NGHIEN CU'U SU BI^N DOI GEN PML/RARa O BENH NHAN LO XE Ml CAP i n e M3
Vu Minh Phu'O'ng Trw&ng Dai hoc Y Ha ngi Nghien cu'u nay nham 1) xae djnh ty le biin ddi gen PML/RARa d benh nhan la xe mi cip thi M3, va 2) md ta cac kiiu cua bien ddi gen PML/RARa d benh nhan la xe mi cip thi M3. Ddi twang nghien cu'u gom 33 benh nhan da dwgc diiu trj d Vien Huyit hge truyen mau trung wang va Khoa Huyet hge truyin mau benh vien Bach mai vdi chin doan la xe mi cip thi M3 theo tieu chuin cua FAB, chwa dwgc diiu trj hda chit tw thing 4/2007 den thing 3/2009. NhQ'ng ddi twang nay dwgc lam phan tich nhiem sac thi va PCR xic djnh gen PML/RARa. Kit qua cho thiy sd benh nhan cd biin doi gen PML/fRARa la 25/33, chiim 76%. Cd 18 benh nhan thi ber chiem 72%, 3 benh nhan the bcr2 chiim 12% va 4 benh nhan thi bcr3, chiim 16%. Tit ca cac trwdng hgp khdng thiy t(15;17) deu cd kieu biin doi gen la bcr2 va ber3, khdng gap bcrl: 9 trwdng hgp cd t(15;17) deu cd kiiu bien ddi gen bcrl.
TCF khoa: la xe mi cip thi M3, biin ddi gen PML/RARa 1. OATVANOE
Chuyen doan t(15;17) thudng gap d la xe mi d p thi M3. Binh thudng gen PML (ma hoa ylu t l phien ma putative novel) nam tren nhilm sac thi (NST) sd 15, gen RARa nim tren NST sd 17.
Chuyin doan giQ-a 2 NST nay tao nen phu'c hgp gen lai (hay cdn ggi la biin ddi gen) PML/RARa. Vj trf dui giy d nhilm sac thi 17 la intron 2. Ngugc lai tren nhilm sac thi so 15 cd 3 vj tri dirt giy la intron 6, exon 6 va intron 3 tao nen ba kieu gen PML/RARa tuang irng la bcrl, bcr2 va ber3. Do su khae nhau ve kfch cd san phim, bcrl va bcr2 dugc xip vao loai phien ma dai (L type), bcr3 xip vao loai phien ma ngan (S type). S type thudng gap d t h i M3v (la mdt dang biin t h i cua M3)
[3]. Tren ca sd nay d l tai dugc nghien cu'u vdi muc tieu:
- Xic djnh ty le biin doi gen PML/RARa d benh nhan la xe mi cap the M3.
Md ta cac kiiu cua biin ddi gen PML/RARa a benh nhan la xe mi cip the M3.
2. 0 6 l TU'gNG VA PHU'ONG PHAP NGHIEN CU'U
2.1. Odi tu'O'ng nghien cii'u
33 benh nhan dugc dilu tri a Vien Huylt hge truyin mau TW va Khoa Huylt hge truyin mau benh vien Bach mai vdi chin doan la xe mi cip thi M3 theo tieu chuin cua FAB, chua dugc dilu tri hda chit tu' thang 4/2007 din thang 3/2009 [2].
2.2. Phu'O'ng phap nghien ciru
Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1859-2376
Cae benh nhan dugc lam xet nghiem phan tfch nhilm sac t h i va xet nghiem PCR xae djnh biin ddi gen PML/RARa 2.2.1. Ky thuit phan tich nhilm s i c t h i Cac ky thuat phan tich nhilm sac t h i dugc tiln hanh tren may AXO 100 (Zeiss).
Ma mdi PML-A1:
PML-A2:
RARA-B:
PML-C1:
PML-C2:
RARA-D:
RARA-E3':
2.2.2. Ky thuit PCR (RT-PCR va nested- PCR) x i c djnh biin doi gen PML/RARa - Sii' dung cac cap m i l dac hieu xae djnh sy biin ddi gen: theo ky thuat cua JIM van Dongen md ta [3]. Trinh t y eae cap mdi cho phan U'ng PCR cua gen PML/RARa dugc chung tdi thilt k l nhu sau:
Chudi nucleotide
CAGTGTACGCCTTCTCCATCA CTGCCTGGAGGCTGTGGAC GCTTGTAGATGCGGGGTAGA TCAAG ATGG AGTCTGAG GAGG AGCGCGACTACGAGGAGAT CTGCTGCTCTGGGTCTCAAT GCCCACTTCAAAGCACTTCT
- Xae djnh eac k i l u b i i n ddi cua gen tren dien di tren agarose, k i t qua dugc PML/RARa dya tren kfch c d cua san p h i m trinh bay tren bang 1.
Bang 1 . Cac kfch ca san phim cua cac biin ddi gen Gen
PML/RARa
Kieu biin doi Bcrl Ber2 Bcr3
Kich c a san phim (bp) 214
178 289
3. K^T QUA NGHIEN CU'U
3.1. Kit qua v§ ty le co biin ddi gen iy benh nhin Ic xe mi c i p t h i M3
Xae dinh dugc 25 b#nh nhan cd biin ddi gen PML/RARa, chilm ty le 76%.
3.2. Kit qua v l ty le cac kilu cua biin d i i gen PML/RARa
Tren cae benh nhan cd kilu biin ddi gen tren, chiing tdi xae djnh sau v l ty le cua cac dudi nhdm. K i t qua v l ty le cac
dudi nhdm dugc trinh bay tren bang 2. K i t qua tren bang 2 cho t h i y ty le kilu gen bcrl la cao nhit, va t h i p nhit la ty le kilu bcr2.
Hinh anh dien di cac san p h i m gen thu dugc sau chay PCR euia cae dudi nhdm bcrl, ber2, bcr3 dugc trinh bay tren eae hinh 1 ^ 3.
Bang 2. Ty le cac kilu cua biin ddi gen PML/RARa (dya vao kfch cd san phim) Kilu biin ddi
Bcrl Bcr2 Ber3
Sd lugng N=25
18 3 4
Tyle (%) 72 12 16
Hinh 1. Hinh anh dien di biin ddi gen PML/FRARa kilu bcrl. Gilng 1: marker; gilng 2:
chirng; gilng 3: chirng am; gilng 4-^8: miu benh nhan duang tfnh kilu bcrl.
Hinh 2. Hinh anh dien di biin doi gen PML/RARa kilu bcr2. Gilng 1: marker; gilng 2:
churng duang; gilng 3: chii'ng am; gilng 4-^8: mlu benh nhan duang tfnh kilu bcr2.
Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1859-2376
Hinh 3. Hinh anh dien di biin ddi gen PML/RARa kilu bcr3. Gilng 1: marker; gilng 2:
chu'ng duang; gilng 3: chirng am; gilng 4 -^ 6: miu benh nhan duang tinh kilu bcr3.
Kit qua doi chilu giura kilu biin ddi nhilm sac thi vdi phan tfch kilu biin ddi
gen PML/RARa dugc trinh bay tren bang 3.
Bang 3. Ddi chilu kit qua phan tfch kilu biin doi gen PML/RARa vdi phan tfch nhilm sic thi
Kilu biin ddi nhilm sac t h i 46,t(15;17)(q22;q21)
46,XX 46,XY 46, XY, 17q-
45, XY, -7 Khdng phan bao
Kilu biin ddi gen (N=16) Bcrl
Bcr3 Bcr2 Ber2 Bcr2 Bcrl
Sd lugng 9 2 1 1 1 2
Tyle 56,25%
12,5%
6,25%
6,25%
6,25%
12,5%
Kit qua tren bang nay cho thiy tit ca cae trudng hgp khdng thiy t(15;17) diu cd kilu biin ddi gen la bcr2 va bcr3, khdng gap bcrl. Vdi 9 trudng hgp cd t(15;17) diu cd kilu biin ddi gen la bcrl.
4. BAN LUAN
Biin ddi gen PML/RARa gom cd 3 kieu biin ddi khae nhau la bcrl, bcr2 va bcr3.
Su khae nhau do 3 vj tri dii't giy khae nhau
cua chuyin doan t(15;17) (q22;q21) tao ra
nhung kilu b i i n ddi gen tuang u'ng la bcrl, bcr2 va bcr3. Theo Jim Van Dongen thi ty le bcr1 la cao nhit (55%), t i l p d i n bcr3 (40%) va hilm gap n h i t la bcr2 (5%) [3].
Theo bang 2 khi phan tfch dudi nhdm cua biin ddi gen PML/RARa chung tdi cung gap ty le cao nhit la bcrl (72%), t i l p d i n la bcr3 (16%), va t h i p nhit la bcr2 (12%). Cac ty le nay cQng thay ddi d cac nghien curu khae nhau (bang 4), nhung nhin chung ty le bcrl v i n cao nhit va t h i p nhit la bcr2. Sy khae nhau nay cd t h i do cd m l u khae nhau. Tren thyc te, khdng nhilu nghien ciru b i t gap dii ca 3 kilu b i i n ddi nay. R i t may nghien cu'u nay da tim thiy dugc ca 3 kilu biin d i i trong nhdm benh nhan nghien cu", nhu tren cac hinh 1
^ 3 .
Bcrl va bcr2 dugc x i p vao kilu phien ma dai (L type) cdn bcr3 dugc x i p vao kilu
phien ma ngan (S type). Vi kilu biin doi bcr2 la tuang doi h i l m gap nen nhilu tac gia khi nghien curu chi quan tam d i n kieu L vdi dai dien la bcrl va kilu S bcrS. Cung da cd mdt s l nghien cii'u quan tam d i n sy khae biet tren lam sang cQng nhu xet nghiem glQ'a k i l u L va kieu S. Thyc t l v i n chua tim dugc m i l lien quan giu'a cac dang nay vdi dac d i i m gidi tfnh, sy roi loan ddng mau hay s l lugng t i i u c i u . Tuy nhien nhu'ng benh nhan loai S cd t h i cd so lugng bach c i u cao han va thudng cd hinh thai la t h i M3v [3]. R i t t i l e trong nhdm nghien CU'U nay chi cd 4 benh nhan S type (bcr3), nen s l lugng qua ft d l cd t h i so sanh.
Chung tdi thong ke ty le ba ddng dang b c r l , 2, 3 cua PML/RARa trong cac nghien CU'U cua cac tac gia khae [1], [4], [5], [6], [7], [8] cuing vdi k i t qua nghien cu'u cua chung tdi tren bang 4.
Bang 4. Ty le cac kieu biin ddi ciia PML/RARa cua mdt sd tac gia Tac gia
Biondi (1992) Santillana (2000)
Chillon (2001) Sucie M (2002) Schnittger (2003)
Ruiz (2004) V M . Phuang (2009)
Vj tri dja ly cua nghien cu'u
Y Peru Tay ban nha
CHSec Dire Mexico Viet nam
s l benh nhan
35 13 37 13 121
43 25
Ty le bcrl (%) 48,5 70,0 67,6 61,5 46,2 62,7 72,0
Ty le bcr2 (%) 17,1
0 5,0
0 0 9,3 12.0
Ty le bcr3 (%) 34,2 24,0 26,4 38,5 53,8 27,9 16,0
Trong nhdm ddi tugng nghien ciru cua chiing tdi ed 3 benh nhan the M3v d i u cd kilu bien ddi la bcrl (thuge L type). Theo nghien cu'u cua Biondi va Schnittger ty le
benh nhan t h i M3v cd k i l u biin ddi bcr3 (S type) la kha cao (5/8 va 23/30 trudng hgp) [1], [8]. Nhung trong nghien curu nay chi cd 3 benh nhan t h i M3v va d i u cd kieu
Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1859-2376
biin ddi bcrl. Cd le vi sd lugng benh nhan t h i M3v qua ft nen khd tim hiiu sau han kilu biin ddi gen cua biin t h i nay.
Nghien cu'u cua Ruiz- Arguelles va cdng s u tren 43 benh nhan la xe mi d p t h i M3 ngudi Mexico ban dja da dua ra k i t qua la ty le bcrl cua nhdm ngudi nay eao han d nhdm benh nhan Caucase va tuang duang vdi nhdm benh nhan ngudi chau A [5]. TO' nghien cu'u nay hg cho rang v i n d l ehung toe cung cd lien quan d i n mdt sd gen gay benh nhu biin ddi gen gay la xe mi c i p t h i M3, va cd le ty le biin ddi gen PML/F^Ra cung nhu kilu biin ddi cung cd su khae nhau giu'a cac chung tdc ngudi.
Bang 3 d i i chilu k i t qua phan tich nhiem sac t h i vdi kilu biin ddi gen t h i y ca 9 trudng hgp cd t(15 ;17) d i u ed kilu bcrl.
Cac trudng hgp khdng cd t(15 ;17) d i u cd kilu biin ddi gen la ber2 hoac bcr3, khdng gap bcr1. Vi so benh nhan cdn ft nen ehiing tdi cung ehua dam dua ra mdi lien quan giu'a kilu b i i n doi gen vdi su xult hien t(15;17). Du sao kilu biin doi bcrl v i n phd biin nhit nen cd t h i gap d nhilu trudng hgp.
5. KET LUAN
- Ty le biin doi gen PML/RARa d benh nhan la xe mi c i p t h i M3 la 76% (25/33 benh nhan).
- Cac kilu cua biin ddi gen PML/RARa d benh nhan la xe mi c i p t h i M3 la: 72%
bcr1, 12% bcr2 va 16% berS. T i t ca cac trudng hgp khdng t h i y t(15;17) d i u cd kilu biin ddi gen la bcr2 va bcr3, khdng gap
bcr1. 9 trudng hgp cd t(15;17) deu cd kieu biin doi gen bcrl.
TAI LIEU THAM KHAO
1. Biondi A., Rambaldi A., Pandoff P.P.
et al. (1992), Molecular monitoring of the myl/retinoic acid receptor alpha fusion gene in acute promyelocytic leukemia by PCR, Blood 80(15): 4 9 2 - 497.
2. Bennett J.M., Catovsky D., Daniel M.T. et al. (1985), Proposed revised criteria for the classification of acute myeloid leukemia. A report of Freneh_
American_British Cooperative Group, Ann Intern Med 103: 620-625.
3. Van Dongen J.I.M., Macintype E.A., Gabert J.A. et al. (1999), Standardized RT-PCR analalysis of fusion gene transcript from chromosome aberrations in acute leukemia for detection of minimal residual disease:
report of the BlOMED-1 Concerted Action: Investigation of minimal residual disease in acute leukemia, Leukemia 13: 1901-1928.
4. Chillon M.C, Sanz R.G., Ana Balanzategui A. et al. (2001), Molecular characterization of acute myeloblastic leukemia accordingh to the new WHO classification: a differrent distribution in Central West Spain, Hematologica 86: 162-166.
5. Ruiz-Argulelles G.J., Garces-Eisele J., Reves-Nunez et al. (2004), More on geographic hematology: the breakpoint cluster regions of the
6.
7.
SUMMARY
Study subtypes of PML/ RARa fusion gene on the patients diagnosed acute
promyelocytic leukemia (AML -M3) The present study aimed at i) determining ratios of subtypes of PML/RARa fusion gene, and ii) describing such subtypes of PML/RARa in patients diagnosed AML-M3. Subjects consisted of 33 patients with acute promyelocytic leukemia (M3) that were recruited to the National Institute of Hematology and Blood Transfusion and at Department of Hematology and Blood Transfusion in Bach Mai hospital that were not in chemical course treatment, from April 2007 to March 2009. They were examined with cytogenetic tests and PCR for PML/RARa fusion transcript The results showed that PML/RARa positive in 76 %. Rates of three subtype (bcrl, bcr2 and bcr3) of PMURARa were 18 patients (72%), 3 patients (12%) and 4 patients (16%), respectively. All patients without t(15;17) had bcr2 and bcr3 subtype. All of 9 patients with t(15;17) had bcrl subtype.
Key words: acute promyelocytic leukemia, M3, PML/RARa
BUOC
D A UNGHIEN CLPU DI§U TRj
T A NCONG BENH
N H A NLO XE Ml CAP DONG TUY CO BI^N DOI GEN CBFp/MYH11
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Santillana S., Campana F., Leon J., Wachtel A. et al. (2000), Characteristics of PML/RAR alpha Fusion Gene Mutations in Patients with Acute Promyelocyte Leukemia (APL) in Peru. ASCO Annual Meeting.
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