JP6614630B2 - 肝細胞癌のリスク評価方法 - Google Patents
肝細胞癌のリスク評価方法 Download PDFInfo
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- JP6614630B2 JP6614630B2 JP2019528942A JP2019528942A JP6614630B2 JP 6614630 B2 JP6614630 B2 JP 6614630B2 JP 2019528942 A JP2019528942 A JP 2019528942A JP 2019528942 A JP2019528942 A JP 2019528942A JP 6614630 B2 JP6614630 B2 JP 6614630B2
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- QOHMWDJIBGVPIF-UHFFFAOYSA-N n',n'-diethylpropane-1,3-diamine Chemical compound CCN(CC)CCCN QOHMWDJIBGVPIF-UHFFFAOYSA-N 0.000 description 1
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- NYDMYYLGAUCDGH-UHFFFAOYSA-N n-methyl-n'-(methylaminomethyl)methanediamine Chemical compound CNCNCNC NYDMYYLGAUCDGH-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- DBSDMAPJGHBWAL-UHFFFAOYSA-N penta-1,4-dien-3-ylbenzene Chemical compound C=CC(C=C)C1=CC=CC=C1 DBSDMAPJGHBWAL-UHFFFAOYSA-N 0.000 description 1
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- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
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- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 229910001487 potassium perchlorate Inorganic materials 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
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- 230000002441 reversible effect Effects 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 1
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
- 229940074404 sodium succinate Drugs 0.000 description 1
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000005208 trialkylammonium group Chemical group 0.000 description 1
- RJNGNWBDDLDAAP-UHFFFAOYSA-N triethyl-[2-(prop-2-enoylamino)ethyl]azanium;chloride Chemical compound [Cl-].CC[N+](CC)(CC)CCNC(=O)C=C RJNGNWBDDLDAAP-UHFFFAOYSA-N 0.000 description 1
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 1
- CCVMLEHYQVSFOM-UHFFFAOYSA-N trimethyl-[2-(prop-2-enoylamino)ethyl]azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CCNC(=O)C=C CCVMLEHYQVSFOM-UHFFFAOYSA-N 0.000 description 1
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical group CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
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Description
〔1〕肝細胞癌のリスクを評価する方法であって:
(1)亜硫酸水素塩処理された、被験体の肝臓組織由来のDNAを増幅する工程であって、該DNAがMGRN1遺伝子のエクソン領域のCpGサイトを含む、工程;
(2)得られた増幅産物をイオン交換クロマトグラフィーにかける工程;
(3)該クロマトグラフィーの検出シグナルのピークの形状に基づいて、該DNAが肝細胞癌の発症リスクが高い被験体から得られたDNAであるか否かを判定する工程、
を含む、方法。
〔2〕肝細胞癌のリスクを評価する方法であって:
(1)亜硫酸水素塩処理された、被験体の肝臓組織由来のDNAを増幅する工程であって、該DNAがMGRN1遺伝子のエクソン領域のCpGサイトを含む、工程;
(2)得られた増幅産物をイオン交換クロマトグラフィーにかける工程;
(3)該クロマトグラフィーの検出シグナルのピークの形状に基づいて、該被験体の肝細胞癌の発症リスクが高いか否かを判定する工程、
を含む方法。
〔3〕前記DNAが、配列番号1で示されるヌクレオチド配列、又は当該配列と少なくとも95%の同一性を有するヌクレオチド配列からなるDNAを含む、〔1〕又は〔2〕記載の方法。
〔4〕前記(3)において、前記検出シグナルのピークの形状が一峰のメチル化DNAのピークである場合は、該DNAを、肝細胞癌の発症リスクが高い被験体から得られたDNAとして選択し、前記検出シグナルのピークの形状が二峰性である場合は、該DNAを、肝細胞癌の発症リスクが低い被験体から得られたDNAとして選択する、〔1〕〜〔3〕のいずれか1項記載の方法。
〔5〕前記(3)が、前記検出シグナルのピークの保持時間を陽性対照又は陰性対照の検出シグナルのピークの保持時間と比較することで、前記メチル化DNAのピークが取得されたことを確認することを含み、
該陽性対照の検出シグナルが、前記被験体の肝臓組織由来のDNAと同じ配列からなりかつ100%メチル化しているDNAが亜硫酸水素塩処理及び増幅されたときに得られるDNAを、イオン交換クロマトグラフィーにかけることによって得られたものであり、
該陰性対照の検出シグナルが、該被験体の肝臓組織由来のDNAと同じ配列からなりかつメチル化していないDNAが亜硫酸水素塩処理及び増幅されたときに得られるDNAを、イオン交換クロマトグラフィーにかけることによって得られたものである、
〔4〕記載の方法。
〔6〕前記イオン交換クロマトグラフィーがアニオン交換クロマトグラフィーである、〔1〕〜〔5〕のいずれか1項記載の方法。
(1)亜硫酸水素塩処理された、被験体の肝臓組織由来のDNAを増幅する工程であって、該DNAがMGRN1遺伝子のエクソン領域のCpGサイトを含む、工程;
(2)得られた増幅産物をイオン交換クロマトグラフィーにかける工程;
(3)該クロマトグラフィーの検出シグナルのピークの形状に基づいて、該DNAがHCCの発症リスクが高い被験体から得られたDNAであるか否かを判定する工程。
(1)亜硫酸水素塩処理された、被験体の肝臓組織由来のDNAを増幅する工程であって、該DNAがMGRN1遺伝子のエクソン領域のCpGサイトを含む、工程;
(2)得られた増幅産物をイオン交換クロマトグラフィーにかける工程;
(3)該クロマトグラフィーの検出シグナルのピークの形状に基づいて、該被験体がHCCの発症リスクが高い被験体であるか否かを判定する工程。
(1)患者及び検体
肝炎ウィルス(HBV又はHCV)感染歴があり、HCCを有する患者から摘出された非がん肝臓組織(N群)36サンプル、ならびに肝炎ウィルス感染歴及びHCCを有さない患者から摘出された健常肝臓組織(C群)36サンプルを検体とした。
前記患者から得た新鮮凍結組織サンプルを、フェノール−クロロホルムにて処理し、次いで透析を施すことによって、高分子量DNAを抽出した(Sambrook,J.ら、モレキュラークローニング:実験マニュアル 第3版、コールドスプリングハーバー出版、NY、6.14〜6.15ページ 参照)。得られたDNA500ngを、EZ DNA Methylation−GoldTMキット(Zymo Research社製)を用い、亜硫酸水素塩処理に供した。
(2)で得られた亜硫酸水素塩処理ゲノムDNAをPCR増幅した。PCRでは、ゲノムDNA中のCpGサイトを含む以下の3つの異なる領域を増幅した。
Liv25領域:MGRN1遺伝子のエクソン領域のCpGサイトを含むDNA領域
Liv27領域:Liv25領域の近傍のCpGサイト(MGRN1遺伝子のイントロン領域のCpGサイト)を含むDNA領域
Liv28領域:異なる遺伝子(KDM4B)のCpGサイトを含むDNA領域
増幅された領域(亜硫酸水素塩処理前の配列)及び増幅に用いたプライマーのヌクレオチド配列を表1に示す。それぞれの領域について、同じ配列でメチル化率0%のDNA(陰性対照)及びメチル化率100%のDNA(陽性対照)を調製し、同様の手順で亜硫酸水素塩処理の後PCR増幅した。
Liv25:
[94℃1分→64℃1分→72℃1分]×5サイクル→[94℃1分→62℃1分→72℃1分]×5サイクル→[94℃1分→60℃1分→72℃1分]×5サイクル→[94℃1分→58℃1分→72℃1分]×35サイクル
Liv27:
[94℃1分→56℃1分→72℃1分]×50サイクル
Liv28:
[94℃1分→56℃1分→72℃1分]×50サイクル
PCR終了後、予めethidium bromideを添加した3%アガロースゲルに、反応液5μLにloading dye solution 1μLを混ぜたものをアプライして電気泳動し、目的のPCR増幅産物が得られたことを確認した。
攪拌機付き反応器中の3重量%ポリビニルアルコール(日本合成化学社製)水溶液2000mLに、テトラエチレングリコールジメタアクリレート(新中村化学工業社製)200g、トリエチレングリコールジメタアクリレート(新中村化学工業社製)100g、グリシジルメタクリレート(和光純薬工業社製)100g及び過酸化ベンゾイル(キシダ化学社製)1.0gの混合物を添加した。攪拌しながら加熱し、窒素雰囲気下にて80℃で1時間重合した。次に、強カチオン性基を有する親水性単量体として、メタクリル酸エチルトリメチルアンモニウムクロリド(和光純薬工業社製)100gをイオン交換水に溶解した。これを同じ反応器に添加して、同様にして、攪拌しながら窒素雰囲気下にて80℃で2時間重合した。得られた重合組成物を水及びアセトンで洗浄することにより、4級アンモニウム基を有する親水性重合体の層を表面に有する被覆重合体粒子を得た。得られた被覆重合体粒子について、粒度分布測定装置(AccuSizer780/Particle Sizing Systems社製)を用いて測定したところ、平均粒子径は10μmであった。
(4)で準備したアニオン交換カラムを用いて、以下の条件でイオン交換クロマトグラフィーを行い、(3)で得られた各PCR増幅産物を分離検出した。
システム:LC−20Aシリーズ(島津製作所社製)
溶離液:溶離液A 25mmol/Lトリス塩酸緩衝液(pH7.5)
溶離液B 25mmol/Lトリス塩酸緩衝液(pH7.5)
+1mol/L硫酸アンモニウム
分析時間:分析時間は15分
溶出法:以下のグラジエント条件により溶離液Bの混合比率を直線的に増加させた。
0分(溶離液B40%)→10分(溶離液B100%)
検体:(2)で得られたPCR増幅産物
流速:1.0mL/min
検出波長:260nm
試料注入量:5μL、陰性対照及び陽性対照は2μL
カラム温度:70℃
HCC患者由来非がん肝臓組織サンプル(N群)のMGRN1遺伝子のエクソン領域のCpGサイトを含むDNA(Liv25領域)から得られたHPLCクロマトグラムのうち、典型的な例を図1に示す。図1の各図には、非メチル化DNA(陰性対照)及び100%メチル化DNA(陽性対照)のクロマトグラムも重ねて表示されている。これらのN群サンプルでは、陽性対照のピークと重なる一峰性のピークのみが出現していた。またこれらのデータの一次微分値(図2)は、1つの極大値を有する曲線であった。クロマトグラムでピークに肩がでるなど、一峰でないサンプルは、一次微分値が2つの極大値を有する曲線となることで判別することができた(図示せず)。
Liv27領域から得られたHPLCクロマトグラムも、Liv25領域と同様に、N群では一峰のピーク、C群では二峰性のピークを有する傾向がみられた。他方、Liv28領域から得られたHPLCクロマトグラムは、N群では二峰性ピーク、C群では一峰のピークを有する傾向がみられた。しかしながら、Liv27領域及びLiv28領域では、N群、C群のいずれにも上記傾向と一致しないピークが得られる場合が少なくなく、ピークの形状だけではHCC患者の組織と健常組織とを精度よく区別できないことが示唆された。
Liv25領域、Liv27領域及びLiv28領域のクロマトグラムを用いてHCCリスク評価を行い、その感度(陽性一致率)及び特異性(陰性一致率)を算出した。Liv25及びLiv27領域では、一峰のピークを有するクロマトグラムが得られたサンプルをHCCリスク陽性、二峰性のピークを有するクロマトグラムが得られたサンプルをHCCリスク陰性と判定した。Liv28領域では、二峰性のピークを有するクロマトグラムが得られたサンプルをHCCリスク陽性、一峰のピークを有するクロマトグラムが得られたサンプルをHCCリスク陰性と判定した。各領域についてのN群とC群におけるHCCリスク陽性及び陰性と判定されたサンプル数、ならびに評価の感度及び特異性を表2に示す。Liv25領域での評価は、感度及び特異性ともに非常に高く、他の領域と比較して顕著に高精度であった。
Claims (5)
- 肝細胞癌のリスクを評価する方法であって:
(1)亜硫酸水素塩処理された、被験体の肝臓組織由来のDNAを増幅する工程であって、該DNAが、配列番号1で示されるヌクレオチド配列又は当該配列と少なくとも95%の同一性を有するヌクレオチド配列からなる、MGRN1遺伝子のエクソン領域のCpGサイトを含むDNAである、工程;
(2)得られた増幅産物をアニオン交換クロマトグラフィーにかける工程;
(3)該クロマトグラフィーの検出シグナルのピークの形状に基づいて、該DNAが肝細胞癌の発症リスクが高い被験体から得られたDNAであるか否かを判定する工程、
を含む、方法。 - 肝細胞癌のリスクを評価する方法であって:
(1)亜硫酸水素塩処理された、被験体の肝臓組織由来のDNAを増幅する工程であって、該DNAが、配列番号1で示されるヌクレオチド配列又は当該配列と少なくとも95%の同一性を有するヌクレオチド配列からなる、MGRN1遺伝子のエクソン領域のCpGサイトを含むDNAである、工程;
(2)得られた増幅産物をアニオン交換クロマトグラフィーにかける工程;
(3)該クロマトグラフィーの検出シグナルのピークの形状に基づいて、該被験体の肝細胞癌の発症リスクが高いか否かを判定する工程、
を含む方法。 - 前記DNAが、配列番号1で示されるヌクレオチド配列、又は当該配列と少なくとも98%の同一性を有するヌクレオチド配列からなるDNAである、請求項1又は2記載の方法。
- 前記(3)において、前記検出シグナルのピークの形状が一峰のメチル化DNAのピークである場合は、該DNAを、肝細胞癌の発症リスクが高い被験体から得られたDNAとして選択し、前記検出シグナルのピークの形状が二峰性である場合は、該DNAを、肝細胞癌の発症リスクが低い被験体から得られたDNAとして選択する、請求項1〜3のいずれか1項記載の方法。
- 前記(3)が、前記検出シグナルのピークの保持時間を陽性対照又は陰性対照の検出シグナルのピークの保持時間と比較することで、前記メチル化DNAのピークが取得されたことを確認することを含み、
該陽性対照の検出シグナルが、前記被験体の肝臓組織由来のDNAと同じ配列からなりかつ100%メチル化しているDNAが亜硫酸水素塩処理及び増幅されたときに得られるDNAを、アニオン交換クロマトグラフィーにかけることによって得られたものであり、
該陰性対照の検出シグナルが、該被験体の肝臓組織由来のDNAと同じ配列からなりかつメチル化していないDNAが亜硫酸水素塩処理及び増幅されたときに得られるDNAを、アニオン交換クロマトグラフィーにかけることによって得られたものである、
請求項4記載の方法。
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