ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.45 No.2 (2008) pp.33-35
㧔ᣂછ⠪⺰ᢥ㧕ஜᏱੱߦ߅ߌࠆ ,#- ㆮવሶߩᄌ⇣⩲⼱ 㤗Ꮧ 㑐 ૫❱ 
The mutation of JAK2 gene in peripheral blood with normal healthy volunteers
Maki SUGAYA , Kaori SEKI 

ABSTRACT :We performed DNA sequencing of the JAK2 exon 12, related to polycythemia vera (PV),after TA-cloning in normal healthy subjects to identify clonal expansion of exon 12 mutations. We

analyzed at least 8 clones obtained from each individual and found that non-clonal nucleotide substitutions

in two normal subjects (N542S and Q534R). Since allele-specific polymerase chain reaction (PCR) isable to amplify only the limited region which contains known mutations with gain-of-function, we need toclarify the biological implications of unknown single nucleotide substitution of the JAK2 exon 12.

Keywords : polycythemia vera (PV), JAK2 exon 12, V617F, TA-cloning(Received September 29, 2008)PV ᖚ⠪߇㧡⒳㘃ߩᄌ⇣ߩ޿ߕࠇ߆ࠍ᦭ߔࠆࠊߌߢߪ㧝㧚✜ ⺰ߥ޿ߎߣ߇ᄢደᢝࠄߦࠃࠅ␜ໂߐࠇࠆߦ⥋ߞߚ(10)‫ߘޕ‬ ⌀ᕈᄙⴊ∝㧔polycythemia via, PV㧕ߪ, ㅧⴊᐙ⚦⢩ߩ⇣Ᏹߦࠃࠆ⿒ⴊ⃿, ⊕ⴊ⃿߅ࠃ߮ⴊዊ᧼ߩჇടߣ⣛⣲ࠍ઻߁∔ᖚߢ޽ࠆ‫ޕ‬ᕆᕈ㛽㜑ᕈ⊕ⴊ∛߿㛽㜑❫⛽∝ߥߤߦ⒖ⴕߔࠆߎߣ߽޽ࠆߚ߼, ⥃ᐥ਄ߩ⸻ᢿ߇㊀ⷐߥ∔ᖚߢ޽ࠆ‫ޕ‬ㄭᐕ, PV ߩᣂߒ޿ಽሶࡑ࡯ࠞ࡯ߣߒߡJanus kinase2(JAK2) ㆮવሶߩᄌ⇣߇ᵈ⋡ߐࠇߡ޿ࠆ‫ޕ‬JAK2ㆮવሶߪࡅ࠻ᨴ⦡૕ 9p24 ߦ 28 ୘ߩ exon ࠍ฽߻ 143.5 kbp ߽ߩ㐳޿ DNA ߣߒߡሽ࿷ߒ, ߘߎ߆ࠄ 1132 ୘ߩࠕࡒࡁ
㉄߆ࠄߥࠆ࠴ࡠࠪࡦࠠ࠽࡯࠯ (EC2.7.10.2) ߇⠡⸶ߐࠇࠆ‫ޕ‬JAK2ߩ617 ⇟⋡ߩࠕࡒࡁ㉄߇ Val (V) ߆ࠄ Phe (F)ߦᄌ⇣ߔࠆ V617F ᄌ⇣ߪ, WHO ߇ឭ໒ߔࠆ PV ߩ⸻ᢿၮḰߢ޽ࠆ(1)‫ߒ߆ߒޕ‬, V617F ᄌ⇣߇⏕⹺ߐࠇߥ޿ߎߢ, ᄢደᢝࠄߣᚒ‫ߪޘ‬V617F ᄌ⇣ࠍᜬߚߥ޿ PV ᖚ⠪ߦ߅ߌࠆ JAK2 exon 12 ㈩೉ࠍ⺞ᩏߒ, ᣂߒ޿ᄌ⇣ࠍⶄᢙหቯߒߚ (11)‫ޕ‬ᣂߚߦ⊒⷗ߐࠇߚᄌ⇣ߪᓥ᧪ߩᄌ⇣ߣߘߩᄌ⇣႐ᚲ߿ᄌ⇣Ⴎၮ߇ૃߡ޿ࠆ߽ߩߩ, hot spotએᄖߩᄌ⇣߽⏕⹺ߐࠇ, PV ᖚ⠪ߦ߅ߌࠆ exon 12 ᄌ⇣ߩᄙ᭽ᕈ߇␜ໂߐࠇࠆߦ⥋ߞߚ‫ޕ‬ᖚ⠪ߦ߅ߌࠆJAK2ㆮવሶᄌ⇣ߪ೎Ⓜߦ⼑ࠆ(11)ߣߒߡ, ᧄⓂߢߪ PV ᖚ⠪ߦ߅ߌࠆᣂߚߥᄌ⇣ࠍ⊒⷗ߒߚㆊ⒟ߦ߅޿ߡ, ஜᏱੱߦ߅޿ߡ⥝๧ᷓ޿ㆮવሶᄌ⇣ࠍ⊒⷗ߒߚߩߢႎ๔ߔࠆ‫ޕ‬㧞㧚ታ㛎ᣇᴺ

PV ᖚ⠪߽ዋߥ߆ࠄߕሽ࿷ߔࠆߎߣ߇᣿ࠄ߆ߦߥࠅ, ߎ ⵍ㛎⠪ࠇࠄᖚ⠪ߦ߅޿ߡ, JAK2ㆮવሶߩ exon 12 ߦ․ᓽ⊛ߥ㧠 ⵍ㛎⠪ߪᐕ㦂 21ޯ23 ᚽ, ↵ᕈ 㧞ฬ, ᅚᕈ 㧞ฬߩ㧠ฬ⒳㘃ߩᄌ⇣߇⏕⹺ߐࠇߚ(2-9)‫ࠄࠇߎޕ‬ᄌ⇣ߪexon14਄ߩߢ޽ࠆ‫⃿ⴊ⿒߽ࠇߕ޿ޕ‬, ⊕ⴊ⃿߅ࠃ߮ⴊዊ᧼ߩᢙ߇ၮᄌ⇣ߢ޽ࠆV617F ߣߣ߽ߦPV ߩಽሶࡑ࡯ࠞ࡯ߣߒߡ

Ḱ୯ౝߦ޽ࠅ, BMI ߆ࠄ್ᢿߒߡㆡᱜ૕㊀ߢ޽ࠆ‫ߚ߹ޕ‬,ቯ⌕ߔࠆߣ⠨߃ࠄࠇ, ⃻⁁ߢߪPVߩ⸻ᢿࡑ࡯ࠞ࡯ߣߒ

⚦⩶߿࠙ࠗ࡞ࠬᗵᨴ߇⹺߼ࠄࠇߕ, ኅᣖᕈߩㆮવ∛ࠍ઻ߡቯ⟵ߐࠇߡ޿ࠆ (2-9)‫ߒ߆ߒޕ‬, ߘࠇߢ߽߹ߛోߡߩࠊߥ޿‫ⵍޕ‬㛎⠪ߢ޽ࠆஜᏱੱ㧠ฬߦߪ੐೨ߦࠗࡦࡈࠜ࡯
ࡓ࠼ࠦࡦ࠮ࡦ࠻ࠍⴕߞߚᓟ, ోⴊ㧣mL ࠍណⴊߒ, એਅ*1㧦‛⾰↢๮ℂᎿቇ⑼ഥᢎ(msugaya@st.seikei.ac.jp)ߩታ㛎ߦ↪޿ߚ‫ޕ‬*2

㧦‛⾰↢๮ℂᎿቇ⑼ቇㇱ↢㧙㧙(KIWTG/WVCVKQPUQH,#-GZQPKPPQTOCNJGCNVJ[XQNWPVGGTU
Panel A shows DNA sequence traces after TA-cloning from peripheral blood. Nucleotides are indicated by capital letters. The traces reveal mutations within

the JAK2 exon 12 (indicated by arrow heads). Panel B shows the alignment of wild-type and mutant exon 12 JAK2 allele are indicated. Nucleotides areindicated by capital letters, and amino acids by bold capital letters. Normal healthy volunteers with JAK2 exon 12 mutations are listed (N-001 and N002).

Clones indicate number of mutational clone detected per clones analyzed.

 ࠬࠢ࡝࡯࠾ࡦࠣࡦᱷၮ (Q) ߆ࠄࠕ࡞ࠡ࠾ࡦᱷၮ (R) ߳ߩ⟎឵߇หቯߐ ᧃ᪳ⴊ߆ࠄ᛽಴ߒߚ DNA ࠍ㍌ဳߣߒߡ PCR-directࠇߚ (Figure 1)‫ޕ‬ߒߚ‫ޕ‬PCR ߪ Expand High Fidelityࠅ⺃⊒ߐࠇߡ߅ࠅ,sequencing ࠍⴕ޿ JAK2 ㆮવሶ㗔ၞߩ DNA ㈩೉ࠍ᳿ቯ
PLUS

PCR system (RocheDiagnositcs, Mannheim, Germany) ࠍ↪޿ߡ 95˚C ߢ 30⑽㑆, 60˚C ߢ 40 ⑽㑆, 72˚C ߢ 30 ⑽㑆ߩ෻ᔕࠍ 35 ࠨ ߎࠇࠄߩࠕࡒࡁ㉄ᄌ⇣ߪ޿ߕࠇ߽ 1 Ⴎၮߩ⟎឵ߦࠃPV ᖚ⠪ߢหቯߐࠇߚᄌ⇣ߣߪ⇣ߥࠆᄌ⇣ߢ޽ߞߚ‫ޕ‬ᖚ⠪ߦ߅ߌࠆᄌ⇣ߢߪ, ⒘ߦࠕࡒࡁ
㉄ᄌ⇣ࠍ઻ࠊߥ޿ silent mutation ߽⏕⹺ߐࠇߚ߇, ஜᏱࠗ ࠢ ࡞ ➅ ࠅ ㄰ ߒ ߚ ‫ ޕ‬ᓧ ࠄ ࠇ ߚ PCR ↥ ‛ ࠍ BigDyeੱߢ⏕⹺ߐࠇߚᄌ⇣ߪ޿ߕࠇ߽ࠕࡒࡁ㉄ᄌ⇣ࠍ⺃ዉߔࠆ

Foster city, USA) ߣ ABI PRISM 3130 Genetic Analyzer

޿ߚ߼, PCR-direct sequencing ߢߪ⊒⷗ߔࠆߎߣ߇ߢ߈

terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,missense mutation ߢ޽ߞߚ‫ࠄࠇߎޕ‬ᄌ⇣ߪሽ࿷㗫ᐲ߇ૐ(Applied Biosystems) ࠍ ↪ ޿ ߡ DNA ㈩ ೉ ࠍ ᳿ ቯ ߒ ,ߥ޿‫⎇ᧄޕ‬ⓥߢߪ TA-ࠢࡠ࡯࠾ࡦࠣᓟߦ㈩೉᳿ቯߔࠆߎ

V617F ᄌ⇣߇ߥ޿ߎߣࠍ⏕⹺ߒߚ‫ޕ‬ߣߦࠃࠅ PCR-direct sequencing ߢߪ⏕⹺ߢ߈ߥ޿㗫ᐲߩૐ޿ᄌ⇣߇⏕⹺น⢻ߣߥߞߚ‫ޕ‬ 㨀㧭ࠢࡠ࡯࠾ࡦࠣ

 PV ߦ߅ߌࠆㆮવሶᄌ⇣ߪㅧⴊᐙ⚦⢩߆ࠄᧃ᪳ⴊ߇↥ JAK2 exon 12 ߩ PCR ↥‛ࠍ pT7 Blue-2 T-ࡌࠢ࠲࡯

㧔Novagen, CA, USA㧕ߦᝌ౉ߒ, ᄢ⣺⩶ JM109 ࠍᒻ⾰ォ឵ߐߖߚ‫ޕ‬ᓧࠄࠇߚᄢ⣺⩶ࠦࡠ࠾࡯߆ࠄ colony-directPCR ߦ ࠃ ࠅ JAK2 exon 12 㗔 ၞ ࠍ Ⴧ ᏷ ߐ ߖ , BigDyeterminator v3.1 Cycle Sequencing Kit ߣ ABI PRISM 3130

↢ߐࠇࠆㆊ⒟ߢ⺃⊒ߒߡ޿ࠆߣ⠨߃ࠄࠇߡ޿ࠆ‫ޕ‬ᐙ⚦⢩߆ࠄߩಽൻߩㆊ⒟ߦ߅޿ߡ, ᣧᦼߩᄌ⇣⊒↢ߪ߶ߣࠎߤߩⴊ⃿⚦⢩߇ᄌ⇣ࠍ߽ߟߎߣࠍᗧ๧ߔࠆ‫৻ޕ‬ᣇ, ಽൻᓟᦼߩᄌ⇣⊒↢ߪዋᢙߩⴊ⃿⚦⢩߇ᄌ⇣ࠍ߽ߟߎߣࠍᗧ๧ߔࠆ‫⎇ᧄޕ‬ⓥߢ⊒⷗ߐࠇߚᄌ⇣ߪᭂ߼ߡ㗫ᐲ߇ૐ޿ߚ߼,Genetic Analyzer ࠍ↪޿ߡ DNA ㈩೉ࠍ᳿ቯߒߚ‫ޕ‬ⴊ⃿⚦⢩ߩಽൻㆊ⒟ߢߪᲧセ⊛ᓟᦼߦ߅ߌࠆᄌ⇣⊒↢ߣ⠨߃ࠄࠇࠆ‫ߚ߹ޕ‬, ㆮવሶᄌ⇣ࠍ᦭ߔࠆ⚦⢩߇ஜᏱੱߢ㧟㧚⚿ᨐߣ⠨ኤ߽ሽ࿷ߒߚߎߣߪ, ߎࠇࠄᄌ⇣ࠍ᦭ߔࠆ⚦⢩ࠍឃ㒰ߔࠆᯏ᭴߇ሽ࿷ߒߥ޿, ޽ࠆ޿ߪሽ࿷ߒߡ߽ឃ㒰⢻߇ૐ޿ߎ 㧠ฬߩஜᏱੱߦ߅ߌࠆ JAK2 ㆮવሶ exon 12 㗔ၞࠍߣࠍᗧ๧ߔࠆ‫ࠄࠇߎޕ‬ᄌ⇣ࠍ᦭ߔࠆ⚦⢩ߦಽൻߔࠆ೨㚟TA-ࠢࡠ࡯࠾ࡦࠣᓟߦ㈩೉᳿ቯߒߚߣߎࠈ, ᄌ⇣ࠍ᦭ߒߚஜᏱੱ߇㧞ฬ⏕⹺ߐࠇߚ‫৻ޕ‬ᣇߩஜᏱੱ (N-001) ߆ࠄߪ 12 ࠢࡠ࡯ࡦߩ߁ߜ 1 ࠢࡠ࡯ࡦ (8.3%) ߦ 542 ⇟⋡૕⚦⢩߇ߤߩࠃ߁ߥഀวߢሽ࿷ߔࠆߩ߆, ߹ߚ, ߘߩ೨㚟૕⚦⢩߇ߤߩ⒟ᐲߩࠢࡠ࡯ࡦࠍ↥↢ߔࠆߩ߆ߪ⥝๧ᷓ޿‫ࠄߥߗߥޕ‬, ᄌ⇣ࠍ᦭ߔࠆ೨㚟૕⚦⢩ߩഀว߇ᄙߊߥߩࠕࠬࡄ࡜ࠡࡦᱷၮ (N) ߆ࠄ࠮࡝ࡦᱷၮ (S) ߳ߩ⟎឵ࠆߎߣߢ PV ߇⊒∝ߔࠆࡕ࠺࡞ߣ, ೨㚟૕⚦⢩߆ࠄߩ࡯ࡦߩ߁ߜ 1 ࠢࡠ࡯ࡦ (10%) ߦ 534 ⇟⋡ߩࠣ࡞࠲ࡒ

࡞ߩ㧞⒳㘃ߩ⊒∝ࡕ࠺࡞߇ᗐቯߢ߈ࠆ߆ࠄߢ޽ࠅ, ࡕ࠺߇หቯߐࠇߚ‫ߚ߹ޕ‬, ઁᣇߩஜᏱੱ (N-002)ߢߪ 10 ࠢࡠࠢࡠ࡯ࡦ⚦⢩ᢙ߇ᄙߊߥࠆߎߣߢ PV ߇⊒∝ߔࠆࡕ࠺㧙㧙

CA, Tefferi A. Prevalence and clinicopathologic correlates࡞ߦࠃߞߡ᦭ലߥᴦ≮ᴺ߽⇣ߥࠆߣ⠨߃ࠄࠇࠆ‫ޕ‬of JAK2 exon 12 mutationin JAK2 V617F-negative ஜᏱੱߦ߅޿ߡ missense mutation ߇⏕⹺ߐࠇߚ߽ߩ

polycythemia vera. Leukemia 2007, 21: 1960-3.

ߩ, ߎࠇࠄᄌ⇣ߪᖚ⠪ߢ⏕⹺ߐࠇߡ޿ࠆᄌ⇣ߣߪోߊ⇣ߥࠆߚ߼, exon 12 ߩᄌ⇣߇ PV ߩ⏕ቯ⸻ᢿߢߥ޿ߎߣ

7. Martinez-Aviles L, Besses C, Alvarez-Larrán A, CervantesF, Hernández-Boluda JC, Bellosillo B. JAK2 exon 12ߪ᣿⏕ߢ޽ࠆ‫ߒ߆ߒޕ‬, ᧄ⎇ⓥߦදജߒߡ㗂޿ߚஜᏱੱmutations in patients with polycythemia vera or idiopathicߪᲧセ⊛⧯ᐕ㦂ߢ޽ࠆߚ߼, 㜞㦂⠪ߢ⊒∝ߔࠆߎߣߩᄙ޿ PV ߦട㦂ߦࠃࠅ⟜ᖚߔࠆน⢻ᕈ߽⠨߃ࠄࠇࠆ‫ࠇߎޕ‬ࠄ੍᷹߽฽߼, ᐙ⚦⢩޽ࠆ޿ߪ೨㚟૕⚦⢩ߩ PV ⸻ᢿ

erythrocytosis. Haematologica 2007, 92: 1717-8.

8. Butcher CM, Hahn U, To LB, Gecz J, Wilkins EJ, ScottHS, et al. Two novel JAK2 exon 12 mutations in JAK2߽⸻ᢿࡑ࡯ࠞ࡯߿࡝੍᷹ࠬࠢࡑ࡯ࠞ࡯ߦߥࠅᓧࠆߣ⠨߃

V617F-negative polycythemia vera patients. Leukemiaࠄࠇߚ‫੹ޕ‬ᓟ, ⸃ᨆน⢻ߥⵍ㛎⠪ᢙࠍჇ߿ߔߎߣߢ, ஜᏱ2008; 22:870-3.

ੱߦࠃࠆᄌ⇣ߣᐕ㦂ߩ㑐ଥࠍ⺞ᩏߔࠆ੍ቯߢ޽ࠆ‫ޕ‬

9. Williams DM , Kim AH, Rogers O, Spivak JL, Moliterno

AR. Phenotypic variations and new mutations in JAK2⻢ ㄉV617F-negative polycythemia vera, erythrocytosis, and

idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641-6.

 ᧄ⎇ⓥࠍㆀⴕߔࠆߦ޽ߚࠅᄢደᢝ৻㚍ᢎ᝼ ᧲੩ක⑼ᄢቇ╙㧝ౝ⑼ ߅ࠃ߮ᄢደᢝ⚐ሶಎᢎ᝼㧔᧲੩ක⑼ᄢቇ㔍

10. Ohyashiki K, Akahane D, Gotoh A, Ito Y, Tauchi T,Miyazawa K, et al. Uncontrolled thromobocytosis inᴦᕈ఺∉∔ᖚ⎇ⓥ࠮ࡦ࠲࡯㧕ࠃࠅᄙᄢߥࠆߏᢎ␜ߏ

polycythemia vera is a risk for thrombosis, regardless ofදജࠍ㗂߈߹ߒߚ‫ߦߎߎޕ‬ᗵ⻢ߩᗧࠍ⴫ߒ߹ߔ‫ޕ‬JAK2 V617F mutational status. Leukemia 2007, 21:2544-2545.

ෳ⠨ᢥ₂

1. Tefferi A, Thiele J, Orazi A, Kvasnica HM, Barbubi T,

11. Ohyashiki H. J, Hisatomi H, Shimizu S, Sugaya M,
Ohyashiki K.

Hanson CA, et al. Proposals and rationale for revision of

the World Health Organization diagnostic criteria forpress)

polycythemia ra, essential thrombocythemia, and primary
myelofibrosis:

recommendations

fromanadinternational expert panel. Blood 2007; 110: 1092-7.

Genetic Diversity of JAK2 exon 12Mutations in polycythemia. Haematologica

hoc

2. Scott LM, Tong W, Levine RL, Scott MA, Beer PA,

Stratton MR, et al. JAK2 exon 12 mutations inpolycythemia vera and idiopathic erythrocytosis. N Engl J
Med 2007, 356: 459-68.

3. Percy MJ, Scott LM, Erber WN, Harrison CN, Reilly JT,
Jones FG, Green AR, et al. The frequency of JAK2 exon

12 mutations in idiopathic erythrocytosis patients withlow serum erythropoietin levels. Haematologica 2007, 92:
1607-14.

4. Li S, Kralovics R, De Libero G, Theocharides A,

Gisslinger H, Skoda RC. Clonal heterogeneity inpolycythemia vera patients with JAK2 exon 12 andJAK2-V617F mutations. Blood 2008, 111: 3863-6.

5. Pietra D, Li S, Brisci A, Passamonti F, Rumi E,Theocharides A, et al.Somatic mutations of JAK2 exon 12

in patients with JAK2(V617F)-negative myeloproliferative
disorders. Blood, 2008, 111: 1686-9.

6. Pardanani A, Pardanani A, Lasho TL, Finke C, Hanson㧙㧙2008 (in