JP6103691B2 - 真性赤血球増加症に関与するjak2の変異の識別方法 - Google Patents
真性赤血球増加症に関与するjak2の変異の識別方法 Download PDFInfo
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Description
第三の特徴として、本発明は、骨髄増殖症候群に冒された、あるいは骨髄増殖症候群を示す可能性のある哺乳動物において、とりわけ赤血球増加症を示す患者または真性赤血球増加症、血小板増加症および/もしくは骨髄線維症の兆候を示している疑いのある患者において、JAK2 V617F変異の有無を検出することを可能にする診断手段を目的としている。
JAK2エクソン12−PCRF センス 5’−GGGTTTCCTCAGAACGTTGA−3’(54804−54823)(SEQ ID No5)
JAK2エクソン12−PCRR アンチセンス 5’−TTGCTTTCCTTTTTCACAAGA−3’(55240−55260)(SEQ ID No6)
JAK2エクソン12SEQF センス 5’CAGAACGTTGATGGCAGTTG−3’(54813−54832)(SEQ ID No7)
JAK2エクソン12SEQR アンチセンス 5’TGAATAGTCCTACAGTGTTTTCAGTTT−3’(55207−55233)(SEQ ID No8)
センス 5’−CAACCTCAGTGGGACAAAGAA−3’(1386−1407)(SEQ ID No9)
アンチセンス 5’−GCAGAATATTTTTGGCACATACA−3’(2019−2041)(SEQ ID No10)
TTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAATATTC(SEQ ID No11)
オリゴ「S」(センス) GGCAGAGAGAATTTTCTGAAC(SEQ ID No15)
オリゴ「R」(アンチセンス) GCTTTCCTTTTTCACAAGATA(SEQ ID No16)
センサーwt GTCTCCACAGACACATACTCCATAA3’−FL(SEQ ID No17)
アンカーJAK2 5’−LC Red640AAAACCAAATGCTTGTGAGAAAGCT3’−PH(SEQ ID No18)
cJAK2F GCACACAGAAACTATTCAGAGTC(SEQ ID No19)
cJAK2S AGCAGCAAGTATGATGAGC(SEQ ID No20)
cJAK2A CTAGTGATCCAAATTTTACAAACT(SEQ ID No21)
cJAK2R GTTTAGCAACTTCAAGTTTCC(SEQ ID No22)
センサーwt GTCTCCACAGACACATACTCCATAA3’−FL(SEQ ID No23)
アンカーJAK2 5’−LC Red640AAAACCAAATGCTTGTGAGAAAGCT3’−PH(SEQ ID No24)
対立遺伝子に特異的な蛍光プローブおよび一本鎖DNAによる認識
PCR反応
センスプライマー配列:AAGCTTTCTCACAAGCATTTGGTTT(SEQ ID No25)
アンチセンスプライマー配列:AGAAAGGCATTAGAAAGCCTGTAGTT(SEQ ID No26)
リポーター1配列(VIC):TCTCCACAGACACATAC(SEQ ID No27)
リポーター2配列(FAM):TCCACAGAAACATAC(SEQ ID No28)
a)患者の核酸のサンプルを得ること、
b)前記核酸のサンプルにおけるJAK2遺伝子のG1849T変異体の有無を検出すること
を含んでおり、
G1849T変異体の存在がPVまたは他のあらゆる骨髄増殖症候群の指標となることを特徴としている。
a)患者のサンプルを得ること、
b)JAK2 V617F変異体の有無を検出すること
を含んでおり、
前記変異体の存在がPVまたは他のあらゆる骨髄増殖症候群の指標となることを特徴としている。
−「siSearch Program」、http://sonnhammer.cgb.ki.se/siSearch/siSearch_1.6.html(「Improved and automated prediction of effective siRNA」,Chalk AM,Wahlesdelt C,and Sonnhammer ELL,Biochemical and Biophysical Research Communications,2004)。
−「SiDirect」、http://design.rnai.jp/sidirect/index.php(Direct:highly effective,target−specific siRNA design software for mammalian RNA interference,Yuki Naito et al,Nucleic Acids Res,Vol.32,No.Web Server issue,Oxford University Press(著作権),2004)。
−「siRNA Target Finder」、Ambion、http://www.ambion.com/techlib/misc/siRNA_tools.html。
−「siRNA design tool」、Whitehead Institute of Biomedical Research、MIT、http://jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/。
http://web.mit.edu/mmcmanus/www/home1.2files/siRNAs.htm、とりわけ、
http://athena.bioc.uvic.ca/cgi−bin/emboss.pl?_action=input&_app=sirna。
−UGGAGUAUGUUUCUGUGGA(SEQ ID No29)、
−GGAGUAUGUUUCUGUGGAG(SEQ ID No30)、
−GAGUAUGUUUCUGUGGAGA(SEQ ID No31)、
から構成されるグループから選択することができる。
平常時では、JAK2はリン酸化していない状態でボックス1に固定されている。Epoとの結合によって受容体の立体構造が変化し、JAK2のリン酸転移が可能となり、それを受けてJAK2はEpo−Rの細胞質内残基をリン酸化し、シグナル伝達のさまざまな正のエフェクター(→)または負のエフェクター(┤)を動員する。
2A:Epo、SCFおよびIL−3を用いた培養。
2B:Epoの不在下での培養。
5A:36+/gpa−のクローニング能力の減少。1〜6日目にSCF−IL3において培養し、5日目にエレクトロポレーションを行い、6日目に選別する(形態/36+/gpa−)。
メチルSCFのみ。13日目(選別後7日目)に計数を行う。
5B:V617F変異を有するJAK2の構造(エクソン12)。
AおよびB:FRETハイブリダイゼーションプローブを用いたLightCycler(登録商標)の融解曲線分析による変異の検出。A:TF−1のDNAにおけるさまざまな希釈度のHELのDNAを用いた実験の結果が示されている。JAK2 V617Fのピーク(57℃)は1%の希釈度でも依然として検出可能である。B:代表的な患者のサンプルの結果を示している(#1:ホモ接合体、#2:ヘテロ接合体、#3:弱い、#4:変異なし)。
C:TaqMan(登録商標)を用いた対立遺伝子に特異的な増幅による変異の検出。HELのDNAの希釈物での実験(100〜1%のHEL:白抜きの四角、TF−1細胞:白抜きの丸)と代表的な患者のいくつかのサンプル(黒の十字)を示している。#a:ホモ接合体の患者、#b:ヘテロ接合体の患者、#c:弱い患者、#d:変異のない患者。
シートの各段階についての患者の数は各欄の端に記載されており(n)、ここではすべての臨床データを示す患者のみをリストしている(n=81)。一次診断試験であるJAK2 V617Fの検出の結果、81人中58人の患者において、PV型の骨髄増殖症候群を診断するためのその他の調査を避けた。
0〜6:JAK2 V617FのsiRNAで処理した(1〜6)または処理していない(0)HEL細胞。
C+:JAK2 V617F RVベクターでトランスフェクトした293HEK細胞。
C−:293HEK。
Je:WT JAK2のsiRNAで処理したK562細胞。
0〜6:JAK2 V617FのsiRNAで処理したK562細胞。
C−:293HEK(JAK2の発現なし)。
−機能的アプローチ(RNA干渉によるPV細胞におけるJAK2の阻害)、
−ゲノム的アプローチ(遺伝子の23個のエクソンのシーケンシング)、および、
−構成的活性化の原因となるJAK2のリン酸化の異常を発見するための生化学的アプローチ
という3つの補足的なアプローチによって、PVの病理におけるJAK2プロテインキナーゼの関与を研究した。
真性赤血球増加症に冒された患者の赤芽球におけるJAK2の機能の研究は、JAK2のmRNAのエクソン15に位置する配列を標的として認識する、JAK2配列に特異的なsiRNA(AMBION、Huntingdon、イギリス)を、エレクトロポレーションによってPVの赤芽球に形質導入することで行った。我々は、JAK2の阻害によって、Epoの不在下でのPV前駆細胞のクローニング能力と「自然発生的な」分化がかなり減少することを示した。JAK2のsiRNAをトランスフェクトした正常な赤芽球前駆細胞は、コントロールsiRNAと比較してクローン化能力が70%減少しており、このことは、JAK2のsiRNAによるトランスフェクションが有効であることを確証している。PVでは、赤芽球前駆細胞においてJAK2を阻害する効果がEpo不在の培養モデルにおいて研究されており、これによって悪性クローン細胞の研究が可能となっている。我々は、JAK2のsiRNAによるトランスフェクション後のPVの赤芽球前駆細胞のクローン化能力、アポトーシスおよび分化を、コントロールsiRNAの場合と比較した。Epoの不在下で培養したPV前駆細胞のクローン化能力の研究により、コントロールsiRNAの場合と比較して、JAK2のsiRNAをトランスフェクトした後にコロニーの数が明らかに減少したことが示された。これらの細胞のアポトーシスについてのフローサイトメトリー分析は、JAK2のsiRNAをトランスフェクトした細胞では、関連のないsiRNAの場合と比較してアポトーシスが顕著に増加したことを示している(70%対53%)。分化に対するJAK2のsiRNAの効果に関する研究(フローサイトメトリーで検出されたグリコフォリンAの回収)では、コントロールsiRNAとJAK2のsiRNAをトランスフェクトした前駆細胞の間ではわずかな違いを示しただけである。
23個のエクソンに対するPCRをゲノムDNAに基づいて正常な被験者に対して行った。そして、細胞培養後にインビトロで得られた赤血球細胞のゲノムDNAを抽出した後に、3人のPV患者について研究した。
・通常のコドン617:バリン(V)をコードするgtc。
・変異したコドン617:フェニルアラニン(F)をコードするttc。
・試験した43のPV中39の変異(90%)。
・2/3のヘテロ接合体。
・13/39、つまり30%のケースが「ホモ接合体」(9pでのヘテロ接合性の喪失と同じ割合)。
・試験した33人の対照中0例の変異。
−うち、15人は正常な被験者。
−18人は二次性赤血球増加症(自然発生的なコロニーなし)。
JAK2 V617Fを検出するための、シーケンシングと二つのSNP遺伝子型決定法の比較。
MPD(すなわち赤血球増加症、血小板増加症、白血球増加症)の疑いがある119のサンプルを分析した。58の血液サンプルをこのために採取し、骨髄バンクの61のサンプルを遡及的に分析した。
標準的なポジティブコントロールであるTF−1細胞系(変異なし)のDNAにおける、ホモ接合性で変異した(JAK2 V617F)ヒト赤白血病細胞系(HEL)のDNAの連続希釈物(1、0.5、0.1、0.05、0.01)を用いた。この細胞系を、ウシ胎児血清を加えたMEM−α培地(Invitrogen)中で成長させた。
JAK2のエクソン12の標的配列の増幅および該配列とのハイブリダイゼーションのために、プライマーとプローブを設計した。変異部位の位置(1849G/T)を、3’をフルオレセインで標識したドナーキャプチャープローブと、5’末端をLightCycler(登録商標)Red640(LCRed640)で標識した、隣接するアクセプターアンカープローブで覆い、該アンカープローブの3’末端は伸長を避けるためにリン酸化した。増幅および融解曲線分析はLightCycler(登録商標)の機器(Roche Diagnostics、Meylan、フランス)で実施した。最終的な反応体積は20μlであり、10ngのDNA、14μlのLightCycler FastStart DNA Master混合物、3mMのMgCl2、0.2μMのプライマー、0.075μMの各プローブが含まれている。簡単に説明すると、サンプルを95℃で10分間加熱し、45サイクル(95℃で10秒、53℃で10秒、72℃で15秒)のPCR増幅の後、95℃で10秒間の変性過程と、それぞれ30秒にわたる63℃と45℃での二回のハイブリダイゼーション過程を行い、そして融解曲線は45℃と70℃(0.1℃/秒)の間に含まれる領域にあった。LightCycler(登録商標)プログラム上での分析は、温度に対する蛍光産物の曲線[Tに対する2(dFl2/Fl1)/dT)]をプロットすることで行った。変異体およびWTのピークはそれぞれ57℃と63℃で見られた。
1849位でのSNPを含む92bpの産物を増幅するために二つのプライマーを設計した。二つのMGB蛍光プローブを、異なる色で蛍光標識して、一つはWT対立遺伝子を標的とし、もう一つは変異対立遺伝子を標的とするよう設計した。96ウェルプレート上で、TaqMan(登録商標)PCRに基づく方法を用いて遺伝子型決定を行った。最終的な反応体積は12.5μlであり、10ngのゲノムDNA、6.25μlのTaqMan(登録商標)Universal Master Mixおよび0.31μlの40×Assays−on−Demand SNP Genotyping Assay Mix(Applied Biosystems)が含まれている。プレートを95℃で10分間加熱し、その後、92℃で15秒の40回の変性サイクルと60℃で1分間の対合/伸長を行った。熱サイクルは7500 Real Time PCR System(Applied Biosystems)で行った。分析はSDSプログラムのバージョン1.3を用いて実施した。最終的な対立遺伝子の識別のための遺伝子型決定は、PCR後の蛍光読み取りに基づいて得られた変異JAK2の蛍光の曲線(Rn)に対する、WTプローブに由来するRnの目視による判定で行った。
何らかの治療を行う前に、診断時に、51%を超えるヘマトクリット値を有する88人の患者を調べ、WHO基準およびPVSG基準の有無について検査した。51%という値はヘマトクリット値の正常域の上限である(Pearson TC et al.,A Polycythemia Vera Updated:Diagnosis,Pathobiology,and Treatment.Hematology(Am.Soc.Hematol.Educ.Program.)2000:51−68)。骨髄細胞をクローン化の測定のために採取し、過剰量の細胞をDNA抽出のために採取した。血清中のエリスロポエチン(Epo)をさまざまな研究所で測定し、その結果、該エリスロポエチンが各研究所の正常域の下限値より低いときには低いとの報告を、同様にして正常あるいは高いとの報告を受けた。同じ患者に由来する末梢顆粒球を先述のように精製し、各時間における血液サンプルを利用できるようにした。骨髄サンプルおよび血液サンプルをインフォームドコンセントを得た後に採取した。
Epoに対する赤血球の応答特性のインビトロでの測定を、同一の研究所(Hotel Dieu、パリ)において、先述した凝固血漿培養法によって行った(Casadevall N,Dupuy E,Molho−Sabatier P,Tobelem G,Varet B,Mayeux P.Autoantibodies against erythropoietin in a patient with pure red−cell aplasia. N.Engl.J.Med.1996;334:630−633)。
マーカーの相関関係はスピアマンの順位相関係数(R)を用いて求めた。
JAK2 V617F変異を検出するためのPCRに基づく遺伝子型決定法の実行可能性と感度
JAK2 V617F変異を検出するためのシーケンシング、LightCycler(登録商標)法およびTaqman(登録商標)法の効率を評価するために、MPDの疑いのある患者に由来する119のサンプルにおいて、3つの方法を平行して利用して該変異の存在を調査した。JAK2 V617F変異は119サンプル中83サンプルで効果的に検出され、35サンプルは3つの方法のいずれによっても変異を示さなかった。1サンプルのみにおいて、シーケンシングではLightCycler(登録商標)とTaqman(登録商標)の二つの方法で明らかになった変異を検出できなかったことから、後者の方法がより感度が良いことが示唆される。
診断時に51%を超えるヘマトクリット値を有する88人の患者の主要な特徴を表1にまとめた。
PVSG基準によりPVとして診断された45人の患者のうち43人(96%)でJAK2 V617Fが存在し、WHO基準で診断された61人の患者のうち57人(93%)で存在した(表1)。しかし、PVSG分類にしたがってPVでないとして分類された29人の患者のうち8人は変異を示したのに対し、WHO分類にしたがってPVでないとされた患者19人は変異を示さなかった。これら8人の患者はPVSG基準によればIEであると考えられ、WHO基準によればPVであると考えられる。SEと診断された患者も正常な赤血球量を有する患者(「AEではない」)も変異を有していなかった。したがって、JAK2 V617Fの有無は、WHO基準を用いた場合には80人中76人の患者で(95%、R=0.879、p<0.0001)、PVSG基準を用いた場合では74人中64人の患者で(86.5%、R=0.717、p<0.0001)、PVの陽性診断に対応する。さらに、WHO基準でPVでないと診断された患者はいずれも変異を有していなかったため、JAK2 V617Fの検出は絶対的赤血球増加症においては100%の予測値を有することになる。
このように、我々は、明白な二次的赤血球増加症の場合を除き、顆粒球におけるJAK2 V617Fを感度の良い方法で検出することが赤血球増加症の診断の最初のステップである(図7)、新規なPV診断カードを提案する。このアプローチにはいくつかの利点があり、常に実行可能というわけではなく結果を検討しなくてはならないことのある、アイソトープを有する赤血球量の測定を避けることができる。また、骨髄の吸引や、時間がかかり標準化されていないEEC分析の実行を避けることもできる。これによりPVの陽性診断のコストが大きく低下するのだが、それは、51%を超えるヘマトクリット値を有しJAK2 V617Fについて陰性の患者のみが、赤血球増加症を特徴づけるために実際に行う全ての調査を受けることになるからである。赤血球増加症におけるJAK2 V617Fの検出だけでPVの診断が支持されても、骨髄の生検の実施は依然として有効であるが、それは、該生検を行うことで骨髄線維症の兆候または芽球細胞の存在を明らかにすることができ、それによりPVの白血病性形質転換が確認されるからである。しかし、骨髄の生検は、細胞遺伝学的分析とともに、随意的な研究として行われるべきものであると考えられる。
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Claims (6)
- 真性赤血球増加症患者または真性赤血球増加症、赤血球増加症、白血球増加症、血小板増加症および骨髄線維症を発症する恐れのある患者のサンプルにおいて、JAK2遺伝子のG1849T変異体(G1849Tは1849位のグアニンがチミンに置換されていることを示す)の有無を判定するためのエクスビボまたはインビトロでの方法であり、
a)患者から得た核酸サンプルにおいてJAK2遺伝子のG1849T変異体の有無を検出すること
b)配列SEQ ID No11、12、23および28から選ばれる少なくとも一つのプローブとハイブリダイゼーションさせること
を含み、
G1849T変異体の存在が真性赤血球増加症、赤血球増加症、白血球増加症、血小板増加症および骨髄線維症の指標となることを特徴とする方法。 - 真性赤血球増加症患者または真性赤血球増加症、赤血球増加症、白血球増加症、血小板増加症および骨髄線維症を発症する恐れのある患者のサンプルにおいて、JAK2遺伝子のG1849T変異体(G1849Tは1849位のグアニンがチミンに置換されていることを示す)の有無を判定するためのエクスビボまたはインビトロでの方法であり、
a)患者から得た核酸サンプルにおいてJAK2遺伝子のG1849T変異体の有無を検出すること
b)配列SEQ ID No4のJAK2のゲノムDNA、cDNA、またはmRNAとハイブリダイズする能力がある、配列SEQ ID No11、12、23および28から選ばれる少なくとも一つのプローブとハイブリダイゼーションさせること
を含み、
G1849T変異体の存在が真性赤血球増加症、赤血球増加症、白血球増加症、血小板増加症および骨髄線維症の指標となることを特徴とする方法。 - 配列SEQ ID No19と21;配列SEQ ID No19と22;配列SEQ ID No20と21;配列SEQ ID No20と22;配列SEQ ID No5と6;配列SEQ ID No15と16;および配列SEQ ID No25と26から選ばれる少なくとも一つのプライマー対を用いたPCR反応による増幅の過程を含むことを特徴とする、請求項1または2に記載の方法。
- 配列SEQ ID No4のJAK2cDNA変異体またはそれに対応するmRNAのG1849T変異を含むゲノム領域を増幅するための、配列SEQ ID No11、12、18、21、23、25および28から選ばれるオリゴヌクレオチドの使用。
- 配列SEQ ID No2のJAK2cDNA変異体またはそれに対応するmRNAのG1849T変異を含むゲノム領域を増幅するための、配列SEQ ID No5、6、15、16および26から選ばれるオリゴヌクレオチドの使用。
- 配列SEQ ID No2または3のJAK2cDNA変異体またはそれに対応するmRNAのG1849T変異を含むゲノム領域を増幅するための、配列SEQ ID No19および22から選ばれるオリゴヌクレオチドの使用。
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