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This method comprises the following step: a) identifying that the 1171st base of sequence No. 1, or the base at the corresponding position, is A.","lang":"en","source":"WIPO_FULLTEXT","data_format":"ORIGINAL"}],"fr":[{"text":"La présente invention concerne un procédé pour fournir des données permettant de diagnostiquer le risque de maladie circulatoire chez un être humain. 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心不全には、各種弁膜症、先天性心疾患~代謝性症候群、拡張型心筋症など、多種の疾患が含まれ、その原因や病態も多彩である。近年のヒトゲノム研究の成果と解析技術の向上で、心不全と直接関連する責任遺伝子および16種以上の憎悪遺伝子群がすでに報告されている(非特許文献1)。しかしながら、本発明者も発表した心不全関連遺伝子(非特許文献2)を用いて、世界でも最多数の遺伝子変異を搭載したマイクロアレイチップを用いても、日本人の心不全リスクの同定率は極めて低かった。 今回申請するSLC39A8遺伝子中の、National Center for Biotechnology Information提供の一塩基多型データベース(http://www.ncbi.nlm.nih.gov/SNP)において登録番号rs13107325で登録されている一塩基多型C1171T(即ちG1171A)は、高血圧(非特許文献3)、単純肥満(非特許文献4)、内臓脂肪型肥満を示すBMI(非特許文献5)、および血中脂質(非特許文献6)との関連が報告されている。しかし、200万以上のSNPと、23000人以上の白人と黒人の症例について米国とオランダで検討がなされたが、一塩基多型C1171Tと心不全との関連は今まで知られていなかった(非特許文献1)。 更に疾患リスクと、ミトコンドリアDNAの一塩基多型も相関することが知られており、ミトコンドリアtRNA Thr 遺伝子中の、上記一塩基多型データベースにおいて登録番号rs3094282で登録されている一塩基多型G15927Aは、家族性難聴の1症例との関連が中国から報告されている(非特許文献7)。しかし、心不全との関連は今まで知られていなかった。なお、この一塩基多型は、PhyloTree(http://PhyloTree.org)によって参照することができるミトコンドリアのハプログループの中でB5bに属する。 本発明は、ヒトの心不全を含む循環器疾患のリスクを診断するための方法の提供を目的とする。 本発明者は、上記課題を解決するため鋭意研究をおこなった結果、亜鉛/カドミウムトランスポーター遺伝子と報告されて来たSLC39A8遺伝子の一塩基多型C1171T、およびミトコンドリアtRNA Thr 遺伝子の一塩基多型G15927Aが、ヒトの心不全を含む循環器疾患のリスクと相関することを初めて見出し、本発明を完成させた。 すなわち、本発明は以下のとおりである。 [1] ヒトの循環器疾患のリスクを診断するためのデータを提供する方法であって、以下の工程: a)配列番号1の位置1171の塩基か、またはそれに相当する位置の塩基がAであることを同定すること を含む、方法。 [2] ヒトの循環器疾患のリスクを診断するためのデータを提供する方法であって、以下の工程: b)配列番号2の位置40の塩基か、またはそれに相当する位置の塩基がAであることを同定すること を含む、方法。 [3] 以下の工程: b)配列番号2の位置40の塩基か、またはそれに相当する位置の塩基がAであることを同定すること をさらに含む、[1]記載の方法。 [4] ヒトのミトコンドリアのハプログループがBである、[1]~[3]のいずれか一項記載の方法。 [5] 循環器疾患が、心不全、高血圧、脂質異常症、内臓脂肪型肥満、代謝性症候群から選択される、[1]~[4]のいずれか一項記載の方法。 [6] 循環器疾患が、心不全である、[5]記載の方法。 [7] 心不全が、肥大型心筋症、拡張型心筋症、肥大型から拡張型へ移行する心筋症、またはミトコンドリア心筋症である、[6]記載の方法。 本発明により、ヒトの心不全を含む循環器疾患のリスクを診断するためのより有用な方法が提供される。 本明細書において使用する「SLC39A8遺伝子」は、GenBankデータベース(http://www.ncbi.nlm.nih.gov/genbank/)にアクセッションナンバーNM_001135146で登録されている遺伝子を指す。この遺伝子は、亜鉛やカドミウム等の微量元素の細胞内輸送に関わると考えられて来た遺伝子である。 本発明の方法において、SLC39A8遺伝子の位置1171の塩基か、またはそれに相当する位置の塩基がAであることを同定することは、SLC39A8遺伝子が一塩基多型C1171Tを有することを意味する。一塩基多型C1171Tは、一塩基多型G1171Aと同義である。SLC39A8遺伝子の位置1171の塩基がAである塩基配列を、配列番号1に示す。 本明細書において使用する「ミトコンドリアtRNA Thr 遺伝子」とは、GenBankデータベースにアクセッションナンバーNC_012920で登録されているミトコンドリア完全ゲノムのうち、位置15888~位置15953にコードされている遺伝子を指す。ミトコンドリアtRNA Thr 遺伝子の転写産物は、スレオニンと結合するトランスファーRNAとなる。 本発明の方法において、ミトコンドリアtRNA Thr 遺伝子のミトコンドリア全体の位置15927の塩基またはそれに相当する位置の塩基がAであることを同定することは、ミトコンドリアtRNA Thr 遺伝子の一塩基多型G15927Aであることを同定することと同義である。ミトコンドリア全体の位置15927の塩基がAであるミトコンドリアtRNA Thr 遺伝子の塩基配列を配列番号2に示す。配列番号2の位置40の塩基がAであることが、一塩基多型G15927Aであることに対応する。 本明細書において、「またはそれに相当する位置の塩基」という語句は、直前に記載された遺伝子の塩基配列中の位置を意味するものであって、該塩基配列について、配列番号に記載されている配列に対するアライメントを行ったときに、該配列番号で注目する位置の塩基にフィットまたはアラインする位置を意味する。そのようなアライメントは、例えばFASTA(例えば、version 3.3t09 May 18, 2001;スコア行列(scoring matrix):blosum62;Henikoff & Henikoff (1992) Proc Natl Acad Sci USA 89:10915-10919;Pearson WR & Lipman DJ (1988) Proc Natl Acad Sci USA 85:2444-2448)、GAP、またはGAP -ENDWeight(「-ENDWeight」は、終末ギャップに対して他のギャップと同様にペナルティーを課す;スコア行列:blosum62 Henikoff & Henikoff (1992) Proc Natl Acad Sci USA 89:10915-10919;Needleman & Wunsch (1970) J Mol Biol 48:443-453)のようなコンピュータソフトウェアツールを用いて行うことができる。 一実施態様において、上記の方法の工程を組み合わせて使用することもできる。すなわち、循環器疾患のリスクを診断するためのデータを提供するための方法は、同定する工程をそれぞれ a)配列番号1の位置1171の塩基か、またはそれに相当する位置の塩基がAであることを同定すること b)配列番号2の位置40の塩基か、またはそれに相当する位置の塩基がAであることを同定すること とした場合に、 ・a) ・b) ・a)およびb) のいずれかの組み合わせを含む。 本発明において塩基を同定する方法は、当業者に周知の任意の方法を用いることができる。該方法は、例えば、限定はされないが、PCR増幅法、一塩基多型アレイチップ法、全ゲノムシーケンシング法などを挙げることができる。 本発明において同定のために用いることのできる遺伝子の供給源は、上記遺伝子またはその転写産物が含まれているものであれば限定されるものではなく、例えば、血液細胞、口腔内粘膜細胞、皮膚組織、体毛、体液などを挙げることができる。 本発明において、被験体には、注目する疾患を発症した者及び発症していない者の双方が含まれる。被検体は、好ましくはミトコンドリアのハプログループBに属し、より好ましくはB5bに属する。 ミトコンドリアのハプログループは、ミトコンドリアゲノムDNAのバリエーションによって分類される。最も一般的な分類は、PhyloTreeによって参照することができる(http://PhyloTree.org)。本発明のG15927A多型は、ハプログループB5bに属する。 本発明における循環器疾患は、これに限定されないが、心不全、高血圧、脂質異常症、内臓脂肪型肥満、2型糖尿病、代謝性症候群を含む。好ましくは、心不全は、心筋症である。心筋症には、肥大型心筋症、拡張型心筋症、肥大型から拡張型へ移行する心筋症、ミトコンドリア心筋症などが含まれる。 上記疾患は、家族性または多因子性疾患でありうる。 本明細書において使用する「リスク」とは、注目する因子が、心不全を含む循環器疾患と関連付けられるか、または循環器疾患の前兆となることが発見されていることを意味する。 本発明はまた、本発明の方法を実施するための一塩基多型アレイチップを提供する。一態様においては、チップには、SLC39A8遺伝子の一塩基多型C1171Tを検出するためのプローブおよび/またはミトコンドリアtRNA Thr 遺伝子の一塩基多型G15927Aを検出するためのプローブが少なくとも固定化されている。 本発明はまた、本発明の方法を実施するためのPCRキットを提供する。一態様においては、キットには、SLC39A8遺伝子の一塩基多型C1171Tを検出するためのプライマー対、ミトコンドリアtRNA Thr 遺伝子の一塩基多型G15927Aを検出するためのプライマー対、ポリメラーゼ、ヌクレオシド三リン酸、標識、緩衝液等が含まれる。 以下、実施例により本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 材料および方法 (1)対象 被検体として、日本人集団の心筋症または重症心不全患者75例を選択し、その血液サンプルを用いた。また、人種間差を見るために、白人の心筋症患者30例の冷凍保存サンプルも用いた。 (2)遺伝子型解析 各血液サンプルを、QIAamp DNA Mini Kit(Qiagen社、日本)を用い、製造業者のプロトコルに従って処理し、ゲノムDNAを抽出した。一塩基多型の遺伝子型の検出は、Genome-Wide Human SNP Array 6.0(Affymetrix社、米国)を用いて行った。このアレイセットは、約900000個の一塩基多型を有するチップからなる。各ゲノムDNA約250ngを、製造業者のプロトコルに従って処理した。遺伝子型判定は、GCOS1.4及びGTYPE4.1ソフトウェアパッケージを用いて解析した。遺伝子型を、DNAサンプルをサンガ―法でシーケンスすることにより確認した。 (3)ミトコンドリアDNAハプログループ解析 各血液サンプルを、mtDNAエキストラクター WBキット(Wako社、日本)を用い、製造業者のプロトコルに従って処理し、ミトコンドリアDNAを抽出した。ミトコンドリアDNAを、シーケンスして配列情報を得た。ハプロタイプは、独自に開発したアルゴリズム(Toyo-oka et al., A novel algorithm from personal genome to the pathogenic mutant causing mitochondrial cardiomyopathy, in “Genes and cardiovascular function”, eds. Ostadal et al., pp85-92, Springer, 2011)を用いて解析した。 結果 (1)一塩基多型解析 一塩基多型解析により、日本人集団75例のうち4例が、SLC39A8の1171番目の塩基がA(C1171T)であることが見出された。一方で、白人集団30例からは、この遺伝子型は検出されなかった。このC1171Tの遺伝子型は、アジア人を対象としたこれまでの一塩基多型解析(HapMap-JPT、HapMap-HCB)では見つかっていなかった(http://www.ncbi.nlm.nih.gov/projects/SNP/SNP_ref.cgi?rs=13107325を参照)。 (2)ミトコンドリアDNAハプロタイプ解析 ミトコンドリアDNAハプロタイプ解析により、C1171T遺伝子型を持つ上記4名は全て、ミトコンドリアtRNA Thr 遺伝子のミトコンドリア全体の15927番目の塩基がA(G15927A)であることが明らかになった。一方で、日本人集団の残りの71例からはこの遺伝子型は見られなかった。また、白人患者集団の30例からも、この遺伝子型は見られなかった。 (3)臨床データとの関連 C1171T遺伝子型を有する患者の臨床所見は、3例が心筋症であり、カテーテル検査を行っており、1人がエコー検査により心筋症の疑いが強いと診断されていた。これらの患者には、従来の白人でのゲノム相関解析でC1171T遺伝子型に関連すると報告された高血圧、内臓脂肪型肥満、脂質異常症や2型糖尿病等の代謝性症候群が全く認められず、血圧も正常、肥満も無く、コレステロールも正常であった。 本発明は、循環器疾患のリスクの診断のために有用である。 配列番号1:一塩基多型C1171Tを有するSLC39A8遺伝子塩基配列 配列番号2:一塩基多型G15927Aを有するミトコンドリアtRNA Thr 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(P.C.T.)","inventorModel":{"inventors":[{"name":{"value":"TOYOOKA TERUHIKO","valueNormalised":"Toyooka Teruhiko"},"inventorship":null}],"inventorships":[],"unmatchedInventorships":[],"activeUserHasInventorship":false},"simpleFamilyId":220527986,"citesPatentCount":0,"countrySpec":{"countryName":"WIPO (P.C.T.)","description":"INTERNATIONAL APPLICATION PUBLISHED WITH INTERNATIONAL Search Report","rule":"","docType":"PATENT_APPLICATION"},"pageTitle":"WO 2014/054103 A1 - Nucleic Acid Detection Method","documentTitle":"Nucleic Acid Detection Method"},"claims":{"source":"xml_claims","claims":[{"lines":[" ヒトの循環器疾患のリスクを診断するためのデータを提供する方法であって、以下の工程:
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