Y HOC VIET NAM THANG 4 - SO PAC BIET/2012
TRIEN KHAI KY THUAT ASOPCR XAC DINH DOT BIEN GEN JAK2 V617F Of BENH NHAN TANG SINH TUY MAN TJNH TAI
KHOA HUYET HOC -TRUYEN MAU BENH VIEN BACH MAI
Nguyen Thien Lu-*, Pham Quang Vinh*'**, Vo Quoc Vinh*, Nguyen Tuan Tung*, Nguyen Thi Lan Hirong*
T O M TAT:
Muc tieu nghien cu>u: Xdc dinh gen ddt bien JAK2 V617F a b#nh tSng sinh tiiy man tinh D6i tu'O'ng va phu-cng phap nghien c i i u : Nghi&n ciru mo ta e i t ngang. S i i dyng k? thu§t ASO-PCR de tim gen dpt bien JAK2 V617F a 93 b$nh nhSn roi loan tang smh tuy man tCr thang 7/2011 - 01/2012, Kk qua. 18/23 (78,2%) b$nh nh§n da h6ng cau nguyen phSt v^ 17/30 (56,6%) b$nh nhSn t§ng tieu cau nguyen ph^t c6 gen d6l bien JAK2V617F Khong phat hi?n gen dpt bien JAK2 V617F a benh nhan la xS mi kinh dong tiiy.
K&l luan: Da trien khai thSnh cdng ky thuat ASO- PCR x^c djnh gen dot bien JAK2 V617F v^ da u-ng dyng trong chan doSn t^i B$nh vi$n Bach Mai
TO' khda: Polycythemia vera: PV, essential thrombocythemia: ET, Allele-specific oligonucleotide Polymerase Chain Reaction:
ASO-PCR
ABSTRACT
DEPLOYING ASO-PCR ASSAY T O DETECT JAK2 V 6 1 7 F GENE M U T A T I O N I N PATIENTS
W I T H C H R O N I C MYELOPROLIFERATIVE DISORDERS AT DEPARTMENT OF HEMATOLOGY A N D BLOOD TRANSFUSION,
BACH M A I HOSPITAL B a c k g r o u n d : JAK2 is a tyrosine kinase that plays an important role in the signaling pathways of many hematopoietic growth factor receptors. A single acquired point mutation-V617F-in JAK2 occurs in 65-97% of the patients with PV and 23-
* Khoa I-Iuy4t hoc- Truyen mdu BV B?ch Mai.
** Truing D^i HQC Y Hd N0I
Phan bi?n khoa h p c : PGS.TS N g u y i n Thj Lan
57% of the patients with ET Objetive: to detect JAK2 V617F mutation in patients with chronic myeloproliferative disorders. Materials and m e t h o d s : case series descriptive study. Using ASO-PCR assay to detect JAK2V617F mutation in 93 patients with chronic myeloferative disorders from 7/2011 to 01/2012 at Baeh Mai Hospital. Results: 18/ 23 patients with PV and 17/30 patients with ET expressing JAK2 V617F mutation,40 patients with chronic myelogenous leukemia not expressing JAK2 V617F mutation.
C o n c l u s i o n : succeeded in setting up ASO-PCR assay to detect JAK2 V617F mutation and applying this assay in diagnose at Baeh Mai Hospital.
I. OAT V X N OE
Dot biSn gen JAK2 V617F dupe cong b6 Idn dSu tien vao nam 2005 da ma ra mot huong di moi trong chan doan va dieu tri benh tang sinh tuy man tinh'"^. Gen JAK2 (Janus iiinase) la mot thanh vien cua hp gen Janus kinase, mot tyrosine kinase tham gia kiem soat su phan bao trong con duong JAK- STAT. Tac dung ue ehe su phan bao cua JAK2 se mk nlu co dot bien V617F tai vung JH2 cua gen nay bien Valine thanh Phenylalanine' . Nhieu nghien cuu cho thay dot biln gen JAK2 tai codon 617 gap ti le cao a benh nhan tang sinh tiiy man: 65-97%
benh da hong cau nguyen phat, 23-57% benh tang tieu cau nguyen phat, 35-57% xa tiiy'"''.
Y HOC VIET NAM THANG 4 - SO D A C BIET/2012 Phan loai va chan doan roi loan tang sinh tuy
man tinh cua To chuc y te the giai 2008 da dua dot bien gen JAK2 la mot tieu chuan chinh trong chan doan benh da hong cau nguyen phat va tang tieu cau nguyen piiat**.
De xac dinh dot bien gen JAK2 V617F co nhieu ky thuat: giai trinh tu chuoi true tiep, real-time PCR. pyrosequencing'.... Tuy nhien. nhiJng ky thuat nay thuong doi hoi phai CO may moc hien dai, dat tien, ton nhieu thai gian, Idiong d l dang trien khai 6 cac phong xet nghiem sinh hpc phan tii lam sang.
De xac dinh dot bien gen JAK2 V617F.
chiing toi lira chpn ky thuat ASO-PCR (allele-speeific oligonucleotide Polymerase Chain Reaction) ma Baxter cong bo lan dau tien vao nam 2005 tren tap ehi Lancet, su dung moi dac hieu voi nucleotide dau 3 ' chi CO the bat cap vai alen co mang dot bien.
Day la ky thuat eo dp nhay va dp dac hieu cao'', gia thanh hop ly, khong doi hoi trang thiet hi phiie tap, de ung dung trong phong xet nghiem sinh hpc phan tu huyet hpc,
II. OOI TUONG VA PHUONG PHAP NGHIEN COU
2J.Doi tirffng nghien cuu:
Nghien cum tien hanh tren 93 benh nhan roi loan tang sinh tiiy man (23 benh nhan da hong cau nguyen phat va 30 benh nhan tang tieu cau tien phat nhilm sac the Phi- , 40 benh nhan lo xe mi kinh dong tuy Phl(+)) dupe chan doan tai Khoa Huyet hoc- Truyen mau BV Bach Mai tu thang 07/2011- 01/2012. theo tieu chu^n WHO 2001.
2.2.Phuffngphdp nghien ciru:
- 4ml mau ngoai vi hoae 2ml djch tuy xuong chong dong EDTA dupe tach bach eau bang li giai hong cau true tiep, sau d6 tien hanh tach ADN (kit Quigen).
- Phan ung PCR gom ADN ban mau, moi, PCR master mix (Invitrogen), nhiet dp bat cap 60"C va chay trong 45 chu ky tren may PCR Mastercycler pro S.
• Ky thuat ASO-PCR sii dung 3 moi de khuech dai gen JAK2 binh thuong (wild-type) va gendptbienJAK2 V617F:
JK-R -5' CTGAATAGTCCTACAGTGTTTTCAGTTTCA 3";
JK-FS-5' AGCATTTGGTTTTAAATTATGGAGTATATT 3 ' JK-FC-5' ATCTATAGTCATGCTGAAAGTAGGAGAAAG 3^
• JK-R la moi ngupc chung cho ca gen JAK2 binh thuong (wild-type) va gen dot bien JAK2 V617F. Moi xuoi thii nhat JK-FS dac hieu vai alien dot bien khueeh dai san phim 203 bp. M6i xuoi thii hai JK-FC khuech dai cho mot san pham 364 bp tir ca alen dot biln va aien binh thuong, dupe sir dung de kiem chirng chat lucmg ADN.
• M6i mhi benh deu co chay chiing am va ehiing duong.
• San pham PCR dupe dien di tren gel agarose 2-3% nhupm Ethidium bromide va quan sat bang may chup gel,
III. KET QUA NGHIEN CLTU
Khi tri^n khai ky thuat chiing toi da phai thay cap moi khuech dai gen dot biln bk cap, voi d6i cac dieu kien phan ling khac nhau de dat nhiet dp qua eao chi co cap m6i khuSeh dai dupe kk qua toi uu: Vai nhiet dp thSp chi co alen binh thuong bat cap. D^ dat duac mot 10
Y HOC VIET NAM THANG 4 - SO DAC BIET/2012
nhiet dp toi uu cho ea 2 cap mfii hoat dpng phai tien hanh do nhiet dp timg buoc, kho khan hon khi do nhiet dp bit cap 6 mot s6 dot bien gen benh ly huyet hpc khac. Ngoai
ra, vi ASO-PCR la ky thuat rk nhay, viec t6i uu hoa dieu kien phan ling de Ioai trir k^t qua duong tinh gia la vSn dh cAn luu y.
364bp 203bp
Anh 1: Mot so ket qua dien di tren agarose tim dot bign JAK2 V617F bfing ky thuat ASO-PCR
1: Chung (-): (nguai binh thuong) L: thang chuSn lOObp,
8: chiing (-): nuac 3,6: mlu BN Da HC va tang ti6u cdu tien phat co gen JAK2(V617F) (+) 2: chiing (+) 4,5: mlu BN khong co gen JAK2(V617F
Bdng 1: Ket qua xet nghiem gen JAK2 V617F a benh nhan da hong cau va tSng tieu cSu
JAK2(V617F) Dircnq tinh Am tinh Tonq
DA H O N G CAU n
18 6 23
%
78,2 21,8 100
TANG Tieu CAU n
17 13 30
%
56,6 43,4 100
Trong nghien cim phat hien 18 /23 truang hpp(78,2%) da hong cau va 17/30 truong hpfp (56,6%) tang ti6u cdu co gen JAK2 V617F duong tinh. Tat ca 40 benh nhan lo xe mi kinh dong tiiy d6u khong phat hien gen JAK2 V617F.
IV. BAN LUAN
6 nhom tang tieu cau nguyen phat gap 56,6% benh nhan co gen JAK2 V617F, cao hon nghien cmi ciia Nguyen Vu Bao Anh (45%), Phan Thi Xinh*^ (52%) va tuong duong voi nghien cuu cua Baxter nhung cao hon nghien ciru ciia cac tac gia nuoc ngoai khac*. Di^u nay cho thay chiing toi da toi im hoa thanh cong quy trinh xet nghiem ASO- PCR tim dot bi^n gen JAK2 V617F trong benh tang ti6u cSu tien phat. Kk qua nghien
ciiu phat hien 78,2% benh nhan da hong eau e6 gen JAK2 V617F, cao hon nghien ciiu ciia Nguyen Vu Bao Anh^ (75%) va nghien ciiu ciia Phan Thi Xinh^ (30%). Ket qiia nay thap hon nghien ciiu ciia Baxter' (97%) nhimg cao hon nghien cim ciia Levine'' (73%). Dieu nay CO the do quan the dot bien gen JAK2 V617F trong benh tSng sinh tiiy man khac nhau o cac nuoc hoac tiiy thuoc vao dp nhay cua ky thuat va loai bach cau ma cac tac gia su dung''. Han nii'a, nhieu tac gia da tim thay dot 11
Y HOC VIET NAM THANG 4 - SO D A C BIET/2012 bien gen JAK2 con xay ra ngoai codon 617
nhu exon 12^ nen viec trien khai them ky thuat xac dinh dot bien gen JAK2 exon 12 6 nhom benh nhan tang sinh tiiy man khong co dot b i k gen JAK2 V617F la ckn thi£t. Nho do, se giiip eae bac sy lam sang nang cao han chat luong chan doan nhom benh nhan tang sinh tiiy man. Trong nhom benh lo xe mi kinh dong tiiy Phl(+), chiing toi khong tim thay benh nhan nao eo dot bien gen JAK2 V617F, tuong tu vol ket qua nghien ciiu ciia Jones''. Dieu nay khang dinh xet nghiem ciia chiing toi dang tin cay, loai trir dupe ket qua duong tinh gia, rat de gap a ky thuat ASO- PCR, mot khi chua toi uu dupe nhiet dp bat cap. Thai gian toi khi co luong benh nhan dii Ian, chiing toi se tien hanh nghien ciiu them ve cac moi lien quan giira tinh trang Iam sang, huyet hpc, dap ling dieu tri ciia benh nhan tang sinh tuy man tinh co gen JAK2 V617F. Nho do, ngoai iing dung trong chan doan, viec xet nghiem gen JAK2 co the se giup cac bac sy lam sang co dinh huong ve tinh trang lam sang, huyet hpc ciing nhu du doan dap ling dieu tri ciia benh nhan.
V. KET LUAN
Chiing toi da trien khai thanh eong ky thuat ASO-PCR d l xac djnh gen JAK2 V617F tai khoa Huydt Hoc-Truyen mau BV Bach Mai. Vai uu diem nhanh, nhay, dp dae hieu cao, gia thanh hpp ly da ling dung hieu qua vao chan doan o benh nhan, gop phan nang cao chk luong ehan doan, di§u tri benh ly huyet hpc.
TAI UEU THAM KHAO
1. Nguyen Vu Bao Anh. iTng dung ky thuat ASO-PCR xac dinh dot biSn gen JAK2
V617F 6 hpi chimg tang sinh tiiy man tai Vien Huyet hpc-Truyen mau TW. Ludn van tot nghiep BSNTBV- chuyen nganh huyil hoc. 2009; tr 30-50.
2. Phan Thi Xinh, Hoang Anh Vu, Nguyen Tan Binh. Ung dung ky thuat ASO-PCR xac dinh dot biln gen JAK2(V6I7F) trong nhijng roi loan tang sinh tiiy. Tap chi y hoc Viel Nam. 2010; s6 2: tr 351-354.
3. Baxter EJ, Scott LM, Campbell FJ el al.
Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005;365:1054-1061 4. David P. Steensma et al. JAK2 V617F in
myeloid disorders: molecular diagnostic techniques and their clinical utility. The Journal of molecular diagnostics. 2006;
Volume; 8, No: 4: 397-411
5. Jones AV, Kreil S, Zoi K, el al.
Widespread occurrence of the JAK2 V617 mutation in chronic myeloproliferative disorders. Blood. 2005;ia6:2162-2168.
6. Levine RL, Wadleigh M, Cools J, el al.
Activating mutation of the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell.
2005;7;387-397.
7. Scott LM, el al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med.
2007;356(5):459-68.
8. Tefferi A and Vardiman JW.
Classification and diagnosis of myeloproliferative neoplasms: The 2008 World Health Organization criteria and point-of-care diagnostic algorithms.
Leukemia. 2008; 22:14-22.
