TCNCYH Phu tru'dng 62 (3) - 2009
D O T BIEN GEN GLUCOSE - 6 - PHOSPHATE DEHYDROGENASE (G6PD) 6 BA NHOM DAN T O C MUOfNG, TAY, THAI 6 VIET NAM
Nguyin Minh H u n g \ Ta Thj T i n h \ Hiroyuki Matsuoka^
'/C/ioa Nghien cdu va Dieu trj Sdt ret - Vien Sdt ret - Ky Sinh Triing ~ Cdn trimg Trung Uang -Khoa MiSn djch - Tnfdng Dai hgc Y Jicbl - Nbat Ban
Thie'u C6PD I a mdt bat thudng dl truyen hay gap nhai, anh hudng den han 400 tneu ngtfdi tren the gidi.
Hiu bet cic trudng hgp thieu C6PD deu do dot bien tren gen G6PD. Muc tieu: phit hien cic diem dot bien tren gen G6PD cua mdt sd nhdm din tdc Viet Nam. Boi tugng va phugng pbap nghien cdu: tich chiet DNA td 39 miu mau ciia ba nhdm dan tge Mudng, Tiy vi Thai. Sau khi diing phin dng PCR (Polymerase Chain Reaction) de nhan ban cic doan exon cua gen C6PD, san pham PCR dugc nhugm vdi fluorescent vi sau dd la dgc tnnh tU DNA tren may sequencing. Ket qua: cd 6 sU thay ddi cua nucleotide tren gen G6PD tai cac vj tn 143 T > C, 487 C > A, 871 G > A, 1360 C > T, 1376 G > T vi 1388 G > A. Dot bien tai cic vi tn 871 G> A (G6PD Viangchan) vi 1360 C>T (G6PD Union) hay gap nbat, ty le tuang ifng li 25,7% vi 53,8%. Ket luan: G6PD Viangchan vi G6PD Union la hai bien the ehii yeu trong nghien edu ve G6PD tai Viet Nam.
Tfl khoa: thie'u hut G6PD
L D A T V A N D E
Glucose - 6 - Phosphate dehydrogenase (G5PD) la enzyme xuc tac phan flng dau tien trong chu trinh Hexose Monophosphate v l t h i i u G6PD I I mpt trong nhflng b i t thfldng di t r u y i n hay gap n h i t d ngfldi.
Gen quy djnh tdng hdp enzyme G6PD nam tren nhanh d i i vung 2 bang 8 cua nhiem s i c t h i X (Xq28), gdm 1 3 exon trong dd exon 1 khdng ma h o i tdng hpp acid amin. Hau h i t c l e trfldng hpp t h i i u G6PD la do sfl thay ddi cua mdt acid amin g l y ra bdi mdt dot b i i n tren gen G5PD va cho den nay hdn 140 b i i n the G6PD da difpc phat hien va cdng be [10]. Dd ddt b i i n tren gen G5PD la benh di t r u y i n nen viec sfl dung cac dot bien d l tim ngudn gde td tien chung cua eac nhdm dan tdc mang n h i i u y nghTa v l mat nhan chung hpc
1 1 , 3 , 8 1 .
Viet Nam cd 54 nhdm d i n tdc khac nhau, thieu G6PD la tfldng dd'i phd bien d c l e d i n tdc da difpc d i i u tra. Ty le t h i i u G6PD tfl 0,5 - 31 % d c l e nhdm dan tec phia B l e va 1,9 - 4 , 4 % d cac
nhdm d i n tdc phfa Nam [7, 9]. Tuy nhien nghien cflu G6PD d mflc dp p h l n tfl mdi chi dflng lai d 8 nhdm dan tdc Kinh, K'ho, Chau Ma, Nung, T l y , Raglai, Pake va Khd Mu [3, 7]. '
D l t i i p tuc tim hieu dang dot b i i n gen d cac nhdm dan tdc cdn lai, chung tdi da t i i n h i n h giai trinh tfl gen cho cac mau thieu G6PD thu thap tfl ngfldi d i n tdc Mfldng ( H o i Binh), Tay (Cao Bang), T h i i (Sdn La). Muc tieu:
Phat hien cac diem dot bien tren gen G6PD cua mgt sd nhom dan tge Viet Nam.
II. DOI TU'ONG VA PHU'ONG PHAP NGHIEN CL/U
1. Dd'i tfldng nghien cflu
Sang lpc t h i i u G5PD cho 1328 dd'i tfldng thudc ba nhdm dan tpc Mfldng (284 ngfldi), Tay (424 ngifdi) va T h i i (620 ngfldi). Cd 242 trfldng hpp thieu G5PD dflpc xac dinh b l n g test nhanh cua Akira Hirono trong dd Mfldng 85 ngifdi, Tay 37 ngfldi va Thai 120 ngfldi.
lOOpl mau dau ngdn tay cua ngfldi thie'u
TCNCYH PhtJ trWdng 62 (3) - 2009
G6PD dflpc thu thap vao g i i y t h i m Whatman 3, de khd tfl nhien, b i o quan d 4°C rdi c h u y i n sang phan tfch tai trfldng Dai hpc Jichi - N h l t B i n .
Cd 39 trfldng hpp difpc phan tfch tim dot bien tren gen G5PD bao gdm Mfldng (3 nam, 15 nfl);
T l y (2 nam, 2 nfl) va Thai (7 nam, 10 nfl).
2. Phfldng phap nghien cu'u
DNA dfldc tach chiet tfl mau thu thap bang kit QUIAamp DNA (Germany). Sfl dung 12 cap mdi de khuyech dai 12 exon cua gen C6PD gid'ng nhfl b l n g 1. Sin pham PCR sau khi dflpc tinh sach se difpc t i i n hanh dpc trinh tfl DNA tren he thd'ng may ABI PRISM 310 Applied Biosystems, Foster City, CA, USA).
Bang 1. Trinh tU moi xudi va moi ngUgc cho pban dng PCR va Sequencing
Exon
2 3 v a 4
5 6 7 8 9 10 11 va
12 13
Ten, trinh tfl cua sense primers
1 a 5' - AGACCCCAGAGGAACTCAAGAAA Bia 5' - TGTCCCCAGCCACTTCT
B3 5' - ACACACGGACTCAAAGAGAG 85 5' - AGCTCTGATCCTCACTCCCC 7a 5' - ACATGTGGCCCCTGCACCACA 9a 5' - TTGGGGTCCCCATGCCCTTG B9a 5' - ACCCAAGGAGCCCATTC E 5' - CTGAGACAGCTGGTGCT B I I 5' - ACTCCACATGGTGGCAGGCAG RBI 2 5' - TGAGGGTGGAGCTACCTCAT
Ten, tr
2b 5 ' - B2 5 ' - B2a5' 8b 5 ' - B6 5' B8 5' R2 5' B105' B12 5' 18a 5'
inh tfl cua antisense primers
GCAACAATTAGTTGGAAAAGCTGA CCGAAGCTGGCCATGCTGGG - TGGTGGGAGCACTGCCTGAGTA GGCCAGGTGAGGCTCCTGAGTA - GTGACTGCTCTGCTCTGCCACCCTG - TGCCTCGTCACAGATGGGCC - ACACAGGGCATGCCCAGTTCTG '-CACCATGTGGAGTCCCCCGG
- ATGAGGTAGCTCCACCCTCA - CGGGGGTGGAGGTGGGTGCCCA
San pham
(bp) 294 358 343 285 242 231 276 342 397 164
Nghien cflu dflpc sfl cho phep cua hdi ddng Y dflc Vien Sd't ret - Ky sinh trung - Cdn trung Trung fldng va trfldng Dai hpc Y Jichi, N h l t ban (giay phep phan tich GE 03 - 25).
I
C A A C A C C T T G A C C A A C A T C T T O A C
I
\^^' V
A l A2C T G C A C A C G A A G T
C T G C G C A C G A A G T
Bl B2
C G A G C T C C G T G A G C A G C T C C T T G A G
C l
A i
C2Vj tri: 871
A l : khdng dot b i i n A2: dot b i i n C > T Reverse DNA: C > A
V j t r i : 1360 B l : khdng dot bien B2: dot b i i n A > G Reverse DNA: T > C
V j t r i : 1376 C l : Khdng dot b i i n C2: Dot b i i n G > T
TCNCYH PhtJ tru'dng 62 (3) - 2009
D2
V j t r i : 1388 D l : khdng dot b i i n D2: ddt b i i n G > A
A G G C T G G
Ai my.
El
A A G C T G G
/•y j \ K^^
E2
Vi tri: 487
E l : khdng dot b i i n E2: ddt bien G > A
I
G T C T CC CC
I
G A C C T A C C C C
Fl
F2
V j t r i : 143 F l : khdng dot b i i n F2: ddt b i i n T > C
Hinb 1. Sau vi tri tbay ddi nucleotide tren gen C6PD
Dot b i i n tai vj trf 871 C > A (G6PD Viangchan) va 1 350 C > T (G5PD Union) chiem da sd vdi ty le tflpng flng I I 25,7% (10/39) va 53,8% (21/39). C l biet, cd 3 trfldng hdp nfl, ngfldi mang tren mdi gen G5PD mdt dot b i i n , dd la cac cap dot b i i n 871 G > A/1 376 G > T va 1 360 C > T/l 375 G > T (bang2).
Bang 2. Cac bien the G6PD d ba nhom dan tge
Ten cua cac bie'n the Viangchan Union Canton
Viangchan/Canton
Union/Canton
Kaiping Mahidol
Thay ddi Nucleotide
871 G > A 1360 C > T 1375 G > T 871 G > A 1376 G > T 1360 C > T 1376 G > T 1388 G > A 487 G > A
Thay ddi Amino acid
V291M R454C R459L V291M R459L R454C R459L R463H G163S
Nh(
Mfldng 3 13
• 2
0
0
0 0
3m dan Tay
1 0 2
1
0
0 0
tdc Thai
5 6 0
0
2
1 2
To'ng cdng (%) 9(23,1) 19 (48,7)
4 (10,2)
1 (2,5)
2(5,1)
1 (2,6) 2(5,1) Aures
Tdng cdng
1 4 3 T > C lle48Th 1 (2,5)
39 (100)
TCNCYH PhtJ tru'dng 62 (3) - 2009
I V , B A N L U A N
Ty le t h i i u G5PD I I Mfldng 29,9% (85/284), Tay 8,7% (37/424) va Thai 19,4% (120/620). G i i i trinh tfl gen G6PD cho 39/242 m l u mau thu thap, chung tdi phat hien difpc 5 dang bien t h i G5PD, trong dd G6PD Viangchan va G5PD Union e h i l m da so', phu hop vdi ket qua phan tfch dot bien gen G5PD trfldc d l y tai Viet Nam [3, 7].
G5PD Viangchan dflpc p h l t hien lan dau tien d ngfldi Lao djnh cfl tai Canada nam 1988 sau dd la ngfldi L l o d Hawaii nam 1993. Theo phan loai cua Td chflc Y t l The gidi (WHO, 1989), G6PD Viangchan nam trong phan Idp 2 vdi hoat tinh enzyme hdng c l u chi bang 1 - 10% so vdi ngfldi binh thfldng. G5PD Viangchan hay gap trong c i c nghien cflu tai T h i i Lan, Lao, Campuehia [5, 8].
Tai Viet Nam, G5PD Viangchan c b i l m da sd' trong cac bien t h i , 11/25 trfldng hdp ngfldi Kinh, 8/9 trfldng hdp ngfldi Raglai va 1 6/1 6 trfldng hdp ngifdi Pakd [3, 7]. Sfl x u i t hien b i i n the G5PD Viangchan trong cdng ddng ngifdi T h i i , Lao, Campuehia, Viet Nam (Pake, Raglai, K'Ho, Kinh, Thai, Tay, Mfldng chflng td hp cd lien quan v l nguon gd'c td tien chung.
G6PD Union l l n dau tien dfldc phat hien tren ngfldi Philippin va r i t phd bien d c l e nfldc nhfl Philipin, Solomon, va Vanuatua [1]. O Viet Nam, C6PD Union gap tren nhdm dan tdc Kinh tai L l m Ddng vdi t i n suit t h i p [7J, nhflng lai dac biet cao d nhdm ngifdi Khd M u , m i i n B l e Viet Nam [3].
Treng nghien cflu cua chung tdi, G6PD Union e h i l m da sd'd ngfldi dan tdc Mfldng va T h i i .
B i i n the G6PD Canton gap d c l e nfldc Ddng Nam A nhfl T h i i Lan [8], Myanma [5] nhifng vdi tan suit t h i p , ngflpc ban vdi sfl phd bien cua nd d Trung Qud'c vdi ty le 2 8 , 2 % [4]. Sfl x u i t hien cua G6PD Canton tai Thai Lan dflpc cho la sfl ddng h o i cua ngfldi Thai ban dia va ngfldi Trung Qud'c di dan tfl tinh Fushan va Quang Ddng [8]. Tai Viet Nam, b i i n t h i nay gap d ngfldi Kinh vdi ty le
25,3% [7]. Trong nghien cflu cua chung tdi, G5PD Canton gap tren c l 3 nhdm d i n tdc Mfldng, T l y , Thai va cd ty le I I 1 7,9% (7/39).
G5PD Kaiping I I b i i n t h i hay gap n h i t tai Trung Qud'c [4]. Tai Viet Nam, G6PD Kaiping x u i t nhifng vdi t i n suit t h i p [7].
Vdi lich sfl hdn 1000 nam bj Trung Qud'c dd hd, c i c bien the G5PD Canton, G5PD Kaiping d Viet Nam cd the bat ngudn tfl ngfldi Trung Qudc.
G6PD Mahidol va G6PD Aures I I hai bien t h i hoan t o l n mdi va lan d i u tien phat hien tren ngfldi Viet Nam.
V . K E T L U A N
Qua nghien cflu cac ca t h i t h i i u G6PD cua ba nhdm d i n tdc Mfldng, Tay, Thai, da xac djnh b i i n the G6PD Viangchan v l G5PD Union la r i t phd b i i n trong cac dang dot b i i n gen G6PD.
T A I L I E U T H A M K H A O
1. Ganczakowski, M (1995). Multiple G5PD variants correlate w i t h malaria endemicity in the Vanuatu Archipelago. Am J Hum Genet, 56, pp. 294 - 301.
2. Hirono, H (1998). An improvement single - step screening method for G6PD deficiency. Jpn J Trop Med Hyg, 25, pp. 1 - 4.
3. Hung N M (2009). G6PD variants in three minority ethnic groups in Central and Northern Vietnam. Trop Med Health, 37, No 1, pp. 1 7 - 20.
4. Jiang W (2006). Structure and function of G5PD deficient variants in Chinese population.
Human Genet. 11 9, pp. 463 - 478.
5. Matsuoka H (2004). G6PD Mahidol (487G
> A) is the most cdmmon variant in the Myanmar population. J Hum Genet. 49, pp. 544 - 547.
6. Matsuoka H (2005). G6PD Viangchan (871 G > A) is the most common variant in the Cambodia population. J Hum Genet, 50, 468 - 472.
7. Matsuoka H (2007). Seven different G6PD
TCNCYH Phu tritdng 62 (3) - 2009
variants including a new variant distributed in 9. Verle~ P (2000). Glucose - 6 - phosphate Lam Dong province, Vietnam. Acta Med, dehydrogenase in northern Vietnam. Trop Med Okayama, 5 1 , pp. 2 1 3 - 2 1 9 . and Int Health, 3, pp. 203 - 206.
8. Nuchprayoon 1 (2002). G6PD Viangchan 10. Vulliamy T and Beutler E (2002).
(871 G > A) is the most common deficiency Hematologically important mutations: glucose - 6 - variant in the Thai population. Hum Mutat, 1 9, (1 85). phosphate dehydrogenase. Blood Cells Moi Dis, 28, (93).
Summary
MUTATIONS ON GLUCOSE - 6 - PHOSPHATE DEHYRDROGENASE (G6PD) GENE IN THREE ETHNIC GROUPS: MUONG, TAY, AND THAI IN VIETNAM
Glucose - 6 - phosphate dehydrogenase (G5PD) is one of the most common hereditary disorders in the world, affecting to more than 400 million people in the world. In most of the cases, G5PD deficiency was caused by mutation (s) on G6PD gene. Objectives: to find out mutant points on G6PD gene of some Vietnam ethnic groups. Methods: genomic DNA was extracted from 39 blood samples belongs to 3 ethnic groii|3s: Muong, Tay, and Thai. PCR (Polymerase Chain Reaction) was used to amplify the all of exons of G6PD gene. PCR products was purified and dyed with fluorescent then reading sequencing of DNA.
Results: there were 6 changes of nucleotide at position 143 T > C, 487 G > A, 871 G > A, 1360 C > T, 1376 G > T and 1388 G > A. The change at positions 871 G > A (G6PD Viangchan) and 1360 C > T (G6P1'3 Union) was quite predominant with prevalence 25.7% and 53.8%, respectively. Conclusion:
(i6PD Viangchan and C6Pf) Union were two common variants in the studies of G6PD among three ethnic groups: Muong, Tay, and Ihai in Vietnam.
Keywords: G6PD deficiency, Vietnam, G6PD Viangchan, G6PD Union
eiEU TRA TY LE THIEU HUT GLUCOSE 6 PHOSPHATE DEHYDROGENASE HONG CAU 6 MOT SO DAN TOC SONG TAI
D A K LAK V A L A M DONG, VIET NAM
Ta Thj TTnh, Le Minh Dao
Vien Sdt ret - Ky sinh triing - Cdn triing Trung ttang
Thieu glucose 6 pholphale dehydrogenase hong cau (G6PP) li mdt benh dl truyen cd Hen quan de'n nhiim si( lhe gidi linh X, mat benh ly rdi loan enzym thtfdng gap nhai d ngifdi. Benh hay gap d nhdng nhom dim Ioc sdng trong vung sdt ret lUu hanh. Viec xac djnh ty le thieu G6PD d cic nhdm din tge tren cd y nghia quan trgng lrong chinh sich thtidc sdt rel. Muc tieu: xic djnh ty le thieu G6PD hong cau d 4 nhdm dan Ioc lai Dak Lak va Lam Dong. Doi Tugng va phuang pbap ngbien cdu: dieu tra dt ngang tren 4 dan tdc E Dc, M'ndng (tai Dak Lak nam 2005) vi Co ho, Chu ru (tai Liim Ddng nam 2006), sd dung phuang phip nhanh (Akira hirano - 1998) de phii hien cic trtfdng hop thieu C6PD. Ket qua: ty le thieu C6PD nhu sau-
