RCA method, an amplification reaction is configured using a primer that anneals to a circular DNA to initiate complementary strand synthesis and a second primer that anneals to a long complementary strand generated by this primer.
In the RCA method, a DNA polymerase having a strand displacement action is used. Therefore, the portion that has become double-stranded by complementary strand synthesis is replaced by a complementary strand synthesis reaction started from another primer annealed to the 5 ′ side. For example, the complementary strand synthesis reaction using circular DNA as a template does not end in one round. Complementary strand synthesis continues while replacing the previously synthesized complementary strand, and a long single-stranded DNA is produced. On the other hand, the second primer anneals to the long single-stranded DNA generated using the circular DNA as a template, and complementary strand synthesis starts. Since single-stranded DNA generated by the RCA method uses circular DNA as a template, the base sequence is a repetition of the same base sequence. Thus, continuous production of long single strands results in continuous annealing of the second primer. As a result, single-stranded portions that can be annealed by the primer are continuously generated without going through a denaturing step. Thus, DNA amplification is achieved.
If the circular single-stranded DNA necessary for the RCA method is generated according to the base type of the polymorphic site, the base type can be determined using the RCA method. For this purpose, a linear single-stranded padlock probe is used. The padlock probe has complementary base sequences on both sides of the polymorphic site to be detected at the 5 ′ end and 3 ′ end. These base sequences are linked at a portion consisting of a special base sequence called a backbone. If the polymorphic site is a base sequence complementary to the end of the padlock probe, the end of the padlock probe hybridized to the allele can be ligated by DNA ligase. As a result, the linear padlock probe is circularized and the reaction of the RCA method is triggered. The reaction of the DNA ligase significantly decreases the reaction efficiency when the terminal portion to be ligated is not completely complementary. Therefore, the base type of the polymorphic site can be determined by confirming the presence or absence of ligation by the RCA method.
The RCA method can amplify DNA but does not generate a signal as it is. If only the presence or absence of amplification is used as an index, the base species cannot usually be determined unless a reaction is performed for each allele. Methods are known in which these points are improved for the determination of base species. For example, using a molecular beacon, the base type can be determined in one tube based on the RCA method. The molecular beacon is a signal generation probe using a fluorescent dye and a quencher, as in the TaqMan method. The 5 'end and 3' end of the molecular beacon are composed of complementary base sequences and independently form a hairpin structure. If both ends are labeled with a fluorescent dye and a quencher, a fluorescent signal cannot be detected in a state where a hairpin structure is formed. If a part of the molecular beacon is set as a base sequence complementary to the amplification product of the RCA method, the molecular beacon hybridizes to the amplification product of the RCA method. Since the hairpin structure is eliminated by hybridization, a fluorescent signal is generated.
The advantage of the molecular beacon is that the base sequence of the molecular beacon can be made common regardless of the detection target by using the base sequence of the backbone portion of the padlock probe. By changing the backbone base sequence for each allele and combining two types of molecular beacons with different fluorescence wavelengths, the base type can be determined in one tube. Since the cost of synthesis of fluorescently labeled probes is high, the ability to use a common probe regardless of the measurement target is an economic advantage.
All of these methods have been developed for genotyping a large amount of samples at high speed. Except for MALDI-TOF / MS, it is usually necessary to prepare a labeled probe or the like in any way for any method. On the other hand, a method for determining a base type that does not rely on a labeled probe has been performed for a long time. As one of such methods, for example, a method using restriction fragment length polymorphism (RFLP), a PCR-RFLP method, and the like can be mentioned.
RFLP utilizes the fact that a restriction enzyme recognition site mutation or insertion or deletion of a base in a DNA fragment caused by restriction enzyme treatment can be detected as a change in the size of the fragment produced after the restriction enzyme treatment. If there is a restriction enzyme that recognizes a base sequence containing a polymorphism to be detected, the base of the polymorphic site can be known by the principle of RFLP.
As a method that does not require a labeled probe, a method of detecting a difference in base using a change in secondary structure of DNA as an index is also known. PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its base sequence (Cloning and polymerase chain reaction-single-strand conformation polymorphism anomalous ensemble genes 11). 1992 Jan 1; 12 (1): 139-146, Detection of p53 gene mutations in human brain tumours by single-strand conformation polymorphism analysis of oligomeric polymorphism. ncogene.1991 Aug 1; 6 (8): 1313-1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr. 1; 4 (5). The PCR-SSCP method is performed by dissociating the PCR product into single-stranded DNA and separating it on a non-denaturing gel. Since the mobility on the gel varies depending on the secondary structure of the single-stranded DNA, if there is a difference in base at the polymorphic site, it can be detected as a difference in mobility.
In addition, examples of methods that do not require a labeled probe include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated according to differences in instability. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of a partially dissociated DNA fragment becomes very slow and is different from the mobility of a complete double-stranded DNA without a dissociated part, so that both can be separated.
Specifically, first, a region containing a polymorphic site is amplified by a PCR method or the like. The amplification product is hybridized with a probe DNA whose base sequence is known to make a double strand. This is electrophoresed in a polyacrylamide gel that gradually increases as the concentration of denaturing agents such as urea moves, and is compared with a control. In a DNA fragment in which a mismatch has occurred due to hybridization with the probe DNA, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the moving speed becomes extremely slow. The presence or absence of mismatch can be detected by detecting the difference in mobility generated in this way.
It is also possible to determine the base species using a DNA array (cell engineering separate volume "DNA microarray and the latest PCR method", Shujunsha, 2000.4 / 20, pp97-103 "Analysis of SNPs using oligo DNA chips" , Shinichi Kanie). In the DNA array, sample DNA (or RNA) is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of polymorphic sites simultaneously.
In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous film such as a nitrocellulose membrane can also be used.
In the present invention, an oligonucleotide-based array developed by Affymetrix can be exemplified as a nucleotide immobilization (array) method. In an oligonucleotide array, oligonucleotides are usually synthesized in vitro. For example, in situ synthesis methods of oligonucleotides using photolithographic technology (Affymetrix) and ink-jet (Rosetta Informatics) technology for immobilizing chemical substances are already known. Can be used.
The oligonucleotide is composed of a base sequence complementary to a region containing SNPs to be detected. The length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bases, preferably 10 to 50 bases, more preferably 15 to 25 bases when the oligonucleotide is immobilized. Furthermore, in general, mismatch (MM) probes are used in the DNA array method in order to avoid errors due to cross hybridization (non-specific hybridization). The mismatch probe constitutes a pair with an oligonucleotide having a base sequence completely complementary to the target base sequence. An oligonucleotide consisting of a base sequence that is completely complementary to a mismatch probe is called a perfect match (PM) probe. In the process of data analysis, the influence of cross-hybridization can be reduced by eliminating the signal observed with the mismatch probe.
A sample for genotyping by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a subject. The biological sample is not particularly limited. For example, a DNA sample can be prepared from chromosomal DNA extracted from tissues or cells of peripheral blood leukocytes, skin, oral mucosa, etc., tears, saliva, urine, feces or hair of the subject. A specific region of chromosomal DNA is amplified using a primer for amplifying a region containing a polymorphic site to be determined. At this time, a plurality of regions can be simultaneously amplified by the multiplex PCR method. The multiplex PCR method is a PCR method using a plurality of primer sets in the same reaction solution. When analyzing a plurality of polymorphic sites, the multiplex PCR method is useful.
In general, in the DNA array method, a DNA sample is amplified by the PCR method and the amplification product is labeled. A labeled primer is used for labeling the amplification product. For example, first, genomic DNA is amplified by a PCR method using a primer set specific to a region containing a polymorphic site. Next, DNA labeled with biotin is synthesized by a labeling PCR method using a primer labeled with biotin. The biotin-labeled DNA synthesized in this way is hybridized to the oligonucleotide probe on the chip. The hybridization reaction solution and reaction conditions can be appropriately adjusted according to conditions such as the length of the nucleotide probe immobilized on the substrate and the reaction temperature. One skilled in the art can design appropriate hybridization conditions. In order to detect the hybridized DNA, avidin labeled with a fluorescent dye is added. The array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.
More specifically, after obtaining the DNA containing the polymorphic site of the present invention prepared from the subject and the solid phase on which the nucleotide probe is immobilized, the DNA and the solid phase are then obtained. Make contact. Furthermore, the base species of the polymorphic site of the present invention is determined by detecting DNA hybridized to the nucleotide probe immobilized on the solid phase.
In the present invention, “solid phase” means a material capable of fixing nucleotides. The solid phase of the present invention is not particularly limited as long as nucleotides can be immobilized. Specifically, solid phases including microplate wells, plastic beads, magnetic particles, substrates and the like may be exemplified. it can. As the “solid phase” of the present invention, a substrate generally used in DNA array technology can be preferably used. In the present invention, the “substrate” means a plate-like material capable of fixing nucleotides. In the present invention, the nucleotide includes oligonucleotides and polynucleotides.
In addition to the method described above, an allele specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allele-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where a polymorphic site to be detected exists. When ASO is hybridized to sample DNA, if a mismatch occurs in the polymorphic site due to polymorphism, the efficiency of hybridization is reduced. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.
The present invention also provides a diagnostic agent for type 2 diabetes, which comprises an oligonucleotide that hybridizes to DNA containing the polymorphic site of the present invention and has a chain length of at least 15 nucleotides. Each of these is used for a test using gene expression as an index or a test using gene polymorphism as an index.
The oligonucleotide specifically hybridizes to DNA containing the polymorphic site of the present invention. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the second edition 1989) means that cross-hybridization does not occur significantly with DNA encoding other proteins. If the specific hybridization is possible, the oligonucleotide does not need to be completely complementary to each of the base sequences described in (1) to (29) to be detected.
The oligonucleotide can be used as a probe or primer in the test method of the present invention. When using this oligonucleotide as a primer, the length is 15 bp-100 bp normally, Preferably it is 17 bp-30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA containing the polymorphic site of the present invention.
The present invention provides a primer for amplifying a region containing the polymorphic site of the present invention, and a probe that hybridizes to a DNA region containing the polymorphic site. In the present invention, the primer for amplifying a region containing a polymorphic site also includes a primer that can initiate complementary strand synthesis toward the polymorphic site using DNA containing the polymorphic site as a template. The primer can also be expressed as a primer for providing a replication origin on the 3 ′ side of the polymorphic site in DNA containing the polymorphic site. The interval between the region where the primer hybridizes and the polymorphic site is arbitrary. For the interval between the two, a suitable number of bases can be selected according to the analysis method of the base at the polymorphic site. For example, in the case of a primer for analysis using a DNA chip, the primer can be designed so that an amplification product having a length of 20 to 500, usually 50 to 200 bases, can be obtained as a region containing a polymorphic site. A person skilled in the art can design a primer according to the analysis method based on the base sequence information about the surrounding DNA region including the polymorphic site. The base sequence constituting the primer of the present invention can be appropriately modified as well as a base sequence completely complementary to the genomic base sequence.
In addition to the base sequence complementary to the genomic base sequence, an arbitrary base sequence can be added to the primer of the present invention. For example, in a primer for a polymorphism analysis method using a type IIs restriction enzyme, a primer to which a recognition sequence for a type IIs restriction enzyme is added is used. Such a primer with a modified base sequence is included in the primer of the present invention. Furthermore, the primer of the present invention can be modified. For example, a fluorescent substance or a primer labeled with a binding affinity substance such as biotin or digoxin is used in various genotyping methods. Primers having these modifications are also included in the present invention.
On the other hand, in the present invention, a probe that hybridizes to a region containing a polymorphic site refers to a probe that can hybridize to a polynucleotide having a base sequence of a region containing a polymorphic site. More specifically, a probe containing a polymorphic site in the base sequence of the probe is preferable as the probe of the present invention. Alternatively, depending on the base analysis method at the polymorphic site, the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, although the polymorphic site is not included in the base sequence of the probe itself, a probe including a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.
In other words, a probe capable of hybridizing to the polymorphic site of the present invention on genomic DNA or a site adjacent to the polymorphic site is preferred as the probe of the present invention. In the probe of the present invention, modification of the base sequence, addition of the base sequence, or modification is allowed in the same manner as the primer. For example, a probe used in the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe of the present invention as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the DNA region surrounding the polymorphic site of the present invention in the genome.
The primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it. The length of the base sequence complementary to the genomic DNA of the primer or probe of the present invention is usually 15 to 100, generally 15 to 50, usually 15 to 30. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.
The present invention also provides a reagent for use in the test method of the present invention. The reagent of the present invention includes the primer and / or probe of the present invention. Various reagents, enzyme substrates, buffers and the like can be combined with the reagent of the present invention according to the method for determining the base species. Examples of the enzyme include enzymes necessary for various analysis methods exemplified as the above-mentioned base species determination method, such as DNA polymerase, DNA ligase, or IIs restriction enzyme. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.
Furthermore, the reagent of the present invention can be accompanied by a control in which the base at the polymorphic site is clear. As a control, a genome or a genomic fragment in which the base type of the polymorphic site is known in advance can be used. The genome may be extracted from cells, or cells or cell fractions may be used. If cells are used as a control, the result of the control can prove that the genomic DNA extraction operation was performed correctly. Alternatively, DNA consisting of a base sequence containing a polymorphic site can be used as a control. Specifically, a YAC vector or a BAC vector containing a genome-derived DNA whose base species at the polymorphic site of the present invention has been clarified is useful as a control. Alternatively, a vector in which only several hundred bases corresponding to the polymorphic site are cut out and inserted can be used as a control.
Furthermore, another aspect of the test agent in the present invention is a test agent for type 2 diabetes comprising a solid phase on which a nucleotide probe that hybridizes with the DNA containing the polymorphic site of the present invention is immobilized. These are used for examinations using the polymorphic site of the present invention as an index. These preparation methods are as described above.

  FIG. 1 shows the structure of the ABCA1 gene in the center. Exons are indicated by vertical lines or boxes. The translation region starts with exon 2 and extends to exon 50. The SNPs used for the analysis are shown as Nos. 1 to 31 in “SNP used for the analysis”, but these JSNPIDs are shown in parentheses (SNP number: JSNP ID (ssj000−)); 1: 1176,2 : 1179, 3: 1184, 4: 1191, 5: 1194, 6: 1195, 7: 1198, 8: 1205, 9: 1209, 10: 1212, 11: 1217, 12: 1235, 13: 1241, 14: 1242. 15: 1259, 16: 1264, 17: 1271, 18: 1276, 19: 1279, 20: 1280, 21: 1289, 22: 1296, 23: 1300, 24: 1304, 25: 1305, 26: 1306, 27 : 1311, 28: 1322, 29: 1326, 30: 1333, 31: 1337). The predicted LD block is shown as a “LD block” with a horizontal line and a white circle (◯). White circles indicate SNPs constituting the haplotype. The upper arrow (▼) indicates the position of the SNP that has been previously associated with serum lipid levels or coronary artery disease (CAD).

数値は平均値±ＳＤ．である。統計学的有意はスチューデントのｔ検定によって決定した。Ｐ＜０．０５はアスタリスク＊で表中に示した。
関連解析は、症例群と正常耐糖能（ＮＧＴ）対照群のジェノタイプをカイ二乗検定によって検定し、ｐ＜０．０１を統計学的に有意であるとした。ＤＭ母集団の高い発生率（ＤＭおよびＩＧＴに関してそれぞれ、１０．５％および１４．５％）を考慮すると（Ｓｅｋｉｋａｗａ，Ａ．ｅｔ ａｌ．Ｊ Ｄｉａｂｅｔｅｓ Ｃｏｍｐｌｉｃａｔｉｏｎ １４，７８−８３（２０００））、試験の検出力は相対危険度２および対立遺伝子頻度０．１で、ＤＭ感受性遺伝子を検出するために５％有意水準で８０％を超える。表３５〜３７は、ＤＭ関連ＳＮＰを含む遺伝子、有意差を示したＳＮＰのＪＳＮＰＩＤ、ＳＮＰｓの有リスク対立遺伝子、ｐ値、オッズ比（ＯＲ）および９５％信頼区間を示す。一般的な意味において、有リスク対立遺伝子の遺伝は、優性または劣性のいずれかに従い、本実施例の試験結果から認めることができる。優性型の対立遺伝子は、そのホモ接合状態およびヘテロ接合状態の双方に関して統計学的に有意となる傾向があるが（表３６）、劣性型の対立遺伝子は、そのホモ接合型の場合に限って統計学的に有意となる傾向がある（表３７）。

〔優性型〕

ＳＮＰｓの詳細は、″ＪＳＮＰ ＩＤ″として記載したＩＤナンバーをもってウェブサイト（ｈｔｔｐ：／／ｓｎｐ．ｉｍｓ．ｕ−ｔｏｋｙｏ．ａｃ．ｊｐ）に公開されている。なお、Ｐ値および９５％ＣＩはカイ二乗検定によって決定した。†はｐ＜０．０１のマルチプルＳＮＰｓを持つ遺伝子を表す。
〔劣性型〕

ＳＮＰｓの詳細は、″ＪＳＮＰ ＩＤ″として記載したＩＤナンバーをもってウェブサイト（ｈｔｔｐ：／／ｓｎｐ．ｉｍｓ．ｕ−ｔｏｋｙｏ．ａｃ．ｊｐ）に公開されている。なお、Ｐ値および９５％ＣＩはカイ二乗検定によって決定した。＊は、対象群の有リスク遺伝子型の数が０であったためＯＲが決定不可であったものを表し、†はｐ＜０．０１のマルチプルＳＮＰｓを持つ遺伝子を表す。
また、症例と対照群のあいだの遺伝距離（ハプロタイプに基づくＦｓｔ）を測定したが、母集団におけるサブストラクチャーは検出されなかった。興味深いことに、推定のＤＭ関連遺伝子であるＭＥＴ遺伝子（表３６）は、ＤＭとの関連の証拠を有しない遺伝的に連鎖していない遺伝子（ＧＲＭ８）と１０Ｍｂ離れていた。遺伝距離を測定することによって、ＭＥＴは、症例群と対照群のあいだで統計学的に有意な差を示した（Ｆｓｔ＝０．００６２５、ｐ＝０．０１６５）のに対し、ＧＲＭ８は２群が識別不能であることが示された（Ｆｓｔ＝０．０００２７、ｐ＝０．９０８７３）。
これらの上記２つの表における遺伝子をｐ値に従って分類した。全体的に、有意な遺伝子の上位５個は、ＭＥＴ、ＮＰＨＳ１、ＴＦＲＣ、ＭＭＰ１９およびＳＬＣ１Ａであり、ｐ値はそれぞれ、０．０００８、０．００１１、０．００２２、０．００２２、および０．００２４であった。統計学的に有意な試験において２つまたはそれ以上のＳＮＰｓを含む遺伝子は、ＤＬＧ５、ＦＲＡＰ１、ＬＡＲＧＥおよびＭＥＴであった。さらに、これまでに報告されたＤＭまたはその関連疾患に対する感受性座に存在する遺伝子は、ＴＨＢＳ３、ＲＧＳ５、ＢＬＺＦ１、ＴＦＲＣおよびＬＡＲＧＥ（Ｖｉｏｎｎｅｔ，Ｎ．ｅｔ ａｌ．Ａｍ．Ｊ．Ｈｕｍ．Ｇｅｎｅｔ．６７，１４７０−１４８０（２０００），Ｌｉｎｄｇｒｅｎ，Ｃ．Ｍ．ｅｔ ａｌ．Ａｍ．Ｊ．Ｈｕｍ．Ｇｅｎｅｔ．７０，５０９−５１６（２００２），Ｌａｎｄｅｒ，Ｅ．Ｓ．＆ Ｋｒｕｇｌｙａｋ，Ｌ．Ｎａｔｕｒｅ Ｇｅｎｅｔ．１１，２４１−２４７（１９９５），Ｗｉｌｔｓｈｉｒｅ，Ｓ．ｅｔ ａｌ．Ａｍ．Ｊ．Ｈｕｍ．Ｇｅｎｅｔ．６９，５５３−５６９（２００１），Ｇｈｏｓｈ，Ｓ．ｅｔ ａｌ．Ａｍ．Ｊ．Ｈｕｍ．Ｇｅｎｅｔ．６７，１１７４−１１８５（２０００），Ｗａｔａｎａｂｅ，Ｒ．Ｍ．ｅｔ ａｌ．Ａｍ．Ｊ．Ｈｕｍ．Ｇｅｎｅｔ．６７，１１８６−１２００（２０００），Ｐｒａｔｌｅｙ，Ｒ．Ｅ．ｅｔ ａｌ．Ｊ．Ｃｌｉｎ．Ｉｎｖｅｓｔ．１０１，１７５７−１７６４（１９９８））であった。
ＤＭに関わりがあることが知られている遺伝子は３個だけであった（ＡＢＣＣ８（ＳＵＲ）、ＬＥＰ、およびＴＮＦ）（Ｅｌｂｅｉｎ，Ｓ．Ｃ．ｅｔ ａｌ．Ｄｉａｂｅｔｅｓ Ｃａｒｅ ２４，４７２−４７８（２００１），Ｏｚａｔａ，Ｍ．ｅｔ ａｌ．Ｊ Ｃｌｉｎ．Ｅｎｄｏｃｒｉｎｏｌ．Ｍｅｔａｂ．８６，３６５９−３６６４（２００１），Ｄａｙ，Ｃ．Ｐ．ｅｔ ａｌ．Ｄｉａｂｅｔｏｌｏｇｉａ ４１，４３０−４３４（１９９８））。本発明において認められた遺伝子の大多数は、グルコース代謝以外の機能または経路に関係している。ＤＭに間接的に関与しているそれらの遺伝子は、以下の領域に割付することができる：脂質代謝（ＡＢＣＡ１、ＦＡＢＰ２、ＬＤＬＣ、およびＶＬＤＬＲ）、免疫系（ＦＲＡＰ１およびＰ２ＲＸ７）、神経系（ＣＴＳＤおよびＳＬＣ１Ａ１）、恒常性（ＳＥＲＰＩＮＡ３およびＳＥＲＰＩＮＢ２）、細胞構造の維持（ＤＬＧ５およびＬＡＲＧＥ）、組織再モデリング（ＭＭＰ１９）等。
［実施例２］ ２型糖尿病に関連する多型部位の連鎖不平衡についての解析
（ａ）有意差を示したＳＮＰの周辺のＳＮＰをタイピングし、これらのＳＮＰ間に連鎖不平衡が成り立つか否かを検討した。ＳＮＰ間に連鎖不平衡が認められた場合の解析結果を表３８〜５１に示す。
（ａ−１）ＭＥＴ

（ａ−２）ＮＰＨＳ１

（ａ−３）ＳＥＲＰＩＮＢ２

（ａ−４）ＡＢＣＡ１

（ａ−５）ＣＴＳＤ

（ａ−６）ＳＴＥ

（ａ−７）ＡＢＣＣ８

（ａ−８）ＭＹＬ２

（ａ−９）ＤＬＧ５

（ａ−１０）ＬＤＬＣ

（ａ−１１）ＬＡＲＧＥ

（ａ−１２）ＢＬＺＦ１

（ａ−１３）ＦＲＡＰ１

（ａ−１４）ＬＥＰ

（ｂ）ＳＮＰ間について連鎖不平衡が認められた場合には、ハプロタイプ型を用いた疾患関連解析を行った。見出された個々のハプロタイプにおいて症例群と正常耐糖能（ＮＧＴ）対照群をカイ二乗検定を用いて解析し、ｐ＜０．０５を有意差ありとした。結果を表５２に示す。

さらに、カイ二乗検定と異なる意味を持つ検定方法として、症例群と正常耐糖能（ＮＧＴ）対照群の分布全体の差を検定する手法（ＦＡＳＴＥＨＰＬＵＳ）（Ｚｈａｏ ＪＨ，Ｓｈａｍ ＰＣ．Ｆａｓｔｅｒ ｈａｐｌｏｔｙｐｅ ｆｒｅｑｕｅｎｃｙ ｅｓｔｉｍａｔｉｏｎ ｕｓｉｎｇ ｕｎｒｅｌａｔｅｄｓｕｂｊｅｃｔｓ．Ｈｕｍ Ｈｅｒｅｄ ２００２；５３（１）：３６−４１）を用いて統計解析を行い、ｐ＜０．０５を有意差ありとした。ＦＡＳＴＥＨＰＬＵＳは全体を比較する方法であり、例えば複数のアリルが疾患感受性／抵抗性を持つ場合などに異なる結果をもたらす可能性がある。個々のハプロタイプ、および、ハプロタイプ領域に含まれるＳＮＰについてＦＡＳＴＥＨＰＬＵＳを適用したところ、カイ二乗検定では有意差が認められなかった以下のハプロタイプおよびＳＮＰが有意に転じた。
以下にＦＡＳＴＥＨＰＬＵＳにて新たに有意と認められた４つのＳＮＰを示す。
（１）ＩＭＳ−ＪＳＴ０３５６５６（ＮＰＨＳ１） ｐ値＝０．０２９３、（２）ＩＭＳ−ＪＳＴ０１７２２２（ＣＴＳＤ） ｐ値＝０．０２８２、（３）ＩＭＳ−ＪＳＴ０７５５６３（ＳＴＥ） ｐ値＝０．０１１９、（４）ＩＭＳ−ＪＳＴ０２０６０７（ＬＡＲＧＥ） ｐ値＝０．０２２２
以下にＦＡＳＴＥＨＰＬＵＳにて新たに有意と認められた５つのハプロタイプを示す。
（１）ＭＥＴ．ｈａｐｌｏ２ ｐ値＝０．０２５５、（２）ＣＴＳＤ．ｈａｐｌｏ１ ｐ値＝０．０４０１、（３）ＳＴＥ．．ｈａｐｌｏ１ ｐ値＝０．０１１５、（４）ＡＢＣＣ８．ｈａｐｌｏ１ ｐ値＝０．００７６、（５）ＭＹＬ２．ｈａｐｌｏ１ ｐ値＝０．００７６
（ｃ）各のハプロタイプの観察例は下記の表５３〜６６に示される。
（ｃ−１）ＭＥＴ

（ｃ−２）ＮＰＨＳ１

（ｃ−３）ＳＥＲＰＩＮＢ２

（ｃ−４）ＡＢＣＡ１

（ｃ−５）ＣＴＳＤ

（ｃ−６）ＳＴＥ

（ｃ−７）ＡＢＣＣ８

（ｃ−８）ＭＹＬ２

（ｃ−９）ＤＬＧ５

（ｃ−１０）ＬＤＬＣ

（ｃ−１１）ＬＡＲＧＥ

（ｃ−１２）ＢＬＺＦ１

（ｃ−１３）ＦＲＡＰ１

（ｃ−１４）ＬＥＰ

表６７にＭＥＴのハプロタイプ解析の結果を示す。

表６８にＭＥＴのディプロタイプ解析の結果を示す。

表６９にＭＥＴのｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表７０にＭＥＴの有意なハプロタイプについて示す。

表７１にＮＰＨＳ１のハプロタイプ解析の結果を示す。

表７２にＮＰＨＳ１のディプロタイプ解析の結果を示す。

表７３にＮＰＨＳ１のｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表７４にＮＰＨＳ１の有意なハプロタイプについて示す。

表７５にＳＥＲＰＩＮＢ２のハプロタイプ解析の結果を示す。

表７６にＳＥＲＰＩＮＢ２のディプロタイプ解析の結果を示す。

表７７にＳＥＲＰＩＮＢ２のｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表７８にＳＥＲＰＩＮＢ２の有意なハプロタイプについて示す。

表７９にＳＴＥのハプロタイプ解析の結果を示す。

表８０にＳＴＥのディプロタイプ解析の結果を示す。

表８１にＳＴＥのｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表８２にＳＴＥの有意なハプロタイプについて示す。

表８３にＤＬＧ５のハプロタイプ解析の結果を示す。

表８４にＤＬＧ５のディプロタイプ解析の結果を示す。

表８５にＤＬＧ５のｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表８６にＤＬＧ５の有意なハプロタイプについて示す。

表８７にＬＤＬＣのハプロタイプ解析の結果を示す。

表８８にＬＤＬＣのディプロタイプ解析の結果を示す。

表８９にＬＤＬＣのｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表９０にＬＤＬＣの有意なハプロタイプについて示す。

表９１にＢＬＺＦ１のハプロタイプ解析の結果を示す。

表９２にＢＬＺＦ１のディプロタイプ解析の結果を示す。

表９３にＢＬＺＦ１のｓｎｐ解析ｐ−ｖａｌｕｅを示す。

表９４にＢＬＺＦ１の有意なハプロタイプについて示す。

〔実施例３〕 解析対象
本発明者らは、ネフリンは膵ベータ細胞で重要な役割を果たし、その機能障害はＤＭの進行をもたらすと推測した。
フィンランド型先天性ネフローゼ症候群（ＮＰＨＳ１）はまれな常染色体劣性遺伝疾患であり、生後すぐにネフローゼ症候群をきたす（Ｒａｐｏｌａ Ｊ著、「Ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」、Ｐｅｄｉａｔｒ Ｎｅｐｈｒｏｌ、１：４４１−４４６，１９８７年）。フィンランド型先天性ネフローゼ症候群の原因である遺伝子はポジショナルクローニングがなされており、その産物はネフリンと名づけられた（Ｋｅｓｔｉｌａ Ｍ，ら著、「Ｐｏｓｉｔｉｏｎａｌｌｙ ｃｌｏｎｅｄ ｇｅｎｅ ｆｏｒ ａ ｎｏｖｅｌ ｇｌｏｍｅｒｕｌａｒ ｐｒｏｔｅｉｎ−ｎｅｐｈｒｉｎ−ｉｓ ｍｕｔａｔｅｄ ｉｎ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」、Ｍｏｌ Ｃｅｌｌ．１：５７５−５８２，１９９８年）。
ネフリン遺伝子（ＮＰＨＳ１）は２６ｋｂであり、２９のエキソンを持ち、タンパク質をコードする３７２６塩基からなるｍＲＮＡが得られている（Ｋｅｓｔｉｌａ Ｍら著、「Ｐｏｓｉｔｉｏｎａｌｌｙ ｃｌｏｎｅｄ ｇｅｎｅ ｆｏｒ ａ ｎｏｖｅｌ ｇｌｏｍｅｒｕｌａｒ ｐｒｏｔｅｉｎ−ｎｅｐｈｒｉｎ−ｉｓ ｍｕｔａｔｅｄ ｉｎ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」、Ｍｏｌ Ｃｅｌｌ．１：５７５−５８２，１９９８年、Ｌｅｎｋｅｒｉ Ｕら著、「Ｓｔｒｕｃｔｕｒｅ ｏｆ ｔｈｅ ｇｅｎｅ ｆｏｒ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ ｏｆ ｔｈｅ Ｆｉｎｎｉｓｈ ｔｙｐｅ（ＮＰＨＳ１）ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｍｕｔａｔｉｏｎ．」、Ａｍ Ｊ Ｈｕｍ Ｇｅｎｅｔ．、６４：５１−６１，１９９９年）。ネフリンは８個のイムノグロブリン様ドメインを含む細胞外部分と１つの３型フィブロネクチンドメイン様モジュール、１回膜貫通ドメインと１つの細胞質Ｃ末ドメインからなり、イムノグロブリンスーパーファミリーの膜貫通タンパクと推定される（Ｋｅｓｔｉｌａ Ｍら著、「Ｐｏｓｉｔｉｏｎａｌｌｙ ｃｌｏｎｅｄ ｇｅｎｅ ｆｏｒ ａ ｎｏｖｅｌ ｇｌｏｍｅｒｕｌａｒ ｐｒｏｔｅｉｎ−ｎｅｐｈｒｉｎ−ｉｓ ｍｕｔａｔｅｄ ｉｎ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」 Ｍｏｌ Ｃｅｌｌ．１：５７５−５８２，１９９８．、Ｌｅｎｋｅｒｉ Ｕら著、「Ｓｔｒｕｃｔｕｒｅ ｏｆ ｔｈｅ ｇｅｎｅ ｆｏｒ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ ｏｆ ｔｈｅ Ｆｉｎｎｉｓｈ ｔｙｐｅ（ＮＰＨＳ１）ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｍｕｔａｔｉｏｎ．」、Ａｍ Ｊ Ｈｕｍ Ｇｅｎｅｔ．６４：５１−６１，１９９９．）。したがって、ネフリンの機能はイムノグロブリンスーパーファミリーの他のメンバーと類似していると考えられ、他のメンバーの機能としては細胞接着、シグナリング、細胞移動等の様々な機能が知られている（Ｂｒｕｍｍｅｎｄｏｒｆ Ｔ，Ｒａｔｈｊｅｎ ＦＤ著、「Ｔｈｅ ｉｍｍｕｎｏｇｌｏｂｉｎ ｓｕｐｅｒｆａｍｉｌｙ．」、Ｉｎ Ｐｒｏｔｅｉｎ Ｐｒｏｆｉｌｅ，ｖｏｌ １，Ｓｈｅｔｅｒｌｉｎｅ Ｐ，Ｈｙｍｐｈｒｉｅｓ Ｍ，Ｌａｔｃｈｍａｎ Ｄ，Ｄｏｗｎｅｓ Ｐ，編、Ｌｏｎｄｏｎ，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ Ｌｉｍｉｔｅｄ，１９９４年、ｐ．９５１−１０５８．）。
ＮＰＨＳ１でネフリンに変異がみられるということから、ネフリンの腎機能維持に果たす役割は重大と考えられ、広汎な研究がなされてきた。ネフリンは有足細胞のスリット膜領域に局在すると報告されており（Ｈｏｌｔｈｏｆｅｒ Ｈら著、「Ｎｅｐｈｒｉｎ ｌｏｃａｌｉｚｅｓ ａｔ ｔｈｅ ｐｏｄｏｃｙｔｅ ｆｉｌｔｒａｔｉｏｎ ｓｌｉｔ ａｒｅａ ａｎｄ ｉｓ ｃｈａｒａｃｔｅｒｉｓｔｉｃａｌｌｙ ｓｐｌｉｃｅｄ ｉｎ ｔｈｅ ｈｕｍａｎ ｋｉｄｎｅｙ．」、Ａｍ Ｊ Ｐａｔｈｏｌ．、１５５：１６８１−１６８７，１９９９年、Ｈｏｌｚｍａｎ ＬＢら著、「Ｎｅｐｈｒｉｎ ｌｏｃａｌｉｚｅｓ ｔｏ ｔｈｅ ｓｌｉｔ ｐｏｒｅ ｏｆ ｔｈｅ ｇｌｏｍｅｒｕｌａｒ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌ．」、Ｋｉｄｎｅｙ Ｉｎｔ．、５６：１４８１−１４９１，１９９９年、Ｒｕｏｔｓａｌａｉｎｅｎ Ｖら著、「Ｎｅｐｈｒｉｎ ｉｓ ｓｐｅｃｉｆｉｃａｌｌｙ ｌｏｃａｔｅｄ ａｔ ｔｈｅ ｓｌｉｔ ｄｉａｐｈｒａｇｍ ｏｆ ｇｌｏｍｅｒｕｌａｒ ｐｏｄｏｃｙｔｅｓ．」、Ｐｒｏｃ ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ．、９６：７９６２−７９６７，１９９９年）、同種親和性の細胞間相互作用を持ち、腎糸球体ろ過のバリア機能に重要な役割をもつと考えられる（Ｋｅｓｔｉｌａ Ｍら著、「Ｐｏｓｉｔｉｏｎａｌｌｙ ｃｌｏｎｅｄ ｇｅｎｅ ｆｏｒ ａ ｎｏｖｅｌ ｇｌｏｍｅｒｕｌａｒ ｐｒｏｔｅｉｎ−ｎｅｐｈｒｉｎ−ｉｓ ｍｕｔａｔｅｄ ｉｎ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」、Ｍｏｌ Ｃｅｌｌ．、１：５７５−５８２，１９９８年、Ｒｕｏｔｓａｌａｉｎｅｎ Ｖら著、「Ｎｅｐｈｒｉｎ ｉｓ ｓｐｅｃｉｆｉｃａｌｌｙ ｌｏｃａｔｅｄ ａｔ ｔｈｅ ｓｌｉｔ ｄｉａｐｈｒａｇｍ ｏｆ ｇｌｏｍｅｒｕｌａｒ ｐｏｄｏｃｙｔｅｓ．」、Ｐｒｏｃ ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ．、９６：７９６２−７９６７，１９９９年、Ｔｒｙｇｇｖａｓｏｎ Ｋ著、「Ｕｎｒａｖｅｌｉｎｇ ｔｈｅ ｍｅｃｈａｎｉｓｍｓ ｏｆ ｇｌｏｍｅｒｕｌａｒ ｕｌｔｒａｆｉｌｔｒａｔｉｏｎ：ｎｅｐｈｒｉｎ，ａ ｋｅｙ ｃｏｍｐｏｎｅｎｔ ｏｆ ｔｈｅ ｓｌｉｔ ｄｉａｐｈｒａｇｍ．」、Ｊ Ａｍ Ｓｏｃ ｎｅｐｈｒｏｌ．、１０：２４４０−２４４５，１９９９年）。
ネフリンは腎糸球体の有足細胞に特異的に発現すると報告されている（Ｋｅｓｔｉｌａ Ｍら著、「Ｐｏｓｉｔｉｏｎａｌｌｙ ｃｌｏｎｅｄ ｇｅｎｅ ｆｏｒ ａ ｎｏｖｅｌ ｇｌｏｍｅｒｕｌａｒ ｐｒｏｔｅｉｎ−ｎｅｐｈｒｉｎ−ｉｓ ｍｕｔａｔｅｄ ｉｎ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」Ｍｏｌ Ｃｅｌｌ．１：５７５−５８２，１９９８．）。しかし、ヒトの膵ベータ細胞において、ネフリン遺伝子およびタンパク質が発現することが最近報告された（Ｐａｌｍｅｎ Ｔら著、「Ｎｅｐｈｒｉｎ ｉｓ ｅｘｐｒｅｓｓｅｄ ｉｎ ｔｈｅ ｐａｎｃｒｅａｔｉｃ ｂｅｔａ ｃｅｌｌｓ．」、Ｄｉａｂｅｔｏｌｏｇｉａ．４４：１２７４−１２８０，２００１年）。これはネフリンが膵ベータ細胞の維持に何らかの機能を持つことを示している。膵臓では、腎臓と同様に、細胞相互作用および／または細胞質Ｃ末ドメインの８つのチロシンから示唆されるように、シグナル伝達の作用が予測される（Ｋｅｓｔｉｌａ Ｍら著、「Ｐｏｓｉｔｉｏｎａｌｌｙ ｃｌｏｎｅｄ ｇｅｎｅ ｆｏｒ ａ ｎｏｖｅｌ ｇｌｏｍｅｒｕｌａｒ ｐｒｏｔｅｉｎ−ｎｅｐｈｒｉｎ−ｉｓ ｍｕｔａｔｅｄ ｉｎ ｃｏｎｇｅｎｉｔａｌ ｎｅｐｈｒｏｔｉｃ ｓｙｎｄｒｏｍｅ．」Ｍｏｌ Ｃｅｌｌ．１：５７５−５８２，１９９８．）。しかし、膵ベータ細胞におけるネフリンの役割を明らかにした報告はない。
ＤＭはありふれた病気であるため、いくつかのネフリン遺伝子の多型が、ＤＭ進行のリスク因子になっていると推測した。そこで、本発明者らは本発明において、ネフリン遺伝子のＳＮＰと２型糖尿病（ＤＭ）が関連しているかどうかを、日本人の地域を基盤としたケースコントロール試験において検討した。
７２例のＤＭ、７５例の耐糖能障害（ＩＧＴ）、２２７例の正常耐糖能（ＮＧＴ）群を第１群として最初に解析した。次に３０例のＤＭ、７６例のＩＧＴ、２４４例のＮＧＴ群を第１群に追加し、第２群として解析した。これらのサンプルはフナガタ研究（Ｏｉｚｕｍｉ Ｔら著、「Ｇｅｎｏｔｙｐｅ Ａｒｇ／Ａｒｇ，ｂｕｔ ｎｏｔ Ｔｒｐ／ａｒｇ，ｏｆ ｔｈｅ Ｔｒｐ６４Ａｒｇ ｐｏｌｙｍｏｒｐｈｉｓｍ ｏｆ ｔｈｅ β_３−ａｄｒｅｎｅｒｇｉｃ ｒｅｃｅｐｔｏｒ ｉｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ ｔｙｐｅ ２ ｄｉａｂｅｔｅｓ ａｎｄ ｏｂｅｓｉｔｙ ｉｎ ａ ｌａｒｇｅ Ｊａｐａｎｅｓｅ ｓａｍｐｌｅ．」、Ｄｉａｂｅｔｅｓ Ｃａｒｅ．、２４：１５７９−１５８３，２００１年）のコホートより選択された。フナガタ研究は東京より約４００ｋｍ北の農業地帯において行われたポピュレーションベーススタディである。また、本研究は山形大学の倫理審査委員会の承認、および、対象者より文書によるインフォームドコンセントを得た上で実施された。全工程は、ガイドラインに従い実施した。なお、研究対象となった地域は何十年かの間ほとんど人の移動はない。したがって、本発明者らの研究は、集団の層別化が擬陽性をもたらすリスクを最小化するという意味で、遺伝的背景を研究するにはふさわしい設定である。
ＤＭ、ＩＧＴ、ＮＧＴの各群は年齢・性別を一致させて解析した（年齢６５．８±９．３ｖｓ．６５．１±９．８ｖｓ．６４．８±８．８、性別比（女性／男性），５６／４６ｖｓ．７７／７４ｖｓ．２６３／２０８）。遺伝子型と共に次の形質も解析した。
身長、体重、７５ｇ経口糖負荷試験、ＨｂＡ１ｃ、ウエスト周囲径、ヒップ周囲径、ウエスト／ヒップ比、ボディマスインデックス（ＢＭＩ）、体脂肪率、収縮期血圧、拡張期血圧、血清総コレステロール（ＣＨＯ）、中性脂肪（ＴＧ）、高比重リポタンパク（ＨＤＬ）コレステロール。尿タンパクは試験紙（ライフスティックス、バイエルメディカル社、東京）による半定量測定を行った。
〔実施例４〕 遺伝子型および遺伝子型解析
ネフリン遺伝子におけるＳＮＰｓ（翻訳開始コドンＡＴＧからヌクレオチド位置２９４のエキソン３におけるＣからＴ（Ｃ２９４Ｔ）（ＪＳＮＰデータベースＩＤ：ＩＭＳ−ＪＳＴ０３５６５６）；イントロン５／エキソン６スプライシング部位からヌクレオチド位置６１上流のイントロン５におけるＣからＧ（−６１Ｃ／Ｇ）（ＩＭＳ−ＪＳＴ０２１０６１）；エキソン１７におけるヌクレオチド位置２２８９のＣからＴ（２２８９Ｔ）（ＩＭＳ−ＪＳＴ０００５４１）が解析された。Ｃ２９４ＴおよびＣ２２８９ＴのＳＮＰｓは、サイレントな多型である。ゲノムＤＮＡを末梢血白血球より抽出した。遺伝子タイピングは、先に記載されているように、フルオロジェニックＰＣＲで行った（Ｒａｎａｄｅ Ｋら著、「Ｈｉｇｈ−ｔｈｒｏｕｇｈｐｕｔ ｇｅｎｏｔｙｐｉｎｇ ｗｉｔｈ ｓｉｎｇｌｅ ｎｕｃｌｅｏｔｉｄｅ ｐｏｌｙｍｏｒｐｈｉｓｍ．」、Ｇｅｎｏｍｅ ｒｅｓｅａｒｃｈ．、１１：１２６２−１２６８，２００１年）。増幅に使用したＴａｑＭａｎプライマーセットは以下のとおりである。
ＳＮＰ Ｃ２９４Ｔには５’−ＣＣＣＣＴＧＣＡＧＧＴＧＡＡＴＴＣＣＡ−３’（配列番号：３１）および５’−ＧＣＡＣＴＣＡＴＡＣＴＣＣＧＣＧＴＣＡＴＣ−３’（配列番号：３２）、ＳＮＰ −６１Ｃ／Ｇには５’−ＣＣＴＧＧＡＴＣＣＣＡＧＡＧＧＡＧＡＴＣＡ−３’（配列番号：３３）および５’−ＣＡＧＧＧＴＴＡＴＡＧＡＧＴＣＡＧＡＧＴＣＡＴＣＡＴＣＴ−３’（配列番号：３４）、ＳＮＰ Ｃ２２８９Ｔには５’−ＧＡＣＣＣＣＡＣＴＧＡＧＧＴＧＡＡＣＧＴ−３’（配列番号：３５）および５’−ＧＡＡＣＡＴＧＣＣＣＧＧＧＡＧＧＡＴ−３’（配列番号：３６）。
ＦＡＭあるいはＶＩＣレポーター分子で標識したＴａｑＭａｎプローブは以下のとおりである。ＳＮＰ Ｃ２９４Ｔには５’−ＴＧＣＡＣＡＴＴＧＡＧＧＣＣＴ−３’（配列番号：３７）および５’−ＣＡＣＡＴＣＧＡＧＧＣＣＴＧ−３’（配列番号：３８）、ＳＮＰ −６１Ｃ／Ｇには５’−ＴＣＡＧＧＣＡＧＡＡＧＡＧＧ−３’（配列番号：３９）および５’−ＴＣＡＧＧＧＡＧＡＡＧＡＧＧＴ−３’（配列番号：４０）、ＳＮＰ Ｃ２２８９Ｔには５’−ＴＧＧＣＡＴＣＡＡＣＡＧＴＧＣ−３’（配列番号：４１）および５’−ＡＴＴＧＧＣＡＴＣＧＡＣＡＧＴＧ−３’（配列番号：４２）。
クエリーの多型塩基は、プローブの三分割した真ん中および下線部において設計した。蛍光の増加は、ＡＢＩ ７９００ ＨＴシーケンスディテクションシステムを用いて計測した。
〔実施例５〕 ＳＮＰｓの遺伝子型とＤＭの関連
本発明者らは３つのＳＮＰｓ（Ｃ２９４Ｔ、Ｃ６５０Ｇ、Ｃ２２８９Ｔ）の遺伝子型とＤＭとの関連を第１サンプル群（ｎ＝３７４）を用いて解析した。関連解析はＦｉｓｈｅｒ’ｓ ｅｘａｃｔ ｐｒｏｂａｂｉｌｉｔｙ ｔｅｓｔにより、ＳＮＰ遺伝子型頻度と優性遺伝形式あるいは劣性遺伝形式を想定したケースとコントロールの比較により行った。表９５に示す通り、調査した全ＳＮＰは優性遺伝様式においてＤＭと有意な関連性が示された。表９５はネフリン遺伝子におけるＳＮＰｓとＤＭの関連を示す。

データは数値（％）で示す。ＩＧＴ群およびＤＭ群における有リスク遺伝子型の頻度をそれぞれＮＧＴ群と比較した。Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。
すなわち、Ｃ２９４ＴにおいてＴＴおよびＣＴの遺伝子型を持つ人、−６１Ｃ／ＧにおいてＧＧおよびＣＧの遺伝子型を持つ人、Ｃ２２８９ＴにおいてＴＴおよびＣＴの遺伝子型を持つ人は、他の遺伝子型を持つ人より糖尿病性が強かった。従って、これらの遺伝子型はＤＭの進行における有リスク遺伝子型であるといえる。逆に、ＤＭ群では健常群より有リスク遺伝子型を持つ人が多くみられた。
〔実施例６］ ハプロタイプ解析
本発明者らは、さらに３ＳＮＰを用いてハプロタイプ解析を行った。
各ＳＮＰ間の連鎖不平衡を、ＬＤサポート（Ｋｉｔａｍｕｒａ Ｙら著、「Ｄｅｔｅｒｍｉｎａｔｉｏｎ ｏｆ ｐｒｏｂａｂｉｌｉｔｙ ｄｉｓｔｒｉｂｕｔｉｏｎ ｏｆ ｄｉｐｌｏｔｙｐｅ ｃｏｎｆｉｇｕｒａｔｉｏｎ（ｄｉｐｌｏｔｙｐｅ ｄｉｓｔｒｉｂｕｔｉｏｎ）ｆｏｒ ｅａｃｈ ｓｕｂｊｅｃｔ ｆｒｏｍ ｇｅｎｏｔｙｐｉｃ ｄａｔａ ｕｓｉｎｇ ｔｈｅ ＥＭ ａｌｇｏｒｉｔｈｍ．」、Ａｎｎ Ｈｕｍ Ｇｅｎｅｔ．、６６：１８３−９３，２００２年）というハプロタイプ予測プログラムによって評価した。各ＳＮＰ間で強い連鎖不平衡を示すＳＮＰは、ハプロタイプブロックとした。ハプロタイプ頻度の決定は、ＬＤサポートを用いて行った。
ハプロタイプと疾患の表現型との関連評価には、マルチカラム表を用いたカイ二乗検定を行った。マルチカラム分割表におけるカイ二乗検定は通常、漸近平衡の正常な分布を示さない。
本発明者らはｃｌｕｍｐというプログラムを用いてＭｏｎｔｅ Ｃａｒｌｏ Ｍａｒｋｏｖ−Ｃｈａｉｎ（ＭＣＭＣ法）によるｐｅｒｍｕｔａｔｉｏｎを適用して解析を行った。
このプログラムのＣｌａｍｐ Ｔ１ ｔｅｓｔは列とカラムにおける辺縁の固定されたセルの数がランダムな分割表を生成する。１０，０００回シミュレートされた表では、カイ二乗統計解析量の帰無仮説下分布（ｎｕｌｌ ｄｉｓｔｒｉｂｕｔｉｏｎ）が得られ、これは実際のデータセットにおけるカイ二乗値に対応する経験ｐ値を求めるのに利用した。
Ｃｌｕｍｐ Ｔ３は存在する２×２の分割表すべてにおけるカイ二乗値の最大値を求めるプログラムである。すなわち、あるカラムとその他のすべてのカラムの和で２×２分割表を作成する。そして、カイ二乗値が最大値を示す表を選択し、ＭＣＭＣ化法によりｅｍｐｉｒｉｃａｌ ｐ値を前述のように計算した。ＭＣＭＣ法は、従来のボンフェローニ補正をした多重２×２分割表検定より、実質的な検定力において有利である。
結果、６ハプロタイプからなる１つのハプロタイプブロックが見つかった。ネフリン遺伝子におけるＳＮＰｓのハプロタイプの頻度および解析の結果を表９６に示す。

データは数値（％）で示す。２ｘ２フィッシャーの直接確率検定（Ｆｉｓｈｅｒ）を示す。Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。
２×６分割表のｃｌｕｍｐ分析のＴ１ ｔｅｓｔによるｅｍｐｉｒｉｃａｌ ｐ値は０．００３４を示した。ｃｌｕｍｐのＴ３ ｔｅｓｔによる結果は、ハプロタイプ１がｅｍｐｉｒｉｃａｌ ｐ値０．００２２（１００００ｐｅｒ ｍｕｔａｔｉｏｎｓ）を示した。さらに、２×２分割表を用いたフィッシャーの直接確率検定では、ハプロタイプ１（ｐ＝０．０００８）はオッズ比（ＯＲ）１以下という、有意な関連性を示した。ボンフェローニの補正後も０．００４７というとても低いｐ値を示した。ハプロタイプ１は非有リスクアリルである各ＳＮＰから構成され、オッズ比１以下で有意差が認められる。以上の結果より、ハプロタイプ１は明らかに疾患抵抗性の指標となる事を示している。逆に、ハプロタイプ１以外のハプロタイプはＤＭ進行における有リスクの指標であると考えられる。実際、ハプロタイプ３はオッズ比が１以上であり有意差を示している。
〔実施例７〕 各個人のハプロタイプコンビネーション（ディプロタイプ形質）の決定
各個人のディプロタイプ形質の決定は、ＬＤサポートを用いて行った。予測の８０％以上の事後確率が認められたものを、ディプロタイプ構築に用いた。ディプロタイプと疾患の表現型との関連評価には、マルチカラム表を用いたカイ二乗検定を行った。
観察された９ディプロタイプは表９７のように２×９分割表に示される。表９７にネフリン遺伝子のハプロタイプのディプロタイプ分析の結果を示す。

データは数値（％）で示す。２ｘ２フィッシャーの直接確率検定（Ｆｉｓｈｅｒ）を示す。Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。
ｃｌｕｍｐのＴ１ ｔｅｓｔによるｅｍｐｉｒｉｃａｌ ｐ値は０．０１１０であり、ｃｌｕｍｐのＴ３ ｔｅｓｔによる結果ではディプロタイプ１／１が最も高いカイ二乗値と最も低いｅｍｐｉｒｉｃａｌ ｐ値０．０００９（１０，０００ｐｅｒｍｕｔａｔｉｏｎ）を示し３た。フィッシャーの直接確率検定においてもディプロタイプ１／１がオッズ比１以下であり、有意差を示した。したがって、有リスクアリルを持たないハプロタイプブロックのみを持つことは、ＤＭの進行を抑止する指標となると考えられる。逆に、ディプロタイプ１／１以外のものは１／１のものよりも２．８７倍（９５％信頼区間１．６１−５．１１）ＤＭを発症しやすいといえる。
〔実施例８〕 ＤＭ群とＮＧＴ群で異なる形質の検討
関連解析の性質として、上記の結果は、ＳＮＰｓはＤＭ群とＮＧＴ群で異なる何らかの形質に関連すると解釈できる。これらの形質は、ＤＭそのものの本来の形質ではなく、むしろＤＭに関連した形質と考えられる。したがって、本発明者らは、ＤＭ群とＮＧＴ群で異なる形質を検討するため、以下の形質を解析した。
身長、体重、７５ｇ経口糖負荷試験、ＨｂＡ１ｃ、ウエスト周囲径、ヒップ周囲径、ウエスト／ヒップ比、ボディマスインデックス（ＢＭＩ）、体脂肪率、収縮期血圧、拡張期血圧、血清総コレステロール（ＣＨＯ）、中性脂肪（ＴＧ）、高比重リポタンパク（ＨＤＬ）コレステロール。尿タンパクは試験紙（ライフスティックス、バイエルメディカル社、東京）による半定量測定を行った。
ＤＭで見られる形質の値の違いによって、統計学的有意差が認められた。フィッシャーの直接確率検定に対するｐ値、ＯＲ、および９５％信頼区間は、２Ｘ２プログラム（ｈｔｔｐ：／／ｌｉｎｋａｇｅ．ｒｏｃｋｅｆｅｌｌｅｒ．ｅｄｕ／ｏｔｔ／ｌｉｎｋｕｔｉｌ．ｈｔｍ）により算出した。表入力の１つにゼロがある場合、″Ｓｈｅｅｈｅ″ＯＲ（Ｓｈｅｅｈｅ ＰＲ著、「Ｃｏｍｂｉｎａｔｉｏｎ ｏｆ ｌｏｇ ｒｅｌａｔｉｖｅ ｒｉｓｋ ｉｎ ｒｅｔｒｏｓｐｅｃｔｉｖｅ ｓｔｕｄｉｅｓ ｏｆ ｄｉｓｅａｓｅ．」、Ａｍ Ｊ Ｐｕｂｉｃ Ｈｅａｌｔｈ．、５６：１７４５−１７５０，１９６６年）が算出される。ＤＭ、ＩＧＴ、およびＮＧＴ群間の形質値の有意差は、分散分析によって評価した。多重比較分析（ｐｏｓｔ ｈｏｃ ａｎａｌｙｓｉｓ）のためにＳｃｈｅｆｆｅ’ｓ Ｆ検定を行った。
予測した通り、体重、ウエスト周囲径、ヒップ周囲径、ウエスト周囲径／ヒップ周囲径、ＢＭＩはＤＭ群でより高く、血清トリグリセライド（ＴＧ）値もまたＤＭ群でＮＧＴ群より高かった（１５３．９±１７９．３ｖｓ．１１０．６±９５．６，ｐ＝０．００５０）。しかし、血清コレステロール値、ＨＤＬコレステロール値には有意な差は認められなかった。また、収縮期および拡張期血圧でも両群に差は認められなかった。ネフリンの機能障害はタンパク尿を起こすことが知られていることから、両群の排泄尿中に１５ｍｇ／ｄｌ以上のタンパクを有する人のタンパク尿の頻度を比べてみたが、有意差は認められなかった（ｐ＞０．９９９９）。EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[Example 1] Identification of type 2 diabetes (DM) -related gene
A related analysis applying the candidate gene approach to identify DM susceptibility genes was performed. 2039 SNPs examined for association with DM were selected from 704 candidate genes. Genotyping was performed by the Taqman method. A test group consisting of 148 cases and 227 normal glucose tolerance (NGT) controls was recruited from the Funagata Diabetes Test (Oizumi, T. et al. Diabetes Care 24, 1579-1583 (2001)). The case (DM) group consisted of 74 patients with impaired glucose tolerance (IGT) and 74 DM patients as judged by the criteria of WHO 1985. Age and sex ratios were matched between the case group and the normal glucose tolerance (NGT) control group (age, 65.3 ± 10.2 vs. 65.1 ± 7.7 years; sex ratio (female / male), 80/68 vs. 128/99). The HbA1c level, which is a criterion for estimating chronic blood glucose levels, was naturally higher in the DM group than in the NGT group (5.88 ± 1.18 vs 4.98 ± 0.32, p <0.0001). Furthermore, clinical characteristics such as body mass index (BMI) and serum glyceride levels were higher in the DM group than in the NGT group (BMI (kg / m ² ), 24.7 ± 3.5 vs 23.3 ± 3.24, p <0.0001; triglycerides (mg / dl), 144.0 ± 152.8 vs 115.2 ± 120.7, p = 0 .047) (Table 34).

Numerical values are mean ± SD. It is. Statistical significance was determined by Student's t test. P <0.05 is indicated in the table with an asterisk *.
In association analysis, the genotypes of the case group and the normal glucose tolerance (NGT) control group were tested by chi-square test, and p <0.01 was regarded as statistically significant. Considering the high incidence of DM population (10.5% and 14.5% for DM and IGT, respectively) (Sekikawa, A. et al. J Diabetes Complication 14, 78-83 (2000)) The power is greater than 80% at a 5% significance level to detect DM susceptibility genes with a relative risk of 2 and an allele frequency of 0.1. Tables 35-37 show genes including DM-related SNPs, JSNPIDs of SNPs showing significant differences, risk alleles of SNPs, p-values, odds ratios (OR) and 95% confidence intervals. In a general sense, the inheritance of a risk allele can be recognized from the test results of this example according to either dominant or recessive. Dominant alleles tend to be statistically significant for both their homozygous and heterozygous states (Table 36), but recessive alleles only in their homozygous form There is a tendency to be statistically significant (Table 37).

[Dominant type]

Details of the SNPs are disclosed on the website (http://snp.ims.u-tokyo.ac.jp) with an ID number described as “JSNP ID”. The P value and 95% CI were determined by chi-square test. † represents a gene having multiple SNPs with p <0.01.
[Recessive type]

Details of the SNPs are disclosed on the website (http://snp.ims.u-tokyo.ac.jp) with an ID number described as “JSNP ID”. The P value and 95% CI were determined by chi-square test. * Represents that the OR could not be determined because the number of risky genotypes in the target group was 0, and † represents a gene having multiple SNPs with p <0.01.
In addition, the genetic distance (Fst based on haplotype) between the case and the control group was measured, but no substructure in the population was detected. Interestingly, the putative DM-related gene, the MET gene (Table 36), was 10 Mb away from the genetically unlinked gene (GRM8) that has no evidence of association with DM. By measuring the genetic distance, MET showed a statistically significant difference between the case group and the control group (Fst = 0.625, p = 0.165), whereas GRM8 had 2 groups Was indistinguishable (Fst = 0.00027, p = 0.0.9873).
The genes in these two tables were classified according to p-value. Overall, the top five significant genes are MET, NPHS1, TFRC, MMP19 and SLC1A, with p-values of 0.0008, 0.0011, 0.0022, 0.0022, and 0.0024, respectively. Met. Genes containing two or more SNPs in statistically significant tests were DLG5, FRAP1, LARGE and MET. Furthermore, the genes present in the susceptibility locus for DM or related diseases reported so far are THBS3, RGS5, BLZF1, TFRC and LARGE (Vionnet, N. et al. Am. J. Hum. Genet. 67, 1470). -1480 (2000), Lindgren, CM et al. Am. J. Hum. Genet.70, 509-516 (2002), Lander, ES & Kruglyak, L. Nature Genet. 247 (1995), Wiltshire, S. et al. Am.J.Hum.Genet.69,553-569 (2001), Ghosh, S. et al.Am.J.Hum.Genet.67, 1174-1185 ( 2000), Watanabe, R.A. .et al.Am.J.Hum.Genet.67,1186-1200 (2000), Pratley, was R.E.et al.J.Clin.Invest.101,1757-1764 (1998)).
Only three genes were known to be involved in DM (ABCC8 (SUR), LEP, and TNF) (Elbein, SC et al. Diabetes Care 24, 472-478 (2001)). Ozata, M. et al. J Clin. Endocrinol. Metab. 86, 3659-3664 (2001), Day, CP, et al. Diabetologia 41, 430-434 (1998)). The majority of genes found in the present invention are related to functions or pathways other than glucose metabolism. Those genes that are indirectly involved in DM can be assigned to the following regions: lipid metabolism (ABCA1, FABP2, LDLC, and VLDLR), immune system (FRAP1 and P2RX7), nervous system (CTSD and SLC1A1), homeostasis (SERPINA3 and SERPINB2), maintenance of cell structure (DLG5 and LARGE), tissue remodeling (MMP19), etc.
[Example 2] Analysis of linkage disequilibrium of polymorphic sites related to type 2 diabetes
(A) SNPs around SNPs showing significant differences were typed, and it was examined whether linkage disequilibrium was established between these SNPs. The analysis results when linkage disequilibrium is recognized between the SNPs are shown in Tables 38 to 51.
(A-1) MET

(A-2) NPHS1

(A-3) SERPINB2

(A-4) ABCA1

(A-5) CTSD

(A-6) STE

(A-7) ABCC8

(A-8) MYL2

(A-9) DLG5

(A-10) LDLC

(A-11) LARGE

(A-12) BLZF1

(A-13) FRAP1

(A-14) LEP

(B) When linkage disequilibrium was observed between SNPs, a disease-related analysis using a haplotype was performed. In the individual haplotypes found, the case group and the normal glucose tolerance (NGT) control group were analyzed using the chi-square test, and p <0.05 was considered significant. The results are shown in Table 52.

Furthermore, as a test method having a meaning different from the chi-square test, a method for testing the difference between the distribution of the case group and the normal glucose tolerance (NGT) control group (FASTEHPLUS) (Zhao JH, Sham PC. Hum Hered 2002; 53 (1): 36-41), statistical analysis was performed and p <0.05 was considered significant. FASTEHPLUS is a method of comparing the whole, and may give different results when, for example, multiple alleles have disease sensitivity / resistance. When FASTEHPLUS was applied to individual haplotypes and SNPs contained in the haplotype region, the following haplotypes and SNPs that were not significantly different in the chi-square test were significantly changed.
The four SNPs newly recognized as significant by FASTEHPLUS are shown below.
(1) IMS-JST035656 (NPHS1) p value = 0.0293, (2) IMS-JST0172222 (CTSD) p value = 0.0282, (3) IMS-JST0755563 (STE) p value = 0.119, (4 ) IMS-JST020607 (LARGE) p value = 0.0222
The five haplotypes newly recognized as significant by FASTEHPLUS are shown below.
(1) MET. haplo2 p value = 0.0255, (2) CTSD. haplo1 p value = 0.0401, (3) STE. . haplo1 p value = 0.115, (4) ABCC8. haplo1 p value = 0.0076, (5) MYL2. haplo1 p value = 0.0076
(C) Examples of observation of each haplotype are shown in Tables 53 to 66 below.
(C-1) MET

(C-2) NPHS1

(C-3) SERPINB2

(C-4) ABCA1

(C-5) CTSD

(C-6) STE

(C-7) ABCC8

(C-8) MYL2

(C-9) DLG5

(C-10) LDLC

(C-11) LARGE

(C-12) BLZF1

(C-13) FRAP1

(C-14) LEP

Table 67 shows the results of haplotype analysis of MET.

Table 68 shows the result of diplotype analysis of MET.

Table 69 shows MET snp analysis p-value.

Table 70 shows significant haplotypes of MET.

Table 71 shows the results of NPHS1 haplotype analysis.

Table 72 shows the results of the NPHS1 diplotype analysis.

Table 73 shows the NPHS1 snp analysis p-value.

Table 74 shows significant haplotypes of NPHS1.

Table 75 shows the results of haplotype analysis of SERPINB2.

Table 76 shows the results of diplotype analysis of SERPINB2.

Table 77 shows the snp analysis p-value of SERPINB2.

Table 78 shows the significant haplotypes of SERPINB2.

Table 79 shows the results of STE haplotype analysis.

Table 80 shows the result of STE diplotype analysis.

Table 81 shows the STE snp analysis p-value.

Table 82 shows significant haplotypes of STE.

Table 83 shows the results of DLG5 haplotype analysis.

Table 84 shows the results of DLG5 diplotype analysis.

Table 85 shows the snp analysis p-value of DLG5.

Table 86 shows significant haplotypes of DLG5.

Table 87 shows the results of LDLC haplotype analysis.

Table 88 shows the results of LDLC diplotype analysis.

Table 89 shows the LDLC snp analysis p-value.

Table 90 shows the significant haplotypes of LDLC.

Table 91 shows the results of BLZF1 haplotype analysis.

Table 92 shows the results of BLZF1 diplotype analysis.

Table 93 shows the snp analysis p-value of BLZF1.

Table 94 shows significant haplotypes of BLZF1.

[Example 3] Analysis target
We speculated that nephrin plays an important role in pancreatic beta cells and that its dysfunction leads to DM progression.
Finnish congenital nephrotic syndrome (NPHS1) is a rare autosomal recessive genetic disease that causes nephrotic syndrome soon after birth (Rapola J, “Congenital nephrotic syndrome”, Pediatr Nephrol, 1: 441-446, 1987). ). The gene responsible for Finnish congenital nephrotic syndrome has been positionally cloned, and its product has been named nephrin (Kestila M, et al. nephrotic syndrome. "Mol Cell. 1: 575-582, 1998).
The nephrin gene (NPHS1) is 26 kb, has 29 exons, and an mRNA consisting of 3726 bases encoding a protein has been obtained (Kestila M et al., “Positionally cloned gene for a novel globular protein-nephrin-is-neutrine-nephrinis-nephrinis-nephrin-is in congenital nephrotic syn .., Mol Cell. 1: 575-582, 1998, Lenkeri et al., and H. enet, 64:. 51-61, 1999 years). Nephrin consists of an extracellular part containing eight immunoglobulin-like domains, one type 3 fibronectin domain-like module, a single transmembrane domain and one cytoplasmic C-terminal domain, and is presumed to be a transmembrane protein of the immunoglobulin superfamily. (Kestila M et al., “Positionally cloned gene for a novel global protein-nephrin-is mutated in congenital nephrotic syndrome, et al., 1998, ul et al. congruental nephrotic syndrome of the Finnish type (N . HS1) and characterization of mutation ", Am J Hum Genet.64: 51-61,1999).. Therefore, the function of nephrin is considered to be similar to other members of the immunoglobulin superfamily, and various functions such as cell adhesion, signaling, and cell migration are known as functions of other members (Brummendorf T , Rathjen FD, “The immunoglobine superfamily.”, In Protein Profile, vol 1, Sheterline P, Hymphries M, Latchman D, Downes P, ed., London, Acade.
Since NPHS1 has mutations in nephrin, the role of nephrin in maintaining renal function is considered critical and extensive research has been conducted. Nephrin has been reported to be localized in the slit membrane region of podocytes (Holthofer H et al., “Nephrin localizations at the polynucleotide filtration area and the charactaristically in the United Kingdom.” 1681-1687, 1999, Holzman LB et al., “Nephrin localize to the slit pore of the globular epithelial cell.”, I ed., Et al., Cit. t the slit diaphragm of global podocytes. ", Proc natl Acad Sci USA., 96: 7962-7967, 1999), and has an important role in the barrier function of renal glomerular filtration. (Kestila M et al., “Positionally cloned gene for a novel global protein-nephrin-is mutated in congenital nephrotic syndrome., 1998, Mol 5-R. Nephrin is specifically located at the slit diaphra m of glomerular podcycles. ", Proc natl Acad Sci USA., 96: 7962-7967, 1999, by Triggvason K.," Unraveling the mechanism of glomer ultrane lt: nephrol., 10: 2440-2445, 1999).
Nephrin has been reported to be specifically expressed in renal glomerular podocytes (Kestila M et al., “Positionally cloned gene for a novel global protein-nephrin-is mutated in congenital nephrotic. 575-582, 1998.). However, it has recently been reported that nephrin genes and proteins are expressed in human pancreatic beta cells (Palmen T et al., “Nephrin is expressed in the pancreatic beta cells.”, Diabetologia 44: 1274-1280, 2001. ). This indicates that nephrin has some function in maintaining pancreatic beta cells. In the pancreas, as in the kidney, signaling effects are predicted, as suggested by eight tyrosine in the cell interaction and / or cytoplasmic C-terminal domain (Kestilla M et al., “Positionally cloned gene for a novel”. (glomerular protein-nephrin-is mutated in congenital nephrotic syndrome. "Mol Cell. 1: 575-582, 1998.). However, there are no reports revealing the role of nephrin in pancreatic beta cells.
Since DM is a common disease, it was speculated that several polymorphisms of nephrin genes are risk factors for DM progression. Therefore, in the present invention, the present inventors examined whether a nephrin gene SNP and type 2 diabetes (DM) are related or not in a case-control study based on a Japanese region.
72 DM, 75 impaired glucose tolerance (IGT), and 227 normal glucose tolerance (NGT) groups were first analyzed as group 1. Next, 30 DMs, 76 IGTs, and 244 NGT groups were added to the first group and analyzed as the second group. These samples were analyzed by Funagata (Oizumi T et al., “Genotype Arg / Arg, but not Trp / arg, of the Trp64Arg polymorphism of the β ₃ -Adrenergic receptor is associated with type 2 diabetes and obesity in a large Japan sample. Diabetes Care. 24: 1579-1583, 2001). The Funagata study is a population-based study conducted in an agricultural area about 400 km north of Tokyo. This study was conducted with the approval of Yamagata University's Ethics Review Board and written informed consent from the subjects. All processes were performed according to the guidelines. In the area studied, there has been almost no movement for decades. Thus, our study is a suitable setting for studying genetic backgrounds in the sense that population stratification minimizes the risk of producing false positives.
Each group of DM, IGT, and NGT was analyzed by matching the age and sex (age 65.8 ± 9.3 vs. 65.1 ± 9.8 vs. 64.8 ± 8.8, sex ratio (female / male ), 56/46 vs. 77/74 vs. 263/208). The following traits were also analyzed along with the genotype.
Height, weight, 75 g oral glucose tolerance test, HbA1c, waist circumference, hip circumference, waist / hip ratio, body mass index (BMI), body fat percentage, systolic blood pressure, diastolic blood pressure, serum total cholesterol (CHO), Neutral fat (TG), high density lipoprotein (HDL) cholesterol. The urine protein was semi-quantitatively measured with a test paper (Lifesticks, Bayer Medical, Tokyo).
[Example 4] Genotype and genotype analysis
SNPs in the nephrin gene (C to T in exon 3 at nucleotide position 294 from translation initiation codon ATG (JSNP database ID: IMS-JST035656); C in intron 5 upstream of nucleotide position 61 from the intron 5 / exon 6 splicing site To G (−61C / G) (IMS-JST021061); C to T (2289T) (IMS-JST000541) at nucleotide position 2289 in exon 17 was analyzed CNPs of C294T and C2289T are silent polymorphisms Genomic DNA was extracted from peripheral blood leukocytes, and genotyping was performed by fluorogenic PCR as described previously (Ranade K et al., “High-through”. ghput genotyping with single nucleotide polymorphism. ", Genome research., 11: 1262-1268, 2001). TaqMan primer sets used for amplification are as follows.
SNP C294T has 5′-CCCCTGCAGGGTGAATTCCA-3 ′ (SEQ ID NO: 31) and 5′-GCACTCATACTCCGCGTCATC-3 ′ (SEQ ID NO: 32), and SNP-61C / G has 5′-CCTGGATCCCAGAGGAGATCA-3 ′ (SEQ ID NO: 33) and 5′-CAGGGTTATAGAGTCAGAGTCCATCATCT-3 ′ (SEQ ID NO: 34), 5′-GACCCCACTGAGGGTGAACGT-3 ′ (SEQ ID NO: 35) and 5′-GAACATGCCCGGGAGGAT-3 ′ (SEQ ID NO: 36) for SNP C2289T.
TaqMan probes labeled with FAM or VIC reporter molecules are as follows. SNP C294T has 5'-TGCACAT T GAGGCCCT-3 ′ (SEQ ID NO: 37) and 5′-CACAT C GAGGCCTG-3 ′ (SEQ ID NO: 38), 5′-TCAGGG for SNP-61C / G C AGAAGAGGG-3 ′ (SEQ ID NO: 39) and 5′-TCAGGG G AGAAGAGGT-3 ′ (SEQ ID NO: 40), 5′-TGGGCATC for SNP C2289T A ACAGTGC-3 ′ (SEQ ID NO: 41) and 5′-ATTGGCATC G ACAGTG-3 ′ (SEQ ID NO: 42).
The polymorphic base of the query was designed in the middle and underlined parts of the probe. The increase in fluorescence was measured using an ABI 7900 HT sequence detection system.
[Example 5] Relationship between genotypes of SNPs and DM
The present inventors analyzed the association between the genotypes of three SNPs (C294T, C650G, C2289T) and DM using the first sample group (n = 374). Association analysis was performed by Fisher's exact probability test by comparing the control with the case assuming the SNP genotype frequency and dominant or recessive inheritance format. As shown in Table 95, all SNPs investigated showed a significant association with DM in the dominant mode of inheritance. Table 95 shows the relationship between SNPs and DM in the nephrin gene.

Data are shown as numerical values (%). The frequency of risk genotypes in the IGT group and DM group was compared with that in the NGT group. P <0.05 and 0.01 are indicated by * and **, respectively.
That is, people with TT and CT genotypes at C294T, people with GG and CG genotypes at -61C / G, people with TT and CT genotypes at C2289T than people with other genotypes Diabetes was strong. Therefore, it can be said that these genotypes are risky genotypes in the progression of DM. Conversely, more people with risk genotype were found in the DM group than in the healthy group.
[Example 6] Haplotype analysis
The inventors further performed haplotype analysis using 3SNPs.
Linkage disequilibrium between each SNP is expressed by LD support (Kitamura Y et al., “Determination of probabilistic distribution. -93, 2002). SNPs showing strong linkage disequilibrium between each SNP were haplotype blocks. The haplotype frequency was determined using LD support.
To evaluate the association between haplotypes and disease phenotypes, a chi-square test using a multi-column table was performed. Chi-square tests in multicolumn contingency tables usually do not show a normal distribution of asymptotic equilibria.
The present inventors performed analysis by applying permutation by Monte Carlo Markov-Chain (MCMC method) using a program called clump.
The Clamp T1 test of this program generates a contingency table in which the number of cells with fixed edges in columns and columns is random. A table simulated 10,000 times yields a null distribution of the chi-square statistic, which is used to determine the empirical p-value corresponding to the chi-square value in the actual data set. used.
Clamp T3 is a program for obtaining the maximum value of chi-square values in all existing 2 × 2 contingency tables. That is, a 2 × 2 contingency table is created with the sum of a certain column and all other columns. Then, a table showing the maximum chi-square value was selected, and the initial p value was calculated by the MCMC method as described above. The MCMC method is advantageous in terms of substantial power over the conventional 2 × 2 contingency table test with Bonferroni correction.
As a result, one haplotype block consisting of 6 haplotypes was found. Table 96 shows the frequency of SNPs haplotypes in the nephrin gene and the results of analysis.

Data are shown as numerical values (%). 2x2 Fisher's exact test (Fisher) is shown. P <0.05 and 0.01 are indicated by * and **, respectively.
The initial p value by T1 test of the clump analysis of the 2 × 6 contingency table was 0.0034. As a result of T3 test of clump, haplotype 1 showed an initial p value of 0.0022 (10000 per mutations). Furthermore, in Fisher's exact test using a 2 × 2 contingency table, haplotype 1 (p = 0.0008) showed a significant association with an odds ratio (OR) of 1 or less. Even after Bonferroni's correction, the p-value was as low as 0.0047. Haplotype 1 is composed of SNPs that are non-risk alleles, and a significant difference is observed at an odds ratio of 1 or less. The above results indicate that haplotype 1 is clearly an indicator of disease resistance. Conversely, haplotypes other than haplotype 1 are considered to be indicators of risk in DM progression. In fact, haplotype 3 has a significant difference with an odds ratio of 1 or more.
[Example 7] Determination of each individual's haplotype combination (diplotype trait)
Each individual's diplotype trait was determined using LD support. Those with a posterior probability of 80% or more of the prediction were used for diplotype construction. A chi-square test using a multi-column table was performed to evaluate the association between the diplotype and the disease phenotype.
The 9 diplotypes observed are shown in a 2 × 9 contingency table as shown in Table 97. Table 97 shows the results of diplotype analysis of the haplotype of the nephrin gene.

Data are shown as numerical values (%). 2x2 Fisher's exact test (Fisher) is shown. P <0.05 and 0.01 are indicated by * and **, respectively.
The initial p value of the clump T1 test is 0.0110, and the result of the clump T3 test shows that the diplotype 1/1 has the highest chi-square value and the lowest initial p value of 0.0009 (10,000 permutation). It was. In Fisher's exact test, the diplotype 1/1 had an odds ratio of 1 or less, indicating a significant difference. Therefore, having only a haplotype block that does not have a risky allele is considered to be an index for inhibiting the progression of DM. Conversely, it can be said that those other than diplotype 1/1 are more likely to develop 2.87 times (95% confidence interval 1.61-5.11) DM than those of 1/1.
[Example 8] Examination of different traits in DM group and NGT group
As a nature of the association analysis, the above results can be interpreted that SNPs are associated with some trait that is different in DM and NGT groups. These traits are not the original traits of DM itself, but rather are traits related to DM. Therefore, the present inventors analyzed the following traits in order to examine different traits in the DM group and the NGT group.
Height, weight, 75 g oral glucose tolerance test, HbA1c, waist circumference, hip circumference, waist / hip ratio, body mass index (BMI), body fat percentage, systolic blood pressure, diastolic blood pressure, serum total cholesterol (CHO), Neutral fat (TG), high density lipoprotein (HDL) cholesterol. The urine protein was semi-quantitatively measured with a test paper (Lifesticks, Bayer Medical, Tokyo).
A statistically significant difference was observed depending on the difference in trait values seen in DM. The p-value, OR, and 95% confidence interval for the Fisher's exact test were calculated by the 2X2 program (http://linkage.rockefeller.edu/ott/linkutil.html). If there is a zero in one of the table entries, “Shehe” OR (by Sheehe PR, “Combination of log relative risk in retrospective studies of disease.”, Am J Public Health., 56: 1617). Calculated. Significant differences in trait values between DM, IGT, and NGT groups were assessed by analysis of variance. A Scheffe's F test was performed for post hoc analysis.
As expected, body weight, waist circumference, hip circumference, waist circumference / hip circumference, BMI were higher in the DM group and serum triglyceride (TG) values were also higher in the DM group than in the NGT group (153.9). ± 179.3 vs. 110.6 ± 95.6, p = 0.050). However, there was no significant difference between serum cholesterol level and HDL cholesterol level. There were no differences between the two groups in systolic and diastolic blood pressure. Since nephrin dysfunction is known to cause proteinuria, we compared the frequency of proteinuria among people with 15 mg / dl protein or more in the excreted urine of both groups. None (p> 0.9999).

データは数値（％）である。ＩＧＴおよびＤＭ群における有リスク遺伝子型の頻度を、それぞれＮＧＴ群と比較した。Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。ＯＲは血清トリグリセリドレベル（ＴＧ）およびボディマスインデックス（ＢＭＩ）で調整した。
以上のように、遺伝子におけるＳＮＰｓとＤＭとの関連が、地域を基盤とした日本人のケースコントロール試験において認められた。
本発明者らは、遺伝子におけるＳＮＰｓがＤＭ発症リスクを評価する有用な指標であることを明らかにし、本発明の結果はさらに、ネフリンが基礎的な機能において重要な役割を果たし、ネフリンの機能障害がＤＭの発症に関連することを示している。
〔実施例１０〕 解析対象
本発明者らは、末梢細胞からＨＤＬへの脂質の輸送を調節するもとのして重要なＡＴＰ−ｂｉｎｄｉｎｇ ｃａｓｓｅｔｔｅ ｔｒａｎｓｏｒｔｏｒ１（ＡＢＣＡ１）が、ＤＭの進行に関与すると推測した。
ＡＢＣＡ１遺伝子の欠損は、Ｔａｎｇｉｅｒ病や家族性低アルファリポタンパク血症（ＦＨＡ）のような遺伝性のＨＤＬ欠損症を起こす（Ｓｃｈｍｉｔｚ Ｇ，Ｂｕｅｃｈｌｅｒ Ｃ：ＡＢＣＡ１：ｒｅｇｕｌａｔｉｏｎ，ｔｒａｆｆｉｃｋｉｎｇ ａｎｄ ａｓｓｏｃｉａｔｉｏｎ ｗｉｔｈ ｈｅｔｅｒｏｍｅｒｉｃ ｐｒｏｔｅｉｎｓ．Ａｎｎ Ｍｅｄ ３４：３３４−３４７，２００２、Ｏｒａｍ ＪＦ，Ｌａｗｎ ＲＭ：ＡＢＣＡ１：ｔｈｅ ｇａｔｅｋｅｅｐｅｒ ｆｏｒ ｅｌｉｍｉｎａｔｉｎｇ ｅｘｃｅｓｓ ｔｉｓｕｕｅ ｃｈｏｌｅｓｔｅｒｏｌ．Ｊ Ｌｉｐｉｄ Ｒｅｓ ４２：１１７３−１１７９，２００１、Ｓｒｉｖａｓｔａｖａ Ｎ：ＡＴＰ ｂｉｎｄｉｎｇ ｃａｓｓｅｔｔｅ ｔｒａｎｓｐｏｒｔｅｒ Ａ１−ｋｅｙ ｒｏｌｅｓ ｉｎ ｃｅｌｌｕｌａｒ ｌｉｐｉｄ ｔｒａｎｓｐｏｒｔ ａｎｄ ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ．Ｍｏｌ Ｃｅｌｌ Ｂｉｏｃｈｅｍ ２３７：１５５−１６４，２００２、Ｓａｎｔａｍａｒｉｎａ−Ｆｏｊｏ Ｓ，Ｒｅｍａｌｅｙ ＡＴ，Ｎｅｕｆｅｌｄ ＥＢ，Ｂｒｅｗｅｒ ＨＢ：Ｒｅｇｕｌａｔｉｏｎ ａｎｄ ｉｎｔｒａｃｅｌｌｕｌａｒ ｔｒａｆｆｉｃｋｉｎｇ ｏｆ ｔｈｅ ＡＢＣＡ１ ｔｒａｎｓｐｏｒｔｅｒ．Ｊ Ｌｉｐｉｄ Ｒｅｓ ４２：１３３９−１３４５，２００１、Ｂｒｏｏｋｓ−Ｗｉｌｓｏｎ Ａ，Ｍａｒｃｉｌ Ｍ，Ｃｌｅｅ ＳＭ，Ｚｈａｎｇ ＬＨ，Ｒｏｏｍｐ Ｋ，ｖａｎ Ｄａｍ Ｍ，Ｙｕ Ｌ，Ｂｒｅｗｅｒ Ｃ，Ｃｏｌｌｉｎｓ ＪＡ，Ｍｏｌｈｕｉｚｅｎ ＨＯＦ，Ｌｏｕｂｓｅｒ Ｏ，Ｏｕｒｌｒｔｔｒ ＢＦＦ，Ｆｉｃｈｔｅｒ Ｋ，Ａｓｈｂｏｕｒｎｅ−Ｅｘｃｏｆｆｏｎ ＫＪＤ，Ｓｅｎｓｅｎ ＣＷ，Ｓｃｈｅｒｅｒ Ｓ，Ｍｏｔｔ Ｓ，Ｄｅｎｉｓ Ｍ，Ｍａｒｔｉｎｄａｌｅ Ｄ，Ｆｒｏｈｌｉｃｈ Ｊ，Ｍｏｒｇａｎ Ｋ，Ｋｏｏｐ Ｂ，Ｐｉｍｓｔｏｎｅ Ｓ，Ｋａｓｔｅｌｅｉｎ ＪＪＰ，Ｇｅｎｅｓｔ Ｊ，Ｈａｙｄｅｎ ＭＲ：Ｍｕｔａｔｉｏｍｓ ｉｎ ＡＢＣ１ ｉｎ Ｔａｎｇｉｅｒ ｄｉｓｅａｓｅ ａｎｄ ｆａｍｉｌｉａｌ ｈｉｇｈ−ｄｅｎｓｉｔｙ ｌｉｐｏｐｒｏｔｅｉｎ ｄｅｆｉｃｉｅｎｃｙ．Ｎａｔ Ｇｅｎｅｔ ２２：３３６−３４５，１９９９、Ｂｏｄｚｉｏｃｈ Ｍ，Ｏｒｓｏ Ｅ，Ｋｌｕｃｋｅｎ Ｔ，Ｌａｎｇｍａｎｎ Ｔ，Ｂｏｔｔｃｈｅｒ Ｌ，Ｄｉｅｄｅｒｉｃｈ Ｗ，Ｄｒｏｂｎｉｋ Ｗ，Ｂａｒｌａｇｅ Ｓ，Ｂｕｃｈｌｅｒ Ｃ，Ｐｏｒｓｃｈ−Ｏｚｃｕｒｕｍｅｚ Ｍ，ｋａｍｉｎｓｋｉ ＷＥ，Ｈａｈｍａｎｎ ＨＷ，Ｏｅｔｔｅ Ｋ，Ｒｏｔｈｅ Ｇ，Ａｓｌａｎｉｄｉｓ Ｃ，Ｌａｃｋｎｅｒ ＫＪ，Ｓｃｈｍｉｔｚ Ｇ：Ｔｈｅ ｇｅｎｅ ｅｎｃｏｄｉｎｇ ＡＴＰ−ｂｉｎｄｉｎｇ ｃａｓｓｅｔｔｅ ｔｒａｎｓｐｏｒｔｅｒ １ ｉｓ ｍｕｔａｔｅｄ ｉｎ Ｔａｎｇｉｅｒ ｄｉｓｅａｓｅ．Ｎａｔ Ｇｅｎｅｔ ２２：３４７−３５１，１９９９、Ｒｕｓｔ Ｓ，Ｒｏｓｉｅｒ Ｍ，Ｆｕｎｋｅ Ｈ，Ｒｅａｌ Ｊ，Ａｍｏｕｒａ Ｚ，Ｐｉｅｔｔｅ ＪＣ，Ｄｅｌｅｕｚｅ ＪＦ，Ｂｒｅｗｅｒ ＨＢ，Ｄｕｖｅｒｇｅｒ Ｎ，Ｄｅｎｅｆｌｅ Ｐ，Ａｓｓｍａｎｎ Ｇ：Ｔａｎｇｉｅｒ ｄｉｓｅａｓｅ ｉｓ ｃａｕｓｅｄ ｂｙ ｍｕｔａｔｉｏｎｓ ｉｎ ｔｈｅ ｇｅｎｅ ｅｏｎｃｏｄｉｎｇ ＡＴＰ−ｂｉｎｄｉｎｇ ｃａｓｓｅｔｔｅ ｔｒａｎｓｐｏｒｔｅｒ １．Ｎａｔ Ｇｅｎｅｔ ２２：３５２−３５５，１９９９、Ｍｉｌｌｅｒ Ｍ，Ｒｈｙｎｅ Ｊ，Ｈａｍｌｅｔｔｅ Ｓ，Ｂｉｒｎｂａｕｍ Ｊ，Ｒｏｄｒｉｇｕｅｚ Ａ：Ｇｅｎｅｔｉｃｓ ｏｆ ＨＤＬ ｒｅｇｕｌａｔｉｏｎ ｉｎ ｈｕｍａｎｓ．Ｃｕｒｒ Ｏｐｉｎ Ｌｉｐｉｄｏｌ １４：２７３−２７９，２００３、Ｓｉｎｇａｒａｊａ ＲＲ，Ｂｒｕｎｈａｍ ＬＲ，Ｖｉｓｓｃｈｅｒ Ｈ，Ｋａｓｔｅｌｅｉｎ ＪＪＰ，Ｈａｙｄｅｎ ＭＲ：Ｅｆｆｌｕｘ ａｎｄ ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ−ｔｈｅ ｃｌｉｎｉｃａｌ ａｎｄ ｂｉｏｃｈｅｍｉｃａｌ ｉｍｐａｃｔ ｏｆ ｖａｒｉａｔｉｏｎｓ ｉｎ ｔｈｅ ＡＢＣＡ１ ｇｅｎｅ．Ａｒｔｅｒｉｓｃｌｅｒ Ｔｈｒｏｍｂ Ｖａｓｃ Ｂｉｏｌ ２００：１３２２−１３３２，２００３）。ＡＢＣＡ１遺伝子の一般的な多型として、血清脂質値、特にＨＤＬコレステロール値、冠動脈疾患（ＣＡＤ）と関連するものは数多く報告されているが（Ｍｉｌｌｅｒ Ｍ，Ｒｈｙｎｅ Ｊ，Ｈａｍｌｅｔｔｅ Ｓ，Ｂｉｒｎｂａｕｍ Ｊ，Ｒｏｄｒｉｇｕｅｚ Ａ：Ｇｅｎｅｔｉｃｓ ｏｆ ＨＤＬ ｒｅｇｕｌａｔｉｏｎ ｉｎ ｈｕｍａｎｓ．Ｃｕｒｒ Ｏｐｉｎ Ｌｉｐｉｄｏｌ １４：２７３−２７９，２００３、Ｓｉｎｇａｒａｊａ ＲＲ，Ｂｒｕｎｈａｍ ＬＲ，Ｖｉｓｓｃｈｅｒ Ｈ，Ｋａｓｔｅｌｅｉｎ ＪＪＰ，Ｈａｙｄｅｎ ＭＲ：Ｅｆｆｌｕｘ ａｎｄ ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ−ｔｈｅ ｃｌｉｎｉｃａｌ ａｎｄ ｂｉｏｃｈｅｍｉｃａｌ ｉｍｐａｃｔ ｏｆ ｖａｒｉａｔｉｏｎｓ ｉｎ ｔｈｅ ＡＢＣＡ１ ｇｅｎｅ．Ａｒｔｅｒｉｓｃｌｅｒ Ｔｈｒｏｍｂ Ｖａｓｃ Ｂｉｏｌ ２００：１３２２−１３３２，２００３、Ｃｌｅｅ ＳＭ，Ｚｗｉｎｄｅｒｍａｎ ＡＨ，Ｅｎｇｅｒｔ ＪＣ，Ｚｗａｒｔｓ ＫＹ，Ｍｏｌｈｕｉｚｅｎ ＨＯＦ，Ｒｏｏｍｐ Ｋ，Ｊｕｋｅｍａ ＪＷ，ｖａｎ Ｗｉｊｌａｎｄ Ｍ，ｖａｎ Ｄａｍ Ｍ，Ｈｕｄｓｏｎ ＴＪ，Ｂｒｏｏｋｓ−Ｗｉｌｓｏｎ Ａ，Ｇｅｎｅｓｔ Ｊ Ｊｒ，Ｋａｓｔｅｌｅｉｎ ＪＪＰ，Ｈａｙｄｅｎ ＭＲ：Ｃｏｍｍｏｎ ｇｅｎｅｔｉｃ ｖａｒｉａｔｉｏｎ ｉｎ ＡＢＣＡ１ ｉｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ ａｌｔｅｒｅｄ ｌｉｐｏｐｒｏｔｅｉｎ ｌｅｖｅｌｓ ａｎｄ ａ ｍｉｄｉｆｉｅｄ ｒｉｓｋ ｆｏｒ ｃｏｅｏｎａｒｙ ａｒｔｅｒｙ ｄｉｓｅａｓｅ．Ｃｉｒｃｕｌａｔｉｏｎ １０３：１１９８−１２０５，２００１、Ｌｕｔｕｃｕｌｔａ Ｓ，Ｂａｌｌａｎｔｙｎｅ ＣＭ，Ｅｌｇｈａｎｎａｍ Ｈ，Ｇｏｔｔｏ ＡＭ Ｊｒ，Ｍａｒｉａｎ ＡＪ：Ｎｏｖｅｌ ｐｏｌｙｍｏｒｐｈｉｓｍｓ ｉｎ ｐｒｏｍｏｔｅｒ ｒｅｇｉｏｎ ｏｆ ＡＴＰ ｂｉｎｄｉｎｇ ｃａｓｓｅｔｔｅ ｔｒａｎｓｐｏｒｔｅｒ ｇｅｎｅ ａｎｄ ｐｌａｓｍａ ｌｉｐｉｄｓ，ｓｅｖｅｒｉｔｙ，ｐｒｏｇｒｅｓｓｉｏｎ，ａｎｄ ｒｅｇｒｅｓｓｉｏｎ ｏｆ ｃｏｒｏｎａｒｙ ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ ａｎｄ ｒｅｓｐｏｎｓｅ ｔｏ ｔｈｅｒａｐｙ．Ｃｉｒｃ Ｒｅｓ ８８：９６９−９７３，２００１、Ｂｒｏｕｓｓｅａｕ ＭＥ，Ｂｏｄｚｉｏｃｈ Ｍ，Ｓｃｈａｅｆｅｒ ＥＪ，Ｇｏｌｄｋａｍｐ ＡＬ，Ｋｉｅｌａｒ Ｄ，Ｐｒｏｂｓｔ Ｍ，Ｏｒｄｏｖａｓ ＪＭ，Ａｓｌａｎｉｄｉｓ Ｃ，Ｌａｃｋｎｅｒ ＫＪ，Ｒｕｂｉｎｓ ＨＢ，Ｃｏｌｌｉｎｓ Ｄ，Ｒｏｂｉｎｓ ＳＪ，Ｗｉｌｓｏｎ ＰＷＦ，Ｓｃｈｍｉｔｚ Ｇ：Ｃｏｍｍｏｎ ｖａｒｉａｎｔｓ ｉｎ ｔｈｅ ｇｅｎｅ ｅｎｃｏｄｉｎｇ ＡＴＰ−ｂｉｎｄｉｎｇ ｃａｓｓｅｔｔｅ ｔｒａｎｓｐｏｒｔｅｒ １ ｉｎ ｍｅｎ ｗｉｔｈ ｌｏｗ ＨＤＬ ｃｈｏｌｅｓｔｅｒｏｌ ｌｅｖｅｌｓ ａｎｄ ｃｏｒｏｎａｒｙ ｈｅａｒｔ ｄｉｓｅａｓｅ．Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ １５４：６０７−６１１，２００１、Ｚｗａｒｔｓ ＫＹ，Ｃｌｅｅ ＳＭ，Ｚｗｉｎｄｅｒｍａｎ ＡＨ，Ｅｎｇｅｒｔ ＪＣ，Ｓｉｎｇａｒａｊａ Ｒ，Ｌｏｕｂｓｅｒ Ｏ，Ｊａｍｅｓ Ｅ，Ｒｏｏｍｐ Ｋ，Ｈｕｄｓｏｎ ＴＪ，Ｊｕｋｅｍａ ＪＷ，Ｋａｓｔｅｌｅｉｎ ＪＪＰ，Ｈａｙｄｅｎ ＭＲ：ＡＢＣＡ１ ｒｅｇｕｌａｔｏｒｙ ｖａｒｉａｎｔｓ ｉｎｆｌｕｅｎｃｅ ｃｏｒｏｎａｒｙ ａｒｔｅｒｙ ｄｉｓｅａｓｅ ｉｎｄｅｐｅｎｄｅｎｔ ｏｆ ｅｆｆｅｃｔｓ ｏｎ ｐｌａｓｍａ ｌｉｐｉｄ ｌｅｖｅｌｓ．Ｃｌｉｎ Ｇｅｎｅｔ ６１：１１５−１２５，２００２、Ｈｏｎｇ ＳＨ，Ｒｈｙｎｅ Ｊ，Ｍｉｌｌｅｒ Ｍ：Ｎｏｖｅｌ ｐｏｌｙｐｙｒｉｍｉｄｉｎｅ ｖａｒｉａｔｉｏｎ（ＩＶＳ４６：ｄｅｌ Ｔ−３９．．．−４６）ｉｎ ＡＢＣＡ１ ｃａｕｓｅｓ ｅｘｏｎ ｓｋｉｐｐｉｎｇ ａｎｄ ｃｏｎｔｒｉｂｕｔｅｓ ｔｏ ＨＤＬ ｃｈｏｌｅｓｔｅｒｏｌ ｄｅｆｉｃｉｅｎｃｙ ｉｎ ａ ｆａｍｉｌｙ ｗｉｔｈ ｐｒｅｍａｔｕｒｅ ｃｏｒｏｎａｒｙ ｄｉｓｅａｓｅ．Ｃｉｒｃ Ｒｅｓ ９３：１００６−１０１２，２００３、Ｔａｎ ＪＨ−Ｈ，Ｌｏｗ Ｐ−Ｓ，Ｔａｎ Ｙ−Ｓ，Ｔｏｎｇ Ｍ−Ｃ，Ｓａｈａ Ｎ，Ｙａｎｇ Ｈ，Ｈｅｎｇ Ｃ−Ｋ：ＡＢＣＡ１ ｇｅｎｅ ｐｏｌｙｍｏｒｐｈｉｓｍｓ ａｎｄ ｔｈｅｉｒ ａｓｓｏｃｉａｔｉｏｎｓ ｗｉｔｈ ｃｏｒｏｎａｒｙ ａｒｔｅｒｙ ｄｉｓｅａｓｅ ａｎｄ ｐｌａｓｍａ ｌｉｐｉｄｓ ｉｎ ｍａｌｅｓ ｆｒｏｍ ｔｈｒｅｅ ｅｔｈｎｉｃ ｐｏｐｕｌａｔｉｏｎｓ ｉｎ Ｓｉｎｇａｐｏｒｅ．Ｈｕｍ Ｇｅｎｅｔ １１３：１０６−１１７，２００３）、糖尿病との関連を示すものはない。
血清ＨＤＬコレステロール値の低下は２型糖尿病患者にしばしば認められ、インスリン抵抗性にも関与するといわれている事から、ＡＢＣＡ１遺伝子と２型糖尿病の関連を推測した。本発明においてＡＢＣＡ１遺伝子上のＳＮＰ（ＩＭＳ−ＪＳＴ１０５３２２）がＤＭと関連することを既に示したが（表３６）、発明者らは更なる解析を行なうために、ＡＢＣＡ１遺伝子のＳＮＰと２型糖尿病（ＤＭ）が関連しているかどうかを、日本人の地域を基盤としたハプロタイプ解析を用いたケースコントロール試験において検討した。
７２例のＤＭ、７５例の耐糖能障害（ＩＧＴ）、２２７例の正常耐糖能（ＮＧＴ）群を解析した。これらのサンプルはフナガタ研究（Ｏｉｚｕｍｉ Ｔら著、「Ｇｅｎｏｔｙｐｅ Ａｒｇ／Ａｒｇ，ｂｕｔ ｎｏｔ Ｔｒｐ／ａｒｇ，ｏｆ ｔｈｅ Ｔｒｐ６４Ａｒｇ ｐｏｌｙｍｏｒｐｈｉｓｍ ｏｆ ｔｈｅ β_３−ａｄｒｅｎｅｒｇｉｃ ｒｅｃｅｐｔｏｒ ｉｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ ｔｙｐｅ ２ ｄｉａｂｅｔｅｓ ａｎｄ ｏｂｅｓｉｔｙ ｉｎ ａ ｌａｒｇｅ Ｊａｐａｎｅｓｅ ｓａｍｐｌｅ．」、Ｄｉａｂｅｔｅｓ Ｃａｒｅ．、２４：１５７９−１５８３，２００１年）のコホートより選択された。フナガタ研究は東京より約４００ｋｍ北の農業地帯において行われたポピュレーションベーススタディである。また、本研究は山形大学の倫理審査委員会の承認、および、対象者より文書によるインフォームドコンセントを得た上で実施された。全工程は、ガイドラインに従い実施した。なお、対象集団をＳＴＲＵＣＴＵＲＥというソフトウェア（Ｐｒｉｔｃｈａｒｄ ＪＫ，Ｓｔｅｐｈｅｎｓ Ｍ，Ｄｏｎｎｅｌｌｙ Ｐ：Ｉｎｆｅｒｅｎｃｅ ｏｆ ｐｏｐｕｌａｔｉｏｎ ｓｔｒｕｃｔｕｒｅ ｕｓｉｎｇ ｍｕｌｔｉｌｏｃａｌ ｇｅｎｏｔｙｐｅ ｄａｔａ．Ｇｅｎｅｔｉｃｓ １５５：９４５−９５９，２０００）で分析したところ層別化は認められなかった（Ｄａｉｍｏｎ Ｍ，Ｊｉ Ｇ，Ｓａｉｔｏｈ Ｔ，Ｏｉｚｕｍｉ Ｔ，Ｔｏｍｉｎａｇａ Ｍ，Ｎａｋａｍｕｒａ Ｔ，Ｉｓｈｉｉ Ｋ，Ｍａｔｓｕｕｒａ Ｔ，Ｉｎａｇｅｄａ Ｋ，Ｍａｔｓｕｍｉｎｅ Ｈ，Ｋｉｄｏ Ｔ，Ｈｔａｙ Ｌ，Ｋａｍａｔａｎｉ Ｎ，Ｍｕｒａｍａｔｓｕ Ｍ，Ｋａｔｏ Ｔ：Ｌａｒｇｅ−ｓｃａｌｅ ｓｅａｒｃｈ ｏｆ ＳＮＰｓ ｆｏｒ ｔｙｐｅ ２ ＤＭ ｓｕｓｃｅｐｔｉｂｉｌｉｔｙ ｇｅｎｅｓ ｉｎ ａ Ｊａｐａｎｅｓｅ ｐｏｐｕｌａｔｉｏｎ．Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ ３０２：７５１−７５８，２００３）。
ＤＭ、ＩＧＴ、ＮＧＴの各群は年齢・性別を一致させて解析し、遺伝子型と共に身長、体重、７５ｇ経口糖負荷試験、ＨｂＡ１ｃ、ウエスト周囲径、ヒップ周囲径、ウエスト／ヒップ比、ボディマスインデックス（ＢＭＩ）、体脂肪率、収縮期血圧、拡張期血圧、血清総コレステロール（ＣＨＯ）、中性脂肪（ＴＧ）、高比重リポタンパク（ＨＤＬ）コレステロール等の形質を解析した。
連鎖不平衡ブロック（ＬＤブロック）はペアワイズ法によりＬＯＤ−ｐ値が０．０１以下のＳＮＰのセットを抽出した。ＬＤブロック毎のハプロタイプとディプロタイプの頻度はＥＭアルゴリズムに基づいたＬＤサポートというソフトウェア（Ｋｉｔａｍｕｒａ Ｙ，Ｍｏｒｉｇｕｃｈｉ Ｍ，Ｋａｎｅｋｏ Ｈ，Ｍｏｒｉｓａｋｉ Ｔ，Ｔｏｙａｍａ Ｋ，Ｋａｍａｔａｎｉ Ｎ：Ｄｅｔｅｒｍｉｎａｔｉｏｎ ｏｆ ｐｒｏｂａｂｉｌｉｔｙ ｄｉｓｔｒｉｂｕｔｉｏｎ ｏｆ ｄｉｐｌｏｔｙｐｅ ｃｏｎｆｉｇｕｒａｔｉｏｎ（ｄｉｐｌｏｔｙｐｅ ｄｉｓｔｒｉｂｕｔｉｏｎ）ｆｏｒ ｅａｃｈ ｓｕｂｊｅｃｔ ｆｒｏｍ ｇｅｎｏｔｙｐｉｃ ｄａｔａ ｕｓｉｎｇ ｔｈｅ ＥＭ ａｌｇｏｒｉｔｈｍ．Ａｎｎ Ｈｕｍ Ｇｅｎｅｔ ６６：１８３−９３，２００２）を用いて予測し、事後確率が８０％以上の場合のみ解析対象とした。
ＥＭアルゴリズム（ｅｘｐｅｃｔａｔｉｏｎ−ｍａｘｉｍｉｚａｔｉｏｎ（ＥＭ）ａｌｇｏｒｉｔｈｍ）を用いたＬＤｓｕｐｐｏｒｔにより個人のハプロタイプ型を決定し、連鎖不平衡が認められた領域について疾患関連解析を行なった。有意差の評価は、並べ替え検定（ｐｅｒｍｕｔａｔｉｏｎ ｔｅｓｔ）により行い、得られた経験的ｐ値（ｅｍｐｉｒｉｃａｌ ｐ）が０．０５未満であった場合に有意差ありと判断した。疾患にリスクを持つハプロタイプを特定するために、Ｅｍｐｉｒｉｃａｌ ｐ＜０．０５を示したＬＤブロックについて、ハプロタイプ毎に疾患群と対照群とのアリル頻度をフィッシャーの直接確率検定により検定し、ボンフェローニの補正を行い、ｐ＜０．０５を有意差ありとした。９５％信頼区域（９５％ＣＩ）も２ＢＹ２プログラム
（ｈｔｔｐ：／／ｌｉｎｋａｇｅ．ｒｏｃｋｅｆｅｌｌｅｒ．ｅｄｕ／ｏｔｔ／ｌｉｎｋｕｔｉｌ．ｈｔｍ）により計算し、その結果を表９９および１００に示す。なお、ハプロタイプ型と各形質との関連をスチューデントのｔ−検定で検定し、ａｔ−ｒｉｓｋディプロタイプはＴＧ、ＢＭＩ、ＨＤＬ−コレステロール値とは独立して有意であることが多重ロジスティック解析により確認している。
ＡＢＣＡ１遺伝子上のＳＮＰｓの連鎖不平衡について検討した結果、図１の通りであった。対象全体では８つのＬＤブロック（１，２，３，４，５，９，１０）、ＮＧＴ＋ＤＭ群では９つのＬＤブロック（１，２，３，４，５，７，８，９，１０）が見られた。ＡＢＣＡ１遺伝子のほぼ全領域が１０のＬＤブロックでカバーされており、ＬＤブロック６は集団により７と８に分かれていた。ＬＤブロック９のＳＮＰ２８（Ｔ／Ｃイントロン４５上）はＳＮＰを用いて行なった疾患関連解析において有意差を示したＳＮＰである。
＜ブロック１とＤＭの関連＞
ケース群（ＩＧＴ＋ＤＭ、またはＤＭ群のみ）とコントロール群（ＮＧＴ）のハプロタイプ分布の検定結果を表９９に示した。ＬＤブロック１はどちらのケースコントロール解析（ＮＧＴ ｖｓ．ＩＧＴ＋ＤＭ、ＮＧＴ ｖｓ．ＤＭ）においても有意差を示したが、ＤＭ群を単独でケース群とした場合（ＮＧＴ ｖｓ．ＤＭ）においてより低いｐ値（ｐ＝０．０００５）を示した。
表１００（ＬＤブロック１に観察された各ハプロタイプの疾患関連解析結果）に示すＬＤブロック１のハプロタイプ３はボンフェローニの補正後もｐ＝０．０００６と高い有意差を示した。ハプロタイプ３のオッズ比は２．３０、９５％信頼区域は１．５−３．５３であり、ハプロタイプ３はａｔ−ｒｉｓｋハプロタイプ、すなわち疾患感受性のハプロタイプであると結論づけられる。ＬＤブロック１を構成するＳＮＰであるＳＮＰ１とＳＮＰ２はいずれもＳＮＰを用いた疾患関連解析では有意差を示さなかった。従って、ハプロタイプ３と連鎖する領域（ＬＤブロック１またはその近傍）の遺伝的多型がＤＭ疾患易罹患性との関連していると考えられる。

上記表９９において、Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。ＮＤは、ケース群またはコントロール群のどちらかにおいて当該ハプロタイプが０と推定されたために解析を”実施していない”ことを指す。

上記表１００において、Ｐ値は、２ｘ２ Ｆｉｓｈｅｒ’ｓ ｅｘａｃｔ ｐｒｏｂａｂｉｌｉｔｙ ｔｅｓｔ（Ｆｉｓｈｅｒ）、およびそれらの補正（Ｂｏｎｆｅｒｒｏｎｉ）により得たものを示す。Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。
＜ＬＤブロック１のディプロタイプ解析＞
ディプロタイプ１／２、１／３、３／３が有意差を示した。１／２は疾患抵抗性であり、１／３、３／３は疾患感受性であった。これはハプロタイプを用いた疾患関連解析の結果ハプロタイプ３が疾患感受性ハプロタイプであったという結果と矛盾しない。さらにハプロタイプ３を持つ個体と持たない個体で検定を行なった結果、ｐ＝０．０００５とやはり有意な結果であり、オッズ比は２．６１であった。ＬＤブロック１の各ディプロタイプの疾患関連解析の結果を表１０１に示す。

Ｐ値は、２ｘ２ Ｆｉｓｈｅｒ’ｓ ｅｘａｃｔ ｐｒｏｂａｂｉｌｉｔｙ ｔｅｓｔ（Ｆｉｓｈｅｒ）、およびそれらの補正（Ｂｏｎｆｅｒｒｏｎｉ）により得たものを示す。Ｐ＜０．０５および０．０１はそれぞれ＊および＊＊によって示す。
＜疾患感受性ハプロタイプ（３）を持つ群の臨床的特徴＞
表３４に示す通り、ＤＭ群はＮＧＴ群に比べてＢＭＩ、血清ＴＧ値が高い傾向がある。従って、ａｔ−ｒｉｓｋよびディプロタイプとＢＭＩ、血清ＴＧ値との関連を検定する必要がある。そこで、ＬＤブロック１がＢＭＩまたはＴＧと独立してＤＭと関連している事を示すために、多重ロジスティック分析を行なったところ、ＴＧおよびＢＭＩ補正後においてもｐ＝０．００１１、オッズ比２．５１、９５％信頼区域１．４５−４．３６と疾患易罹患性において有意差を示した。
ＡＢＣＡ１遺伝子とＨＤＬコレステロール値の関連についての報告があることから、ａｔ−ｒｉｓｋハプロタイプであるハプロタイプ３を持つ個体と持たない個体の血清ＨＤＬコレステロール値の比較を行なった。ハプロタイプ３を持つ個体のＨＤＬコレステロール値は６２．８±１５．５ｍｇ／ｄｌと、ハプロタイプ３を持たない個体のＨＤＬコレステロール値５８．３±１４．７ｍｇ／ｄｌより高い傾向があった（ｐ＝０．０１４０）。そこで、ＨＤＬコレステロール値を多重ロジスティック解析で補正した後にハプロタイプ３を持つ群と持たない群の疾患関連解析を行なったところ、ｐ＝０．０００４、オッズ比２．７０、９５％信頼区域１．５６−４．６７と疾患感受性において有意差を示した。従って、ＡＢＣＡ１遺伝子はＤＭの発症にＨＤＬコレステロール値への影響とは異なる機序で影響を与えていると考えられる。
他の形質についてはＨｂＡ１ｃのようなＤＭと直接関連する項目以外はケースとコントロール群で差は認められなかった。Taking the above results together, these SNPs may be associated with obesity and high TG. To eliminate these possibilities, the inventors performed multivariate logistic analysis and attempted to show that these SNPs were independent of BMI and TG. The present inventors used C2289T as SNPs and analyzed in the second sample group (n = 724). P <0.05 was considered significant. Even after correction for TG and BMI values, the risk genotypes of SNPs showed significant differences in comparison between the IGT group, DM group, and NGT group (Table 98). Table 98 shows the contribution of risky genotypes of the nephrin gene SNP (C2289T) in the progression of DM.

Data are numerical values (%). The frequencies of risk genotypes in the IGT and DM groups were compared to the NGT group, respectively. P <0.05 and 0.01 are indicated by * and **, respectively. OR was adjusted by serum triglyceride level (TG) and body mass index (BMI).
As described above, an association between SNPs and DM in genes was observed in a case-control study of Japanese based on the region.
The present inventors have clarified that SNPs in genes are useful indicators for evaluating the risk of developing DM, and the results of the present invention further show that nephrin plays an important role in basic functions, and nephrin dysfunction Are related to the onset of DM.
[Example 10] Analysis object The present inventors speculated that ATP-binding cassette transducer 1 (ABCA1), which is important for regulating lipid transport from peripheral cells to HDL, is involved in the progression of DM. .
ABCA1 gene deficiency causes hereditary HDL deficiencies such as Tanger disease and familial hypoalphalipoproteinemia (FHA) (Schmitz G, Buechler C: ABCA1: regulation, trafficking and association with heteroprotein. Med 34: 334-347, 2002, Oram JF, Lawn RM: ABCA1: the gatekeeper for eliminating excision cholesterol. J Lipid Res 42- 1173-1179, 2001, Srivasta TP lll lipid transport and atherosclerosis.Mol Cell Biochem 237: 155-164, 2002, Santa Marina-Fojo S, Remley AT, Neufeld EB, Brewer HB: Regulation cell. , Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Loopp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HOF, Lovers O, Orlrtt hbourne-Excoffon KJD, Sensen CW, Scherer S, Mott S, Denis M, Martin D, Frohlich J, Morgan K, Koop B, Pimstone S, Kastele JJP, Gene J Density lipoproteininfiency.Nat Genet 22: 336-345, 1999, Bodzioch M, Orso E, Klucken T, Langmann T, Botcher L, Diedrich W, Drobrik W, Drobnik P, umez M, kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis C, Lackner KJ, Schmitz G: The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 22: 347-351, 1999, Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deutsche JF, Brewer HB, Duverger N, Denefel G, Assmang. the gene encoding ATP-binding cassette transporter Nat Genet 22: 352-355, 1999, Miller M, Rhyne J, Hamlette S, Birnbaum J, Rodriguez A: Genetics of HDL regulation in humans. Curr Opin Lipidol 14: 273-279, 2003, Singaraja RR, Brunham LR, Visscher H, Kastelein JJP, Hayden MR. Efflux and aerosole biosimi- tica vinci Arteriscler Thromb Vasc Biol 200: 1322-1332, 2003). As a general polymorphism of ABCA1 gene, serum lipid level, particularly HDL cholesterol level, coronary artery disease (CAD) has been reported a lot (Miller M, Rhyne J, Hamlette S, Birnbaum J, Rodriguez A). : Genetics of HDL regulation in humans.Curr Opin Lipidol 14: 273-279,2003, Singaraja RR, Brunham LR, Visscher H, Kastelein JJP, Hayden MR: Efflux and atherosclerosis-the clinical and biochemical impact of variations in the ABCA1 gene. Arterisc er Thromb Vasc Biol 200: 1322-1332, 2003, Clee SM, Zwinderman AH, Engelt JC, Zwarts KY, Molhuizen HOF, Romp K, Jukema JW, van WijlMD. J Jr, Kastelein JJP, Hayden MR: Common genetic variation in ABCA1 is associated with altered lipoproteins 1 and a concentrated risky. tuculta S, Ballantyne CM, Elghannam H, Gotto AM Jr, Marian AJ: Novel polymorphisms in promoter region of ATP binding cassette transporter gene and plasma lipids, severity, progression, and regression of coronary atherosclerosis and response to therapy.Circ Res 88: 969 -973, 2001, Brousseau ME, Bodzioch M, Schaefer EJ, Goldkamp AL, Kielar D, Probst M, Ordovas JM, Aslanidis C Lackner KJ, Rubins HB, Collins D, Robins SJ, Wilson PWF, Schmitz G: Common variants in the gene encoding ATP-binding cassette transporter 1 in men with low HDL cholesterol levels and coronary heart disease. Atherosclerosis 154: 607-611,2001, Zwarts KY, Clee SM, Zwinderman AH, Engert JC, Singaraja R, Loubser O, James E, Roomp K, Hudson TJ, Jukema JW, Kastelein JJP, Hayden MR: ABCA1 regulatory variants influence coronary arty disease independent of effects on plasma lipid levels. Clin Genet 61: 115-125, 2002, Hong SH, Rhyne J, Miller M: Novel polypyrimidine vari ration quotient flip cit ol cit ol cit ol cit ol cit ri pi n pi n ti n pi n ti pi n pi n ti pi n pi n ti n pi n ti n pi n ti n pi n ti pi n ti pi n ti pi n ti pi n ti pi n ti pi n ti pi n ti pi n ti pi n ti n pi premature coronary disease. Circ Res 93: 1006-11022, 2003, Tan JH-H, Low PS, Tan Y-S, Tong MC, Saha N, Yang H, Heng C-K: ABCA1 gene polymorphisms and theirarity associations. dissease and plasma lipids in males from three tree populations in Singapore. Hum Genet 113: 106-117, 2003), no indication of an association with diabetes.
Since a decrease in serum HDL cholesterol level is often observed in patients with type 2 diabetes and is said to be involved in insulin resistance, the association between ABCA1 gene and type 2 diabetes was estimated. In the present invention, it has already been shown that SNP (IMS-JST105322) on the ABCA1 gene is associated with DM (Table 36). However, in order to carry out further analysis, the inventors have found that the SNP of the ABCA1 gene and type 2 diabetes ( (DM) was examined in a case-control study using haplotype analysis based on the Japanese region.
Seventy-two cases of DM, 75 cases of impaired glucose tolerance (IGT), and 227 cases of normal glucose tolerance (NGT) were analyzed. These samples boat research (Oizumi T et al., "Genotype Arg / Arg, but not Trp / arg, of the Trp64Arg polymorphism of the β 3 -adrenergic receptor is associated with type 2 diabetes and obesity in a large Japanese sample. " Diabetes Care., 24: 1579-1583, 2001). The Funagata study is a population-based study conducted in an agricultural area about 400 km north of Tokyo. This study was conducted with the approval of Yamagata University's Ethics Review Board and written informed consent from the subjects. All processes were performed according to the guidelines. It should be noted that the target population was a software called STRUCTURE (Pritchard JK, Stephens M, Donnelly P: Inference of population structure using multi-type genomic data. Genetics 155: 95-9D. M, Ji G, Saitoh T, Oizumi T, Tominaga M, Nakamura T, Ishii K, Matsuura T, Inageda K, Matsumine H, Kido T, Htay L, Kamani N, Mata T for type 2 DM su (Septibilities genes in a Japan population. Biochem Biophys Res Commun 302: 751-758, 2003).
Each group of DM, IGT, and NGT was analyzed by matching the age and gender, along with genotype, height, weight, 75 g oral glucose tolerance test, HbA1c, waist circumference, hip circumference, waist / hip ratio, body mass index ( BMI), body fat percentage, systolic blood pressure, diastolic blood pressure, serum total cholesterol (CHO), neutral fat (TG), high density lipoprotein (HDL) cholesterol and other traits were analyzed.
For the linkage disequilibrium block (LD block), a set of SNPs having an LOD-p value of 0.01 or less was extracted by the pairwise method. The frequency of haplotypes and diplotypes for each LD block is a software called LD support based on the EM algorithm (Kitamura Y, Moriguchi M, Kaneko H, Morisaki T, Toyama K, Kamaniti N: Determination of Probability. prediction was made using foreach subject from genetic data using the EM algorithm. Ann Hum Genet 66: 183-93, 2002), and was only analyzed when the posterior probability was 80% or more.
The haplotype of an individual was determined by LD support using an EM algorithm (expectation-maximization (EM) algorithm), and a disease-related analysis was performed on a region where linkage disequilibrium was observed. The significant difference was evaluated by a permutation test, and when the obtained empirical p value (imperial p) was less than 0.05, it was judged that there was a significant difference. In order to identify haplotypes at risk for disease, LD blocks with Empirical p <0.05 were tested for the allylic frequency of the disease group and the control group for each haplotype by Fisher's exact test. Correction was made and p <0.05 was considered significant. The 95% confidence region (95% CI) was also calculated by the 2BY2 program (http://linkage.rockefeller.edu/ott/linkutil.htm) and the results are shown in Tables 99 and 100. The relationship between haplotypes and each trait was tested by Student's t-test, and it was confirmed by multiple logistic analysis that at-risk diplotypes were significant independently of TG, BMI, and HDL-cholesterol levels. ing.
As a result of examining the linkage disequilibrium of SNPs on the ABCA1 gene, it was as shown in FIG. The entire target has 8 LD blocks (1, 2, 3, 4, 5, 9, 10), and the NGT + DM group has 9 LD blocks (1, 2, 3, 4, 5, 7, 8, 9, 10). It was seen. Almost the entire region of ABCA1 gene was covered with 10 LD blocks, and LD block 6 was divided into 7 and 8 by population. SNP 28 (on T / C intron 45) of LD block 9 is a SNP that showed a significant difference in the disease-related analysis performed using SNP.
<Relationship between block 1 and DM>
Table 99 shows the haplotype distribution test results for the case group (IGT + DM or DM group only) and the control group (NGT). LD block 1 showed a significant difference in either case control analysis (NGT vs. IGT + DM, NGT vs. DM), but a lower p-value in the case where the DM group alone was used as the case group (NGT vs. DM). (P = 0.0005).
The haplotype 3 of LD block 1 shown in Table 100 (disease-related analysis results of each haplotype observed in LD block 1) showed a highly significant difference with p = 0.006 even after Bonferroni correction. The odds ratio of haplotype 3 is 2.30 and the 95% confidence range is 1.5-3.53, and it is concluded that haplotype 3 is an at-risk haplotype, ie a disease-susceptible haplotype. Neither SNP1 nor SNP2, which are SNPs constituting LD block 1, showed a significant difference in disease-related analysis using SNP. Therefore, it is considered that the genetic polymorphism in the region linked to haplotype 3 (LD block 1 or its vicinity) is associated with susceptibility to DM disease.

In Table 99 above, P <0.05 and 0.01 are indicated by * and **, respectively. ND indicates that the analysis is “not performed” because the haplotype was estimated to be 0 in either the case group or the control group.

In Table 100 above, the P value is obtained by 2 × 2 Fisher's exact probability test (Fisher) and their correction (Bonferroni). P <0.05 and 0.01 are indicated by * and **, respectively.
<Diplotype analysis of LD block 1>
Diplotypes 1/2, 1/3, and 3/3 showed significant differences. 1/2 was disease resistance and 1/3 and 3/3 were disease susceptibility. This is consistent with the result that haplotype 3 was a disease-susceptible haplotype as a result of disease-related analysis using haplotypes. Further, as a result of testing with individuals having haplotype 3 and individuals having no haplotype 3, p = 0.005 was still a significant result, and the odds ratio was 2.61. Table 101 shows the results of disease-related analysis of each diplotype of LD block 1.

P value shows what was obtained by 2x2 Fisher's exact probability test (Fisher) and those corrections (Bonferroni). P <0.05 and 0.01 are indicated by * and **, respectively.
<Clinical characteristics of group with disease susceptibility haplotype (3)>
As shown in Table 34, the DM group tends to have higher BMI and serum TG values than the NGT group. Therefore, it is necessary to test the association of at-risk and diplotype with BMI and serum TG values. Therefore, in order to show that LD block 1 is related to DM independently of BMI or TG, multiple logistic analysis was performed. As a result, p = 0.0001, odds ratio of 2. after TG and BMI correction. 51, 95% confidence range 1.45-4.36 and a significant difference in disease susceptibility.
Since there is a report about the association between ABCA1 gene and HDL cholesterol level, the serum HDL cholesterol level of individuals with and without haplotype 3 which is at-risk haplotype was compared. The HDL cholesterol level of individuals with haplotype 3 tended to be 62.8 ± 15.5 mg / dl, which was higher than the HDL cholesterol level of individuals without haplotype 3, 58.3 ± 14.7 mg / dl (p = 0) .0140). Therefore, when the HDL cholesterol level was corrected by multiplex logistic analysis and a disease-related analysis was conducted between the group having haplotype 3 and the group not having haplotype 3, p = 0.004, odds ratio 2.70, 95% confidence range 1.56 It showed a significant difference between -4.67 and disease susceptibility. Therefore, it is considered that the ABCA1 gene affects the onset of DM by a mechanism different from the effect on the HDL cholesterol level.
For other traits, there was no difference between the case and control group except for items directly related to DM such as HbA1c.

Industrial applicability

According to the present invention, a test method and a test reagent for type 2 diabetes (DM) are provided. It is possible to predict the onset of type 2 diabetes by detecting the polymorphism of the gene according to any one of (1) to (29) above, the expression of the gene, or the activity of the protein encoded by the gene. It is highly anticipated that it will be possible and will enable more economical and efficient strategies for the prevention and treatment of type 2 diabetes.
In addition, the knowledge provided by the present inventors is considered to contribute to the elucidation of the onset mechanism of DM.

Claims (13)

A test method for type 2 diabetes, comprising detecting a mutation in a gene according to any one of the following (1) to (29) for a subject.
(1) MET, (2) NPHS1, (3) SLC1A1, (4) SERPINB2, (5) FABP2, (6) ZNF263, (7) TNFA, (8) THBS3, (9) ABCA1, (10) SERPINB10, (11) ALDOB, (12) CTSD, (13) STE, (14) ABCC8, (15) SERPINA3, (16) RCV1, (17) TFRC, (18) MMP19, (19) MYL2, (20) VLDLR, (21) P2RX7, (22) DLG5, (23) LDLC, (24) AQP8, (25) LARGE, (26) BLZF1, (27) RGS5, (28) FRAP1, (29) LEP The test method according to claim 1, wherein the mutation is a single nucleotide polymorphism mutation. The subject is characterized by determining the base type of the polymorphic site in the gene according to any one of (1) to (29) of claim 1 or a DNA region in the vicinity of the gene. Inspection method. The method according to claim 3, wherein the polymorphic site is a polymorphic site described in the following (1a) to (29a), respectively.
(1a) a MET gene or a site on a DNA region in the vicinity of the gene, the positions 765, 1383, 1890, 2099, 2814, 3293, 3752 in the nucleotide sequence set forth in SEQ ID NO: 1; 3774, 3927, 4690, 7419, 8219, 8201, 10001, 10318, 13766, 13789, 14258, 15427, 16275, 16597, 16932, 19768, 19835, 230032 , 20240, 20537, 20885, 21546, 24209, 24271, 24376, 24642, 26028, 26226, or 34209
(2a) the NPHS1 gene or a site on the DNA region in the vicinity of the gene, the positions 172, 186, 211, 2554, 2558, 2558, 10001, 10001 in the nucleotide sequence set forth in SEQ ID NO: 2; 10176th, 11840th, 13759th, 14645th, 14967th, 16198th, 16439th, 17635th, 17685th, 17794th, 17805th, 17848th, 17849th, 18491th, 19955th, 20955th, 20613th 21533, 21582, 21811, 21812, 22190, 22965, 22982, 23660, 23780, 24040, 24172, 25334, or 26585 polymorphic sites
(3a) SLC1A1 gene or a site on the DNA region in the vicinity of the gene, and positions 1142, 2732, 2734, 7220, 7001, 12001, 12276, 12922 in the nucleotide sequence set forth in SEQ ID NO: 3. Or any polymorphic site at position 18049
(4a) SERPINB2 gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence of SEQ ID NO: 4, positions 2853, 2891, 5218, 5317, 7560, 8054, 8771, 10001 , 10329, 11781, 12063, 12705, 13125, 13691, 13913, 13916, 13917, 14123, 14169, 14453, 14456, 14483, 16526, 16556, 17643, 17725, 17824, 18076, 18227, 18500, 18687, 18741, 18744, 18875, 19045, 19141, 19149, 19190, 19429, 19548, 195 7th, 19624th, 19645th, 19814th, 200049th, 20082th, 20097th, 20243th, 22243th, 22122th, 22516th, 22561th, 22816th, 23007th, 23904th, 24005th, 24418th Any of the polymorphic sites at positions 24578, 24855, 24855, 25505, 25538, 25564, 25586, 26017, 26234, 26557, 26621, 30155, 30267, or 30607
(5a) the FABP2 gene or a site on the DNA region in the vicinity of the gene, and the positions 2970, 3174, 3175, 4525, 4556, 4556, 4558, 4636 in the nucleotide sequence set forth in SEQ ID NO: 5; 4646, 4774, 5726, 5736, 5852, 7203, 7253, 7310, 8239, 8251, 8296, 8312, 8974, 10001, 10107, 10424, 10464 , 10491, 10794, 11922, 11957, 12820, 16006, 16256, 16309, 1631, 16360, 16923, 17877, 18408, 18649, 18715, 18783, or Any polymorphic part in 19878
(6a) a ZNF263 gene or a site on the DNA region in the vicinity of the gene, and positions 876, 1552, 3213, 3269, 6401, 6425, 6602 in the nucleotide sequence set forth in SEQ ID NO: 6; 9595th, 10001th, 10252th, 10269th, 10294th, 11377th, 12469th, 13642th, 13643th, 13879th, 14367th, 16133th, 17084th, 1765th, or 19263 Mold part
(7a) a TNFA gene or a site on the DNA region in the vicinity of the gene, the positions 175, 2401, 2773, 4452, 4671, 4672, 5184 in the nucleotide sequence set forth in SEQ ID NO: 7; 5609, 5820, 5941, 5942, 5942, 6245, 6315, 6316, 6487, 6603, 6730, 6867, 6931, 6935, 6958, 7953, 8022 , 8133, 8482, 8628, 8650, 8656, 8827, 8829, 8865, 8938, 8945, 8994, 9205, 9269, 9275, 10001, 10002, 1023, 10363 , 10399, 10680, 10765, 10816, 110 2nd, 11565, 11194, 12669, 12936, 12963, 13024, 13128, 13154, 13289, 13538, 13539, 13594, 13632, 13646, 13688, 13822 13855, 13863, 13966, 13993, 14370, 14542, 14625, 15030, 15064, 15581, 15730, 15975, 16626, 16713, 16734, 17240, 17369 Position, polymorphic site at positions 17698, 17711, or 19348
(8a) THBS3 gene or a site on the DNA region in the vicinity of the gene, which is 3098, 3103, 3143, 3178, 4543, 6977, 7158 in the nucleotide sequence set forth in SEQ ID NO: 8; Polymorphic site at any of the positions 10001, 10445, 11728, 13012, 13514, 16404, 18088, 18708, 18776, 18989, 19465, 19769, or 19786
(9a) ABCA1 gene or a site on the DNA region in the vicinity of the gene, wherein the positions 993, 1779, 1813, 3450, 4287, 4287, 4382, 7270 in the nucleotide sequence set forth in SEQ ID NO: 9; 7528, 8383, 8783, 8798, 9798, 9254, 10001, 10999, 11122, 11720, 12473, 12560, 12591, 12664, 12916, 13403, 13605, 13691 14457, 14737, 15278, 15345, 15345, 15636, 15636, 15824, 16793, 17022, 17037, 17283, 17576, 18167, 18619, 18632, 18842 Rank, 1884 18926, 19404, 19404, 19809, 20447, 20514, 20514, 20515, 21963, 22888, 23081, 23157, 23777, 23533, 24389, 25814, 26198, 26655, 26902, 27367, 27535, 27701, 28175, 28467, 28564, 28669, 28774, 28839, 28937, 28944, 29037, 30091, 30613, 30975 , 32803, 32803, 33435, 34263, 34264, 34812, 35306, 35339, 35473, 36217, 37596, or 37855
(10a) SERPINB10 gene or a site on the DNA region in the vicinity of the gene, and positions 145, 4845, 6757, 6859, 7151, 7827, 8066 in the nucleotide sequence set forth in SEQ ID NO: 10; Any polymorphic site at positions 10001, 12885, 13227, 13432, 13563, 15439, 15447, 15729, 15759, 15585, 16094, 16330, or 18565
(11a) ALDOB gene or a site on a DNA region in the vicinity of the gene, wherein the nucleotide position is 99, 380, 396, 399, 712, 934, 1534 in the nucleotide sequence of SEQ ID NO: 11. 1612, 1948, 2079, 2101, 4161, 4318, 4318, 4318, 5091, 6623, 6718, 7796, 7859, 8190, 8203, 9104, 9621, 9642 , 10001, 10489, 11443, 11592, 11593, 11616, 11616, 11617, 12281, 12457, 12802, 13993, 14026, 14800, 16654, 16822, 17133, 17239 , 18216, 18709, 19610 One of the polymorphic sites, or 19,613 positions
(12a) a site on the CTSD gene or a nearby DNA region of the gene, and positions 813, 815, 844, 861, 954, 1026, 1159 in the nucleotide sequence set forth in SEQ ID NO: 12; 1299, 5464, 5600, 7144, 7707, 7726, 7739, 7778, 7803, 7803, 7841, 7842, 7886, 7939, 7963, 8058, 8333, 9948 , 10001, 11990, 12342, 12807, 16266, or 16771
(13a) A site on the STE gene or a DNA region in the vicinity of the gene, and in positions 57, 468, 1881, 2955, 3460, 3460, 3976, 4537 in the nucleotide sequence set forth in SEQ ID NO: 13. 4538, 4722, 4867, 4873, 4947, 5025, 7043, 7676, 8581, 9035, 9347, 9686, 9882, 9894, 10001, 10003, 12142 , 13520, 13551, 13797, 14492, 14753, 14933, 15684, 17022, 18264, 18568, 18958, 19194, 19330, 19354, 19355, 19567, 19785 Rank, 20511th, 20612th, 2 627 of, 20,936 positions, 20,961 positions, 20,971 positions, 21213-position, 21336 positions, 21,609 positions, or polymorphic sites 21914 position, or 21,983 of
(14a) ABCC8 gene or a site on the DNA region in the vicinity of the gene, and positions 6978, 7358, 7359, 7649, 7755, 7755, 8068, 8214 in the nucleotide sequence set forth in SEQ ID NO: 14. 8215, 8308, 9658, 9761, 9861, 9811, 9828, 10001, 10095, 10125, 11658, 11684, 11708, 11767, 11883, 111998, 12109, 12308 13620th, 13664th, 13912th, 14297th, 14309th, 14320th, 14323th, 14367th, 14393th, 14475th, 1459th, 14722th, 15294th, 15601th, 15657th, 16160th, 16230 Rank, 16 67 position, 16417 positions, 17,695 positions, 18213-position, 18815 positions, 19,093 positions, 20082-position, 20211-position, 20461 positions, 21,547 positions, 21995-position, or polymorphic sites 22825 position, or 23,901 of
(15a) SERPINA3 gene or a site on the DNA region in the vicinity of the gene, which is a position 937, 1145, 1878, 2345, 2559, 3559, 3250, 3513 in the nucleotide sequence set forth in SEQ ID NO: 15. 4402, 5776, 6659, 7453, 7934, 7934, 8000, 8049, 8204, 8543, 8554, 8935, 9928, 10001, 10233, 10472, 10479, 10525 , 10544, 10629, 13339, 13348, 16287, 16389, 16626, or 17880
(16a) a site on the RCV1 gene or a nearby DNA region of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 16, positions 25, 49, 469, 557, 592, 2730, 2754, 5761, 6154, 6183, 8510, 8529, 8542, 8590, 9838, 10001, 1012, 10133, 10649, 10702, 10866, 12560, 15632, 15527 , 15853, 15994, 16617, 16790, 16841, 17010, 17348, 17656, 17656, 17816, 17820, 17882, 17882, 18061, 18101, 19304, or Any polymorphic site at position 19485 17a) TFRC gene or a site on a DNA region in the vicinity of the gene, and in the base sequence described in SEQ ID NO: 17, positions 836, 1019, 2511, 4016, 4068, 4169, 4169, 4281 , 5398, 5436, 5450, 5730, 5937, 6044, 6313, 6713, 6759, 6794, 6833, 7528, 8047, 8048, 8080, 9248, 9361, 9417, 9734, 10001, 10001, 11461, 11845, 11852, 12086, 12596, 12864, 12930, 13969, 13969, 14119, 14280, 16833, or 17047 Any polymorphic site
(18a) a position on the MMP19 gene or a DNA region in the vicinity of the gene, the positions 6329, 7329, 7715, 8138, 8507, 8551, 8551, 8574 in the nucleotide sequence set forth in SEQ ID NO: 18. Any polymorphic site at positions 10001, 11138, 11343, 13151, 13868, or 18923
(19a) MYL2 gene or a site on the DNA region in the vicinity of the gene, and the nucleotide sequence described in SEQ ID NO: 19 is position 3147, position 3213, position 4686, position 7343, position 7789, position 7801, position 10001, position 10123, 11264th, 11992th, 15532th, 15681, 15957, 15988, 16641, 18386, 18782, 20354, 21531, 23245, 27740, 30669, 30670, 32077, 32732 32907, 32955, 42589, 43029, 47354, 64777, 71869, 71196, 74274, 75551, 76343, 76344, 78270, 78339, 78689, 78743, 78 06, 78807, 79215, 79319, 79357, 79460, 79463, 790034, 80034, 80062, 800076, 80401, 80475, 80599, 80715, 80865, 80943, 80952 80974, 80991, 81130, 81373, 81428, 81543, 81682, 82422, 82545, 82792, 82929, 83265, 83266, 83394, 83463, 83500, 83578 , 83612, 83650, 84790, 8492, 84945, 85214, 85383, 85384, 85550, 85984, 86109, 86539, 86542, 87169 87170, 87353, 87671, 87953, 89554, 88057, 88178, 88240, 88258, 88271, 88272, 88307, 88512, 88695, 88778, 88798, 89124 Or any polymorphic position at position 91081
(20a) a VLDLR gene or a site on a DNA region in the vicinity of the gene, and positions 6297, 6733, 7994, 9242, 9952, 9901, 11801, 11838 in the nucleotide sequence set forth in SEQ ID NO: 20; Any polymorphic site at positions 12995, 13224, 13277, 18702, 18896, or 19214
(21a) the P2RX7 gene or a site on the DNA region in the vicinity of the gene, and the 99th position, the 694th position, the 2765th position, the 2765th position, the 3101st position, the 3117th position, the 3127th position, 3309th, 3346th, 3802th, 3916th, 4206th, 4430th, 5937th, 6705th, 7378th, 8312th, 8399th, 9928th, 10001, 100th, 10042th, 10277th, 10440th, 10461th , 10479, 10527, 10534, 10550, 10701, 10909, 11066, 11198, 11575, 11631, 11765, 11976, 12119, 12158, 12207, 12272, 12272 Rank, 12498, 1256 , 12791, 12856, 12860, 13207, 13383, 13590, 13860, 13869, 13880, 13881, 13984, 14048, 14059, 14210, 14806, 14921, 15331, 15488, 15558, 15651, 15651, 15686, 15791, 16051, 16082, 16137, 16492, 16649, 16784, 16788, 17072, 17076, 17379, 17527 17593, 17623, 17719, 17998, 18005, 18048, 18288, 18412, 18588, 18640, 19340, 19545, 19676, or 19685 One of the polymorphic sites of
(22a) a DLG5 gene or a site on the DNA region in the vicinity of the gene, and the 2nd position, 121st position, 1438th position, 1669th position, 2180th position, 2436th position, 2543th position in the nucleotide sequence set forth in SEQ ID NO: 22; 2619, 3715, 3727, 5327, 5256, 5335, 5339, 5688, 5752, 6304, 6627, 7195, 7342, 7603, 8273, 8905, 8988, 9013 , 9057, 10001, 10372, 10453, 10468, 10468, 10512, 11038, 11055, 11597, 11681, 11719, 11922, 12034, 12049, 12176, 12554 , 13481, 13864, 14390 15044, 15598, 16176, 16870, 17475, 17649, 18077, 18585, 18619, 18677, 19222, 19731, 19750, 20758, 20962, 21073, 21680 , 21867, 22385, 22874, 23191, 24016, 24520, 24993, 25460, 26155, 26804, 26980, 27882, 29234, 29469, 30254, 30742, 30742, 30756, 30756, 31671, 31874, 31940, 32626, 33393, 33581, 34240, 35294, 35666, 36587, 36653, 36 41st, 37408th, 37583, 37612, 38462, 38470, 38470, 38485, 39146, 39146, 40612, 40641, 41242, 42217, 42539, 45934, 46033, or 46895 Any polymorphic site
(23a) a site on the LDLC gene or a nearby DNA region of the gene, and in the base sequence described in SEQ ID NO: 23, positions 1580, 1754, 1882, 3062, 3507, 4507, 4800, 4905, 5569, 5608, 5643, 5665, 5593, 5864, 5884, 5981, 6169, 6808, 6858, 7194, 7283, 7728, 7835, 8049, 8049 , 8265, 9209, 9445, 9646, 9647, 9716, 9716, 9775, 10001, 10027, 10054, 10193, 10208, 10826, 10908, 10997, 11336, 11754 , 11895, 12031, 12327, 2464, 12561, 14051, 15366, 15778, 17723, 17772, 17783, 17811, 17815, 17862, 17900, 17962, 18184, 18189, 18297, 18411 , 18512, 18563, 19125, 19152, 200092, 21227, 21506, 21617, 21855, 23090, 25700, 26468, 26638, 27682, 27755, 27888, 27973 Position, 27995, 28123, 28127, 28144, 28153, or 28271 polymorphic site
(24a) AQP8 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence set forth in SEQ ID NO: 24, positions 6722, 9643, 9934, 10001, 10002, 13682, 14629, 15376, Polymorphic site at either position 15808 or 16082
(25a) a LARGE gene or a site on a DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 25, positions 1816, 3116, 3117, 3118, 3154, 3157, 3191, 3197, 3205, 3291, 3622, 3652, 3652, 3792, 4488, 5076, 5474, 5481, 5492, 5503, 5654, 5672, 6563, 6843, 6908 7119, 7538, 7538, 7721, 8403, 8626, 10001, 12636, 13103, 14794, 15370, 15448, 15599, 15564, 16045, 16261, 16277 Rank, 16861, 17360, 17611 17714, 17809, 18221, 18221, 18376, 18536, 19063, 22294, 23404, 23612, 25125, 25134, 25662, 25705, 25751, 27407, 28166 , 28567, 30302, 30316, 30353, 30354, 30391, 33594, 33650, 33754, 36690, 37753, 37895, 37900, 39104, 39745, 41404, 41646, 41711, 42020, 42080, 42974, 43052, 43240, 43621, 44734, 44958, 46920, 47431, 47573, 48101, 48 Position 27, 48306-position, 48,807 positions, 49049 positions, 49,848 positions, 49,860 positions, 49,903 positions, 50,114 positions, 50,223 positions, 52,057 positions, 52,543 positions, one of the polymorphic sites of 52,693 positions, or 53,061 of
(26a) a BLZF1 gene or a site on the DNA region in the vicinity of the gene, and positions 1283, 4288, 7520, 7599, 7740, 7745, 8745 and 9050 in the nucleotide sequence set forth in SEQ ID NO: 26; 9862, 10001, 10124, 10168, 10168, 10339, 11017, 13737, 13747, 18354, 20031, 20072, 22094, 24082, 24305, 24478, 24552, 25273 , 25351, 29096, 29100, 2976
(27a) RGS5 gene or a site on the DNA region in the vicinity of the gene, which is a position of 733, 848, 1039, 1351, 1569, 1831, 2078 in the nucleotide sequence set forth in SEQ ID NO: 27; 2707, 2794, 2904, 2995, 2995, 3122, 3520, 4351, 4824, 5189, 5205, 5206, 5525, 5779, 6487, 7712, 10001, 10208 , 10235, 10868, 11092, 11617, 11666, 12216, 12326, 12741, 13396, 13890, 14702, 15823, 16705, or 19086
(28a) the FRAP1 gene or a site on the DNA region in the vicinity of the gene, and positions 3455, 7826, 8715, 8783, 9042, 9390, 9462 in the nucleotide sequence set forth in SEQ ID NO: 28; 9709, 10001, 10635, 10748, 12,794, 12849, 12,915, 13,169, 17020, 18586, 21586, 21123, or 21128 polymorphic sites,
Or
(28a-2) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, and in the base sequence described in SEQ ID NO: 30, positions 9598, 10001, 10306, 11181, 11480, 13593, 13721 Position, 13792, 15314, 15623, 15782, 16708, 16748, or 19630 polymorphic sites
(29a) a LEP gene or a site on a DNA region in the vicinity of the gene, the positions 244, 575, 679, 1487, 1644, 2343, 2715 in the nucleotide sequence set forth in SEQ ID NO: 29; 2763, 3098, 3589, 3880, 5787, 5865, 6436, 7392, 7995, 8161, 8544, 9230, 9795, 10001, 101199, 10503, 111975 , 14981, 15681, or 19300 The method according to claim 3, wherein the polymorphic site is a polymorphic site described in the following (1b) to (29b), respectively.
(1b) A site on the MET gene or a DNA region in the vicinity of the gene, which is located at position 14258 or 19768 in the nucleotide sequence set forth in SEQ ID NO: 1. (2b) On the NPHS1 gene or a DNA region in the vicinity of the gene A site at position 10001 or 17848 in the base sequence described in SEQ ID NO: 2 (3b) is a site on the SLC1A1 gene or a DNA region in the vicinity of the gene, and described in SEQ ID NO: 3 10001 position in the nucleotide sequence (4b) SERPINB2 gene or a site on the DNA region in the vicinity of the gene, the 10001 position in the nucleotide sequence set forth in SEQ ID NO: 4 (5b) FABP2 gene or in the vicinity of the gene A site on the DNA region, in the base sequence set forth in SEQ ID NO: 5 0001 position (6b) on the ZNF263 gene or in the vicinity DNA region of the gene, and at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 6 (7b) on the TNFA gene or in the vicinity DNA region of the gene A site at position 10001 in the base sequence set forth in SEQ ID NO: 7 (8b) a site on the THBS3 gene or a nearby DNA region of the gene, and 10001 in the base sequence set forth in SEQ ID NO: 8 Site (9b) is a site on the ABCA1 gene or a DNA region adjacent to the gene, and the site at position 17283 (10b) SERPINB10 gene or a DNA region adjacent to the gene in the nucleotide sequence set forth in SEQ ID NO: 9 Which is a site at position 10001 in the base sequence set forth in SEQ ID NO: 10 Position (11b) ALDOB gene or a site on the DNA region in the vicinity of the gene, and a site at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 11 (12b) CTSD gene or a site on the DNA region in the vicinity of the gene In the nucleotide sequence of SEQ ID NO: 12, the site at position 11990 or 16771 (13b) is a site on the STE gene or a DNA region in the vicinity of the gene, and in the nucleotide sequence of SEQ ID NO: 13. 18958-position or 21213-position (14b) ABCC8 gene or a site on the DNA region in the vicinity thereof, and the 14475-position in the nucleotide sequence set forth in SEQ ID NO: 14 (15b) SERPINA3 gene or the gene A site on a nearby DNA region, which is described in SEQ ID NO: 15 The site at position 10001 (16b) in the base sequence of the RCV1 gene or a site on the DNA region near the gene, and the site at position 10001 in the base sequence set forth in SEQ ID NO: 16 (17b) the TFRC gene or the gene A site on the neighboring DNA region, the site at position 10001 in the nucleotide sequence shown in SEQ ID NO: 17 (18b) MMP19 gene or a site on the neighboring DNA region of the gene, described in SEQ ID NO: 18 10001 position in the base sequence (19b) MYL2 gene or a site on the DNA region in the vicinity of the gene, and the 10001 position in the base sequence described in SEQ ID NO: 19 (20b) VLDLR gene or in the vicinity of the gene A site on the DNA region, the nucleotide sequence of SEQ ID NO: 20 10001 position (21b) P2RX7 gene or a site on the DNA region near the gene, and the 10001 position (22b) DLG5 gene or a DNA region near the gene in the nucleotide sequence set forth in SEQ ID NO: 21 An upper site, a site (23b) at position 10001 or 37583 in the nucleotide sequence described in SEQ ID NO: 22, a site on the LDLC gene or a DNA region in the vicinity of the gene, and described in SEQ ID NO: 23 18563 position (24b) in the nucleotide sequence of AQP8 gene or a site on the DNA region in the vicinity of the gene, and the 10001 position (25b) LARGE gene in the nucleotide sequence set forth in SEQ ID NO: 24 A site on a nearby DNA region, the salt of SEQ ID NO: 25 A site at positions 10001, 13103, or 15448 in the sequence (26b) is a site on the BLZF1 gene or a nearby DNA region of the gene, and is a site at position 2731 (27b) RGS5 in the nucleotide sequence set forth in SEQ ID NO: 26 A site on a gene or a DNA region in the vicinity of the gene, the site at position 10001 (28b) in the nucleotide sequence shown in SEQ ID NO: 27, a site on the FRAP1 gene or a DNA region in the vicinity of the gene; : Site at position 17020 (28b-2) in the FRAP1 gene or a DNA region adjacent to the gene in the base sequence described in 28, and site at position 10001 in the base sequence described in SEQ ID NO: 30 (29b) A site on the LEP gene or a nearby DNA region of the gene, Site of position 10503 in the nucleotide sequence set forth in SEQ ID NO: 29 The base type in the polymorphic site according to (1b) to (29b) of claim 5 is determined to be type 2 diabetes when the following species are (1c) to (29c), respectively. The method described in 1.
(1c) A site on the MET gene or a nearby DNA region of the gene, wherein the base species at position 14258 in the base sequence described in SEQ ID NO: 1 is T, or the base species at position 19768 is T
(2c) A site on the NPHS1 gene or a nearby DNA region of the gene, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 2 is A, or the base type at position 17848 is A
(3c) The SLC1A1 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 3 is G
(4c) The SERPINB2 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 24578 in the nucleotide sequence set forth in SEQ ID NO: 4 is A
(5c) a site on the FABP2 gene or a DNA region in the vicinity of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 5 is A
(6c) a ZNF263 gene or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 6 is G
(7c) A site on the TNFA gene or a nearby DNA region of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 7 is A
(8c) a THBS3 gene or a site on a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 8 is C
(9c) The ABCA1 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 17283 in the nucleotide sequence set forth in SEQ ID NO: 9 is T
(10c) The SERPINB10 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 10 is G
(11c) A region on the ALDOB gene or a DNA region in the vicinity of the ALDOB gene, wherein the base species at position 10001 in the nucleotide sequence set forth in SEQ ID NO: 11 is T
(12c) A site on the CTSD gene or a nearby DNA region of the gene, wherein the base type at position 11990 in the base sequence described in SEQ ID NO: 12 is A, or the base type at position 16771 is C
(13c) A site on the STE gene or a nearby DNA region of the gene, wherein the base species at position 18958 in the base sequence described in SEQ ID NO: 13 is G, or the base species at position 21213 is C
(14c) the ABCC8 gene or a site on the DNA region in the vicinity of the gene, wherein the nucleotide type at position 14475 in the nucleotide sequence set forth in SEQ ID NO: 14 is A
(15c) The SERPINA3 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 15 is A
(16c) The RCV1 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 16 is T
(17c) a site on the TFRC gene or a nearby DNA region of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 17 is G
(18c) The MMP19 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 18 is A
(19c) a site on the MYL2 gene or a nearby DNA region of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 19 is T
(20c) a VLDLR gene or a site on a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence set forth in SEQ ID NO: 20 is T
(21c) a P2RX7 gene or a site on a DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 21 is C
(22c) a site on the DLG5 gene or a DNA region in the vicinity of the gene, wherein the base type at position 10001 in the base sequence described in SEQ ID NO: 22 is G, or the base type at position 37583 is C
(23c) a region on the LDLC gene or a nearby DNA region of the gene, wherein the base type at position 18563 in the base sequence set forth in SEQ ID NO: 23 is G
(24c) a site on the AQP8 gene or a nearby DNA region of the gene, the base species at position 10001 in the base sequence set forth in SEQ ID NO: 24 is G
(25c) a site on the LARGE gene or a nearby DNA region of the gene, wherein the base type at position 10001 is T, the base type at position 13103 is G, or the base at position 15448 in the base sequence described in SEQ ID NO: 25 The seed is G
(26c) a BLZF1 gene or a site on a DNA region in the vicinity of the gene, wherein the nucleotide type at position 20031 in the nucleotide sequence set forth in SEQ ID NO: 26 is C
(27c) the RGS5 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 27 is G
(28c) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, wherein the base type at position 17020 in the base sequence set forth in SEQ ID NO: 28 is G
(28c-2) The FRAP1 gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 30 is G
(29c) the LEP gene or a site on the DNA region in the vicinity of the gene, wherein the base species at position 10503 in the base sequence set forth in SEQ ID NO: 29 is G A method for testing type 2 diabetes, comprising the following steps (a) and (b).
(A) a step of determining a base type for a polymorphic site on the gene according to any one of the following (1) to (14) or a DNA region adjacent to the gene in a subject;
(1) MET, (2) NPHS1, (3) SERPINB2, (4) ABCA1, (5) CTSD, (6) STE, (7) ABCC8, (8) MYL2, (9) DLG5, (10) LDLC, (11) LARGE, (12) BLZF1, (13) FRAP1, (14) LEP
(B) The base species determined in the step (a) is the base of the polymorphic site in the gene showing the haplotype according to any one of the following (1 ′) to (14 ′) or a DNA region in the vicinity of the gene (1 ′) polymorphic sites on the MET gene, the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the base sequence set forth in SEQ ID NO: 1, Haplotypes that are G, T, and T,
A haplotype that is a polymorphic site on the MET gene, wherein the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the nucleotide sequence set forth in SEQ ID NO: 1 are G, C, and C, respectively Or
A haplotype (2 ′) NPHS1 gene on the MET gene, wherein the base types of the polymorphic sites at positions 19768 and 24209 in the nucleotide sequence set forth in SEQ ID NO: 1 are T and T, respectively. A haplotype that is a polymorphic site, wherein the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2 are A, C, and A, respectively
A haplotype that is a polymorphic site on the NPHS1 gene, wherein the base types of the polymorphic sites at positions 10001, 16198, and 17848 in the base sequence set forth in SEQ ID NO: 2 are G, G, and G, respectively Or
A haplotype that is a polymorphic site on the NPHS1 gene and whose base types of the polymorphic sites at positions 10001, 16198, and 17848 in the nucleotide sequence set forth in SEQ ID NO: 2 are G, C, and G, respectively ( 3 ′) a haplotype that is a polymorphic site on the SERPINB2 gene, wherein the base species of the polymorphic sites at positions 10001 and 24578 in the base sequence set forth in SEQ ID NO: 4 are C and A, or
Haplotype (4 ′) on the ABCA1 gene, wherein the polymorphic sites on the SERPINB2 gene, the base types of the polymorphic sites at positions 10001 and 24578 in the nucleotide sequence set forth in SEQ ID NO: 4 are T and T, respectively. Of the polymorphic sites at positions 10001, 15824, 17283, 20515, and 28564 in the base sequence set forth in SEQ ID NO: 9, respectively, C, A, C, T And a haplotype that is G, or
Haplotype (5 ′) on the CTSD gene, which is a polymorphic site on the ABCA1 gene and the base types of the polymorphic sites at positions 10001 and 11000 in the nucleotide sequence set forth in SEQ ID NO: 71 are G and C, respectively. A haplotype that is a polymorphic site and the base types of the polymorphic sites at positions 10001, 11990, and 16771 in the base sequence set forth in SEQ ID NO: 12 are C, G, and T, respectively (6 ′) STE The polymorphic sites on the gene, the base types of the polymorphic sites at positions 10001, 18958, 19354, 19567, and 20971 in the base sequence set forth in SEQ ID NO: 13, are A, G, and T, respectively. A haplotype that is T, C, or
Haplotype on the STE gene, wherein the base types of the polymorphic sites at positions 20971 and 21213 in the nucleotide sequence set forth in SEQ ID NO: 13 are C and C, respectively (7 ′) on ABCC8 gene A haplotype (8 ′) polymorphic site on the MYL2 gene in which the base type of the polymorphic site at position 14475 in the base sequence described in SEQ ID NO: 14 is A, Haplotype (9 ′) polymorphism on the DLG5 gene in which the base types of the polymorphic sites at positions 10001, 18386, 42589, and 83500 in the base sequence described in 19 are C, T, T, and C, respectively The base species of the polymorphic sites at positions 10001, 24016, 29469, and 37583 in the base sequence set forth in SEQ ID NO: 22 , G respectively, C, haplotype is G, and C,
A haplotype that is a polymorphic site on the DLG5 gene, wherein the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence set forth in SEQ ID NO: 22 are C, G, and C, respectively Or
A haplotype that is a polymorphic site on the DLG5 gene, wherein the base types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence set forth in SEQ ID NO: 22 are C, G, and T, respectively. 10 ′) a haplotype that is a polymorphic site on the LDLC gene, wherein the base species of the polymorphic site at positions 10001 and 18563 in the nucleotide sequence set forth in SEQ ID NO: 23 are G and A, respectively
Haplotype (11 ′) on the LARGE gene, which is a polymorphic site on the LDLC gene, wherein the base types of the polymorphic site at positions 10001 and 18563 in the nucleotide sequence set forth in SEQ ID NO: 23 are G and G, respectively. Of the polymorphic sites at positions 10001, 13103, 15448, 36690, and 43240 in the nucleotide sequence set forth in SEQ ID NO: 25, respectively, are T, G, G, T , And A haplotype (12 ′) polymorphic sites on the BLZF1 gene, wherein the base types of the polymorphic sites at positions 10001 and 20031 in the base sequence set forth in SEQ ID NO: 26 are G and T, respectively. A haplotype (13 ′) polymorphic site on the FRAP1 gene, position 17020 in the nucleotide sequence set forth in SEQ ID NO: 28 A haplotype whose base type of the polymorphic site is G (14 ′) a polymorphic site on the LEP gene, wherein the base type of the polymorphic site at positions 10001 and 10503 in the base sequence set forth in SEQ ID NO: 29 is Haplotypes that are G and A respectively A polymorphic site in the gene according to (1) to (14) of claim 7 (a) or a DNA region in the vicinity of the gene is a polymorphic site according to any of the following (1) to (14), respectively. The inspection method according to claim 7, wherein
(1) The MET gene or a site on the DNA region in the vicinity of the gene, which is the position 765, position 1383, position 1890, position 2099, position 2814, position 3293, position 3752 in the nucleotide sequence set forth in SEQ ID NO: 1. 3774, 3927, 4690, 7419, 8219, 8201, 10001, 10318, 13766, 13789, 14258, 15427, 16275, 16597, 16932, 19768, 19835, 230032 , 20240, 20537, 20885, 21546, 24209, 24271, 24376, 24642, 26028, 26226, or 34209 (2) NPHS1 gene or the gene A site on a nearby DNA region Number: 172, 186, 211, 2554, 2558, 2558, 10001, 10061, 10176, 11840, 13759, 14645, 14967, 16198, 16439 in the nucleotide sequence described in No. 2 17635, 17585, 17794, 17805, 17848, 17491, 18491, 19955, 200088, 20613, 21533, 21582, 21811, 21812, 22190, 22965, 22982 , 23660 position, 23780 position, 24040 position, 24172 position, 25334 position, or 26585 position polymorphic site (3) a region on the SERPINB2 gene or a DNA region adjacent to the gene, SEQ ID NO: 4 The nucleotide sequence described in 2853, 2891, 5218, 5317, 7560, 8054, 8771, 10001, 10329, 11781, 12063, 12705, 13125, 13691, 13913, 13916, 13317 14123, 14169, 14453, 14456, 14483, 16526, 16556, 17643, 17725, 17824, 18076, 18227, 18500, 18687, 18741, 18744, 18875 , 19045, 19141, 19149, 19190, 19429, 19548, 19567, 19624, 19645, 19814, 20049, 20082, 20097, 20243, 22 122nd, 22495th, 22516th, 22691th, 22816th, 23007th, 23904th, 24005th, 24418th, 24578th, 24855th, 24885th, 25505th, 25538th, 25564th, 25586th, 26017th , Positions 26234, 26557, 26621, 30155, 30267, or 30607 (4) ABCA1 gene or a site on the DNA region in the vicinity thereof, and SEQ ID NO: 9 993 position, 1779 position, 1813 position, 3450 position, 4287 position, 4382 position, 7270 position, 7528 position, 8383 position, 8383 position, 8798 position, 9254 position, 10001 position, 10999 position, 11122 position in the described base sequence, 11720, 12473, 12 60th, 12591th, 12664th, 12916th, 13403th, 13605th, 13691th, 14457th, 14737th, 15278th, 15345th, 15345th, 15636th, 15636th, 15824th, 16793th, 17022th 17037, 17283, 17576, 18167, 18619, 18632, 18842, 18842, 18926, 19404, 19404, 19809, 20447, 20514, 20515, 21963, 22988 , 23081, 23157, 23577, 23953, 24389, 25814, 26198, 26655, 26902, 27367, 27535, 27701, 28175, 28467 28564, 28669, 28774, 28839, 28937, 28944, 29037, 30091, 30613, 30975, 32803, 32803, 33435, 34263, 34264, 34812, 35306 A polymorphic site at any of positions 35339, 35473, 36217, 37596, or 37855, or a site on the ABCA1 gene or a nearby DNA region of the gene, the nucleotide sequence set forth in SEQ ID NO: 71 1206, 1905, 1979, 2008, 2054, 2286, 2303, 2808, 3176, 3413, 3415, 3548, 3669, 3756, 3983, 4521, 4544 Rank, 4577, 46 9th, 5226, 5833, 5833, 5422, 6422, 6532, 6583, 6696, 7043, 7198, 7324, 7395, 7484, 7716, 7787, 8197, 8350 , 9541, 9643, 9793, 10001, 10348, 10415, 10741, 10826, 11000, 11024, 11129, 11185, 11286, 11452, 11471, 11653, 11746 , 11858, 12114, 12225, 12520, 12725, 12896, 12899, 12905, 13118, 13492, 13589, 13836, 14226, 14391, 14489, 14536, 14813 , 15071, 15215, 15377, 15399, 15644, 15687, 15830, 16103, 16112, 16139, 16754, 17303, 18237, 18379, 18618, 18903, 18921 , 19014, 19034, 19074, 19149, 19165, 19167, 19365, 19373, 19376, 19386, 19487 or 19659 (5) CTSD gene or A site on the DNA region in the vicinity of the gene, and the 813, 815, 844, 861, 954, 1026, 1159, 1299, 1299, 5464 in the nucleotide sequence set forth in SEQ ID NO: 12; 5600, 7144, 770 , 7726, 7739, 7778, 7803, 7841, 7842, 7886, 7939, 7963, 8058, 8333, 9948, 10001, 11990, 12342, 12807, The polymorphic site of either position 16266 or position 16771 (6) STE gene or a site on the DNA region in the vicinity of the gene, position 57, position 468, position 1881 in the nucleotide sequence set forth in SEQ ID NO: 13 2955, 3460, 3976, 4537, 4538, 4722, 4867, 4873, 4873, 4947, 5025, 7043, 7676, 8581, 9035, 9347, 9867, 9686, 9882 , 9894, 10001, 10003, 12142, 13520, 3551, 13797, 14492, 14753, 14933, 15684, 17022, 17022, 18264, 18568, 18958, 19194, 19330, 19354, 19355, 19567, 19785, 20511 20612, 20627, 20936, 20961, 20971, 21971, 21213, 21336, 21609, 21914, or 21983, polymorphic sites (7) ABCC8 gene or a nearby DNA region of the gene It is the above site, and in the base sequence described in SEQ ID NO: 14, positions 6978, 7358, 7359, 7649, 7755, 8668, 8214, 8215, 8308, 8308, 9658, 9761, 9811, 9828, 10001, 10055, 10125, 11658, 11647, 11708, 11767, 11883, 111998, 12109, 12308, 13620, 13664, 13912, 14297, 14309, 14320 , 14323, 14367, 14393, 14475, 14591, 14722, 15294, 15601, 15657, 16160, 16230, 16367, 16417, 17695, 18213, 18815, 19093 Polymorphic site of any of the positions 20082, 20211, 20461, 21547, 211995, 22825, or 23901 (8) a site on the MYL2 gene or a DNA region adjacent to the gene, In the nucleotide sequence set forth in SEQ ID NO: 19, positions 3147, 3213, 4686, 7343, 7343, 7892, 10001, 10123, 11264, 111992, 15532, 15681, 15957, 15988, 16641 , 18386, 18782, 20354, 21531, 23245, 27740, 30669, 30670, 32077, 32732, 32907, 32955, 42589, 43029, 47354, 64777, 71869, 71961, 74274, 75551, 76343, 76344, 78270, 78339, 78689, 78743, 78806, 78807, 79215, 79319, 79357, 794 0, 79463, 80034, 80062, 80076, 80401, 80475, 80599, 80715, 80865, 80943, 80952, 80974, 80991, 81130, 81373, 81428 , 81543, 81682, 82422, 82545, 82729, 83929, 83265, 83266, 83394, 83463, 83500, 83578, 83612, 83650, 84790, 84921, 84945 , 85214, 85383, 85384, 85550, 85984, 86109, 86539, 86542, 87169, 87170, 87353, 87671, 87953, 87954, 8 Polymorphic site at any of positions 8057, 88178, 88240, 88258, 88271, 88272, 88307, 88512, 88695, 88778, 88798, 89124, or 91081 (9) DLG5 A site on a gene or a DNA region in the vicinity of the gene, and the 2nd, 121st, 1438th, 1669th, 2180th, 2436th, 2543th, 2619th, 3715 in the nucleotide sequence set forth in SEQ ID NO: 22 , 3727, 5256, 5335, 5339, 5688, 5752, 6304, 6627, 7195, 7342, 7603, 8273, 8905, 8988, 9013, 9057, 10001, 10372, 10453, 10468, 104 8th, 10512th, 11038th, 11055th, 11055th, 11597th, 11681th, 11719th, 11922th, 12034th, 12049th, 12176th, 12554th, 13481th, 13864th, 14390th, 15044th, 15598th , 16176, 16870, 17475, 17649, 18077, 18585, 18619, 18677, 19222, 19731, 19750, 20758, 20962, 21073, 21680, 21867, 22385 , 22874, 23191, 24016, 24520, 24993, 25460, 26155, 26804, 26980, 27980, 29234, 29469, 30254, 30742, 0742, 30756, 30756, 31671, 31874, 31940, 32626, 33393, 33581, 34240, 35294, 35666, 36587, 36653, 36941, 37408, 37583 Any of the polymorphic sites at positions 37612, 38462, 38470, 38485, 39905, 39146, 40612, 40641, 41242, 42217, 42539, 45934, 46033, or 46895 (10) LDLC gene or a site on the DNA region in the vicinity of the gene, and in the nucleotide sequence set forth in SEQ ID NO: 23, positions 1580, 1754, 1882, 3062, 3507, 4507, 4800, 4905, 5569, 56 08, 5643, 5665, 5663, 5864, 5884, 5981, 6169, 6808, 6858, 7194, 7283, 7728, 7835, 8049, 8049, 8249, 8265 , 9209, 9445, 9646, 9647, 9716, 9775, 10001, 10001, 10027, 10054, 10193, 10208, 10826, 10908, 10997, 11336, 11754, 11895 , 12031, 12327, 12464, 12561, 14051, 15366, 15778, 17723, 17772, 17783, 17811, 17815, 17862, 17900, 17962, 18184, 18189, 1 297, 18411, 18512, 18563, 19125, 19152, 190092, 210092, 21227, 21506, 21617, 21855, 23090, 25700, 26468, 26638, 27682, 27755 Any of polymorphic sites at positions 27888, 27973, 27995, 28123, 28127, 28144, 28153, or 28271 (11) a site on the LARGE gene or a nearby DNA region of the gene, 1816 position, 3116 position, 3117 position, 3118 position, 3154 position, 3157 position, 3191 position, 3197 position, 3205 position, 3291 position, 3621 position, 3622 position, 3652 position, 3792 position, 4488, 5076, 5 74, 5481, 5492, 5503, 5654, 5672, 6563, 6843, 6908, 7119, 7538, 7538, 7721, 8403, 8626, 10001, 12636 , 13103, 14794, 15370, 15448, 15599, 15765, 16045, 16261, 16277, 16661, 17360, 17611, 17714, 17809, 18221, 18221, 18376 , 18536, 19063, 22942, 23404, 23612, 25125, 25134, 2566, 25705, 25751, 27407, 28166, 28567, 30302, 30316, 30353, 30354, 30391, 33594, 33650, 33754, 36690, 37753, 37895, 37900, 39104, 39745, 41404, 41646, 41711, 42020, 42080, 42974 43052, 43240, 43621, 44734, 44958, 46920, 47431, 47573, 48101, 48127, 48306, 48807, 49807, 49848, 49860, 49903, 50114 Any of polymorphic sites (12) BLZF1 gene or a nearby DNA region of the gene at position 50223, 5257, 52543, 52893, or 53061, which is described in SEQ ID NO: 26 1283 position, 4288 position, 7520 position, 7599 position, 7740 position, 8745 position, 9050 position, 9862 position, 10001 position, 10124 position, 10168 position, 10339 position, 11017 position, 13737 position, 13747 position, 18354 position Polymorphic site at any of positions 20031, 20172, 22094, 24082, 24305, 24478, 24552, 25273, 25351, 29596, 29100, 2964 (13) FRAP1 gene or the It is a site on the DNA region in the vicinity of the gene, and the positions 3455, 7826, 8715, 8783, 9042, 9390, 9390, 9462, 9709, 10001 and 10635 in the nucleotide sequence set forth in SEQ ID NO: 28 Rank, 10748, 127 A polymorphic site of any of positions 4, 12849, 12915, 13169, 17020, 18586, 21123, or 21128 (14) a site on the LEP gene or a nearby DNA region of the gene, In the base sequence described in SEQ ID NO: 29, positions 244, 575, 675, 1487, 1644, 2343, 2715, 2763, 3098, 3589, 3880, 5787, 5865, 6436 , 7392, 7995, 8161, 8544, 9230, 9795, 10001, 10199, 10503, 11975, 14981, 15681, or 19300 The polymorphic site in the gene according to (1) to (14) of claim 7 (a) is the polymorphic site according to any of the following (1) to (14), The inspection method described.
(1) A polymorphic site on the MET gene, which is a polymorphic site at positions 10001, 14258, 19,768, or 24209 in the nucleotide sequence set forth in SEQ ID NO: 1. (2) Polymorphic site on the NPHS1 gene A polymorphic site at positions 10001, 16198 or 17848 in the base sequence described in SEQ ID NO: 2 (3) a polymorphic site on the SERPINB2 gene, in the base sequence described in SEQ ID NO: 4 A polymorphic site at position 10001 or 24578 (4) is a polymorphic site on ABCA1 gene, and is located at positions 10001, 15824, 17283, 20515, or 28564 in the nucleotide sequence set forth in SEQ ID NO: 9. A polymorphic site or a polymorphic site on the ABCA1 gene, which is 10001 in the nucleotide sequence set forth in SEQ ID NO: 71 And polymorphic site at position 11000 (5) polymorphic site on CTSD gene, polymorphic site at positions 10001, 11990, or 16771 in the nucleotide sequence set forth in SEQ ID NO: 12 (6) STE gene The polymorphic site above, which is the polymorphic site at positions 10001, 18958, 19354, 19567, 20971, or 21213 in the nucleotide sequence set forth in SEQ ID NO: 13 (7) Polymorphism on ABCC8 gene A polymorphic site at position 10001 or 14475 in the nucleotide sequence set forth in SEQ ID NO: 14, or a polymorphic site on the MYL2 gene, which is located at 10001 in the nucleotide sequence set forth in SEQ ID NO: 19 Polymorphic site at position # 18386, 42589, or 83500 (9) a polymorphic site on the DLG5 gene, SEQ ID NO: Polymorphic site at position 10001, 24016, 29469, or 37583 in the base sequence described in 22, (10) polymorphic site on the LDLC gene, position 10001 in the base sequence described in SEQ ID NO: 23, Or polymorphic site at position 18563 (11) polymorphic site on LARGE gene, polymorphic site at positions 10001, 13103, 15448, 36690, or 43240 in the nucleotide sequence set forth in SEQ ID NO: 25 (12) a polymorphic site on the BLZF1 gene, the polymorphic site at position 10001 or 20031 in the base sequence described in SEQ ID NO: 26, and (13) a polymorphic site on the FRAP1 gene, which is SEQ ID NO: : Polymorphic site at position 10001 or 17020 in the nucleotide sequence described in 28 (14) on the LEP gene A mold part, SEQ ID NO: 29 10001 of the nucleotide sequence set forth in or 10503 position polymorphic sites Furthermore, the test | inspection method in any one of Claims 3-9 including the process of preparing DNA containing a polymorphic region from the biological sample of a subject. A diagnostic agent for type 2 diabetes, comprising an oligonucleotide having a chain length of at least 15 nucleotides, which hybridizes to the DNA comprising the polymorphic site according to any one of (1a) to (29a) of claim 4. A diagnostic agent for type 2 diabetes comprising a solid phase to which a nucleotide probe that hybridizes with the DNA containing the polymorphic site according to any one of (1a) to (29a) of claim 4 is fixed. A test agent for type 2 diabetes comprising a primer oligonucleotide for amplifying a DNA comprising the polymorphic site according to any one of (1a) to (29a) of claim 4.

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