TAP CHI NGHIEN CU'U Y HOC
AP DUNG QUY TRJNH XAC DINH DOT BIEN GEN MA HOA Y^U T 6 VIII GAY BENH HEMOPHILIA A
Ta Minh Hidu, Tran Van Khanh, Ta Thanh Van Trudng Dai hpc Y Hi Ndi
Benh rol lo^n ddng miu dl tmyen Hemophilia A hay b$nh wa chay miu do dot biin gen F8 ma hda cho yiu to VIII. Dang dot biin gen F8 ehiim ti le cao nhit li dot biin diem (47,5 %), dot biin dio doan Intron 1 vi intron 22 (36,7 %). Cho din nay d Vipt Nam chwa cd nghien cim toin dien vi phin tich ddt biin gen F8, giiip cho viec phan tich phat hien ngwdi linh mang gen benh vi chin doan trwdc sinh benh Hemophilia A. Muc tieu: (1) Ap diing quy trinh ky thuit sinh hoe phin tir de phat hien dot biin gen F8 gay benh Hemophilia A, (2) Thwc hien quy trinh xie dnh ngwdi mang gen va chin doan trwdc sinh cho ngudi cd nguy ca cao. Doi tuong vi phuong phip:
Mpt thai phi,! cd con trai da dwpfc chin doin bj benh Hemophilia A dwa tren tieu ehuin lam sang va can lim sang dien hinh. DNA b0nh nhan dwac tich chiit vi phin tich dot biin bang ky thuit PCR vi glil trinh tw. Thai phu diKiie phin tich xie dmh li ngwdi mang gen dot biin vi chin doin trwdc sinh. Kit qua: Phit hien dot biin del2605C a benh nhan Hemophilia A, thai phu (nguoi me benh nhan) la ngwdi linh mang gen benh, thai nhi mang dpt biin del2605C, em gal cua benh nhin la ngwdi linh khdng mang gen bpnh. Kit luan: (1) Ap dung thanh cdng quy trinh xie dnh dot biin gen F8 gay bpnh Hemophilia A; (2) Ap dung thanh cdng quy trinh xie dinh ngudi mang gen vi ehin doin trwdc sinh cho thai phu (ngudi linh mang gen bpnh).
TO khoa: Hemophilia A, y l u td VIII, dot b i l n gen
I. OAT
VANDt
Hemophilia Id benh rdi loan ddng mdu dl truyin hay edn gpi Id benh ua ehdy mdu. Qud trinh ddng mdu phy thude vdo sy tuang tde ciia nhilu thdnh phin trong mdu. Vai trd eua he thong ddng mdu tao ra khoi fibrin bin vu-ng tai vj tri ton thuang vd ed tae dung elm mdu.
Mdt trong so cdc ylu to ddng mdu khdng ed hode bj thiiu hyt din din sy chay mdu kdo ddi khi thdnh maeh bj ton thuang vl nguydn do ndo dd. Bdnh hemophilia do thilu hut hode bit thudng chirc nang eiia ede ylu to ddng mdu:
yeu to VIII gdy benh hemophilia A, yiu to IX gdy bdnh hemophilia B, ylu to XI gdy bdnh hemophilia C. Hemophilia Id bdnh rdi loan ddng mdu di truyin hay gap nhit. Theo thong k§ cua to chirc Hemophilia t h i gidi, hidn nay c6 khoang 250.000 bdnh nhdn mic bdnh hemophilia vd ehi cd khoang 50.000 dugc diiu trj ddc hidu. Cho d i n nay elu true gen md hda ylu t l VIII (gen F8) vd ca c h i bdnh hpc phdn tu- b0nh hemophilia A dd duge nghidn cuu diy du vd rd rdng. Gen F8 Id mdt trong nhO-ng gen ldn nhit eg t h i , cd kfch thudc 186kb gom 26
exon. Khi gen F8 dot biin, t l bdo bj glam hoae mit kha ndng tong hgp ylu td VIII, gdy nen bdnh hemophilia A. Cd nhiiu dang dot biln gen F8 gdy benh hemophilia A, trong dd pho biln nhit Id dot biln dilm (47,5%) vd dao doan intron (36,7%) [6,7]. Hien nay, kT thudt gidl trinh ty todn bd 26 exon md hda gen F8 duge coi nhu tieu ehuin vdng phdt hien dot biln gdy bdnh Hemophilia A, ky thudt Long Distance PCR (LD-PCR) dugc sir dung d l phdt hidn dot biln dao doan intron 1 vd 22.
Thyc t l eho thiy vide ehln doan sdm benh hemophilia A bing ky thudt phdn tfch gen gliip ede bdc sy Idm sdng dua ra hudng dliu trj sdm, ehfnh xde vd eai thien ehit lugng eude sdng eho benh nhdn. Kit qua phdn tfch gen eung Id tiln d l quan trpng giiip ehan dodn trude sinh ddi vdi cdc doi tugng ed nguy eg eao (ngudi Idnh mang gen benh) vd dua ra tu vin di truyin nhim ngdn ngira vd giam ty Id sinh eon mIe bdnh. Cde nghien eiru v i bdnh hemophilia d nude ta mdi ehi tdp trung chu ylu vdo ty Id mIe bdnh, ddc dilm Idm sdng vd
TAP CHI NGHIEN CCPU Y HOC •
can Idm sdng, dilu trj, nhirng tdc ddng tam ly ddi vdi benh nhdn vd thanh vidn gia dinh [1, 2, 3, 4, 5]; chua cd nghien CLPU todn dien v i phdn tfch ddt biln gen F8 va phdt hien ngudi lanh mang gen benh. Xuit phat tip thue t l tren, de tai nghien ciru ndy dugc thyc hidn nhim muc tieu: Nghien eiru xdy dyng quy trinh xde djnh dot biln gen ma hda ylu to VIII gdy benh Hemophilia A.
II.
D 6 | T U ' Q N G V APHU'QNG
P H A P1. Ddi tu'ang
Nhdm ehu-ng: 3 ngudi nam binh thudng khde manh.
- Nhdm nghien ciru:
+ 1 benh nhan nam da dugc ehan dodn mIe. benh Hemophilia A dya vdo ede trieu chipng Idm sdng vd can Idm sang diln hlnh.
+ Ngudi me ciia benh nhan dang mang thai (thai phy) eIn thyc hien sdng lpc trude sinh theo sa dd pha he sau ddy:
Hinh 1. Sa do pha he gia dinh benh nhan Hemophilia A 2. Phu'ang phap
- KT thudt tdeh chiit DNA.
DNA duge tdeh chiit tir bach elu mdu ngoai vi theo quy trinh phenol/chloroform.
Ndng dp vd tinh sach eua DNA duge do tren mdy Nano-Drop, nhCrng miu DNA dat gid tri
OD280 / OD260 > 1,8 dugc sir dung d i phdn tfch.
KT thudt LD-PCR xae djnh dot biln dao doan intron 22.
* Nguydn t i c ky thuat: SO' dyng cap moi PQ duge thilt k i bam ddc hidu vdi mdt vung intron 22 va cap moi AB dugc thilt k l dae hieu eho 2 vung int22h2 va int22h3 (vung tuong dong vdi h-inh ty intron 22 gen F8). San phim khulch dai LD-PCR sir dung 2 cap mdi tren vdi mdu DNA ngudi blnh thudng eho 2 bang ed kfch thude 12 kb (tuang ipng vdi P+Q) vd 10 kb (tuang ipng vdi A+B). Dot biln dao doan intron 22 lam djeh ehuyen vj trf bdm mdi Q v i phfa d i u mut telomere va vj trf bam mil B sdt lai gdn vdi vj trf bam mil P San phim khulch dai LD-PCR thu duge 2 bang ed kfch thude 11 kb (P+B) & (Q+A), vd 10 kb (A+B). Trudng hgp ngudi Idnh mang dot biln dao doan Intron 22 se cho 3 bang san phim LD-PCR do mang mdt alen thudng vd mdt alen ddt biln.
* Tiln hdnh phan ipng: Cde thdnh phin phan ipng LD-PCR gom: 250 ng DNA; 50 nmol/L Tris.HCI, pH9.2; 2.25 mmol/L MgCb;
7.5% DMSO; 16mmol/L (NH4)2S04; 250 mmol/L dGTP vd deazad GTP; 500 mmol/L dNTPs vd 3.3 U Expand Long TaqDNA polymerase. Chu ki nhiet phan O'ng LD-PCR:
94°C/2 phut, 30 ehu ki (94°C/12 gidy, 65°C/30 giay, 68° C/12 phut).
KT thuat giai trinh ty xde djnh dot biln diim gen F8
Todn bd 26 exon eiia gen F8 duge khulch dai bdng ky thudt PCR va duge tiln hdnh giai trinh ty theo 2 chiiu d i xac djnh dot biln gdy bdnh. Do cd kich thude Idn nhit 3106 bp, exon 14 dugc thiit k l 5 cap mdi d i khulch dai todn bd chiiu ddi exon, nhu hlnh 2.
TAP CHI NGHIEN CLPU Y HOC
To cenrromere
To cenfromere
Int22h-1 (irVroqenic) 2 6 ^ 1
P Q I 2 k b
26 23 22 I
Xqfer
lnt22h-2 lnr22h-3 (proximal) (distal)
^ Xqre
B A 10 kb
^^t
B A 10 kb
H i ^
Tdi to hgp tuang dong
To centromere
2 6
1 1 kb P B
23 1 ,
• A m ~
lnl22h-3 (distal)
10 kb A B
t i A lnt22h-2 f
(proximal
I I kb Q A
Xqter Int22h-I (introgenic)
Ngudi Benh Ngudi lanh binh thudng nhan mang gen PQ
PB+AQ AB
k a t t e ^
VSaauMf JL^il
12kb 11 kb MOkb
I" n nil iirt 111 i I
14ab^
i4ef-<—
"^ "^ '^ "^ iTohr*— ^<^^<— _ ^ 1411'^—
Hinh 2. S a dd t h i l t k l cac doan mdi k h u l c h dai toan bp 26 exon gen F8
. K^T QUA
1. K i t qua xac dmh dot b i l n o- benh nhan Hemophilia A
Sau khi loai trir trudng hgp ddt biln dao doan intron 22 bing kT thudt LD-PCR, miu
TAP CHl NGHIEN CLPU Y HOC •
DNA cua bdnh nhan Hemophilia A dugc giai trinh tu todn bd 26 exon. Ket qua cho thay miu benh nhdn bj ddt biln mit nucleotid C tai vi tri 2605 cua exon 14.
Ngudi binh thudng
2 6 0 5 C
2 5 3 3 4 1
2 6 0 5 C del
. / Benh nhdn
2 9 3 6 4^
M
Hinh 3. Kit qua giai trinh tir exon 14 gen FS cua benh nhan va ngu'ai binh thu'd-ng Chu thich: l^ui ten chl vj trf nucleotide bj dot biin
2. Kit qua xdc djnh ngu'O'i lanh mang gen benh
Mdu DNA eiia ngudi me vd ngudi chj bdnh nhdn dugc tdeh chiit, khulch dai vd giai trinh ty exon 14, SU' dung cap mdi 14cd. Kit qua giai trinh ty eho thiy ngudi me la ngudi lanh mang gen dot biln del 2605C vd ngudi chj gdi Id ngudi Idnh khdng mang gen dot biin.
2605C 2605C i Chj gai San phu ^
Hinh 4. Ket qua giai trinh tu' exon 14 gen F8 cua miu ngu'ai me va mdu chj gdi ciia benh nhan Hemophilia A bj dot biln del 2605C
3. Kit qua chin doan tru'O'c sinh thai phu (ngu'ai me benh nhan)
Do duge xdc djnh Id ngudi mang gen bdnh ndn thai pHy thudc didn chi djnh ehln dodn trude sinh. Mdu t l bdo oi 18 tuin tuoi duge tdeh chilt DNA bdng phuang phdp Chelex true tiip khdng qua nudi ely t l bdo. Miu DNA thai nhi dugc tiln hdnh khulch dai dogn gen exon 14 bdng cdp mdi 14cd vd giai trinh ty. Kit qua giai trinh tu cho thiy mdu'DNA thai nhi mang ddt biln del 2605C.
2605C
Ngiroi binh thiroTng Thai nhi
r O T C S T C T T C C C i i O G A G T C C T i C 33 4 1 4»'
2605C del
•T T T GT - G ; T G T C T T G C GAC A'
as 37
/WM/VA/W/WWAMW
Hinh 5. Kit qua giai trinh tuf exon 14 gen FS mdu DNA thai nhi vd mdu chifng
TAP CHi NGHIEN CLPU Y HQC
IV. BAN LUAN
Nghien ciru ndy l i n d i u tidn dp dyng ky thudt LD- PCR vd giai trinh ty todn bd gen ma hda y l u to VIII d i xac djnh ddt biln gdy benh Hemophilia A vd da thu duge nhCrng kit qua sau: Bing ky thudt giai trinh ty gen, todn bd chilu ddJ ede doan md hda gen F8 da dugc giai ma. Dya tren trinh ty GeneBank (NM_000132), so sanh kit qua giai trinh ty gen eiia benh nhdn (eon trai thai phy) vdi eua ngudi binh thudng eho thiy: tai vj tri 2605,nueleotid C eua bdnh nhdn da bj mit. Dot bien del2605C nay gay tech khung djch md, lam thay doi todn bd trinh ty ede acid amin k l tip vj trf dot biln din hit chieu ddi phan tip protein ylu td VIII, gay ra benh Hemophilia A.
Kit qua giai trinh ty gen ciia mdu DNA thai nhi cung cd kit qua tuang ty: tai vj tri 2605, nucleotid C eua thai nhi bj m i t giong nhu cua ngudi anh trai, dong thdi nghien eu-j da xdc djnh thai nhi mang gidi tfnh nam. TCP nhirng kit qua trdn, thai phy dugc tu vin dinh chl thai. Do bdnh Hemophilia A Id benh di truyin nen dot biln del2605C la dot biln ehi diem eho vide xae djnh trang thai mang gen b§nh eua ede thdnh vidn trong gia dinh. Kit qua giai trinh tu gen tai vung dd xde djnh duge dot biin tren miu DNA eiia thai phy ed hlnh anh cde dinh chong len nhau sau diim. dot biln. Dilu ndy chl ra ring thai phy d trang thdi di hgp tip (mang gen bdnh); ben canh dd, 'miu DNA cua chj gdi bdnh nhdn cd trinh ty exon 14 hoan todn triing khdp vdi ngudi blnh thudng, nhu vdy chj gai bdnh nhan Id ngudi Idnh khdng mang gen benh. Lin d i u tidn tai Viet Nam, todn bp vung md hda eiia gen F8 eua benh nhdn Hemophilia A dugc giai trinh ty, trang thdi gen F8 cua ede thdnh vidn trong gia dinh b$nh nhdn Hemophilia A duge xde djnh.
Mdc du nghidn eiPu mdi ehi dipng lai d gia dinh eiia mdt bdnh nhdn Hemophilia A Vidt Nam, nhung nhirng kit qua thu duge eho thiy
sy thdnh edng bude diu trong vide dp dyng quy trinh phdt hien ddt biln gen md hda ylu td VIII, quy trinh xdc djnh ngudi mang gen vd ehln doan trude sinh bdnh Hemophilia A.
V. K^T LUAN
Ap dyng thdnh edng quy trinh xde djnh dot biln gen F8 gdy benh Hemophilia A. Ap dyng thanh edng quy trinh xdc djnh ngudi mang gen va ehln dodn trude sinh cho thai phy (ngudi lanh mang gen benh).
TAI
LIEU THAM
K H A O1. Vu Thj Minh Chau, (2001). Nghien ciPU dae diem lam sdng ciia benh nhan Hemophilia A gap d Vien Huylt hpc - Truyin mau. Luan van tdt nghiep bde sT ehuyen khoa d p II, Trudng Dal hpe Y Ha Ndi.
2. Tru'ang Thj Kim Dung (2001). Diiu c h l vd sir dyng kit tua lanh gldu ylu td VIII. Y hpe Viet Nam 3, 25 - 29.
3. Nguyin Minh Hiep va cs (1998). Dac dilm Idm sang benh Hemophilia d tre em tai Vien Nhi khoa. Cdng trinh NCKH ciia nghidn eiPU sinh, Nhd xuit ban Y hpe, 214 -219.
4. Cung Thj Ty va cs, (1997). Tinh hlnh benh Hemophilia d mdt sd dja phuang miln ble Viet nam. D l tdi nghidn ciPU elp bd dd nghiem thu nam.
5. Nguyin Thj Van (1999). Nghien cipu dae diim ton thuang khdp vd ea tren benh nhdn Hemophilia A. Ludn vdn thac sT y hpc, Trudng Dai hpc Y Ha Ndi.
6. Oldenburg, J., Ananyeva, N. M. &
Saenko, E. L. (2004). Molecular basis of haemophilia A. Haemophilia 10 (Suppl. 4), 133-139.
7. Graw J, Brackmann HH, Oldenburg J, Schneppenheim R, SpannagI M, Schwaab R. (2005). Haemophilia A: from mutation analysis to new therapies. Nat Rev Genet. 6 488-501.
TAP CHI NGHIEN CU'U Y HOC •
Summary
MUTATION DETECTION OF F8 GENE CAUSING HEMOPHILIA A
Hemophilia A is the most severe inherited bleeding disorder to affect human and is among the most common disorders of this kind. Hemophilia A is caused by F8 gene mutations which reduced levels of VIII coagulation factor. Point mutations account for as much as 47.5 %> whereas inversion intron 1 and 22 cause 36.7 % of all Hemophilia A eases. In Vietnam, so far there has been no full studies of mutation analysis for hemophilia A patients as well as carriers and prenatal diagnosis.
Objectives: Construeing of mutation analyis procedure of hemophilia A. Methods: This study based on analysis of a Hemophilia A family's genetic profile. Genomic DNA was extracted from venous blood collected in EDTA using phenol procedure. The 26 exons of F8 gene were amplified and checked for mutations by using Long PCR and Sequencing procedure. Results: The Hemophilia A patient and unborn fetus were found to have del2605C mutation. His mother is a carrier whereas his sister has a normal status. Conclusion: Successfully contructed a procedure to detect muations in F8 gene.
Keywords:. Hemophilia A, F8 mutation
NGHIEN CU'U PHAT HIEN NGU'ai LANH MANG GEN BENH VA
C H A N D O A N
TRU'aC SINH BENH THOAI HOA C a
T O YTrdn Van Khanh, Ta Thanh Van Truong Dal hpc Y Ha Ndi
Benh thoai hda ca tuy (spinal muscular atmphy: SMA) li bpnh ly dl tmyen lan tren nhiem sac the thudng.
Benh gay nen do dot bien gen SMN1 nam tren nhiim sic thi sd 5. Bpnh chia lim 3 the khie nhau tuy vio miec dp bleu hien bpnh tren iam sang. Phit hipn ngwdi mang gen bpnh vi chin doin trwdc sinh se lim giam tile mae bpnh. Muc tieu: Xay dung quy trinh phat hipn nguoi mang gen bpnh vi chin doin truoc sinh bpnh thoii hda ca tuy (SMA). Ddi twang vi phuong phip nghien ciru: 10 gia dinh va 2 thai phu ed tiin sir sinh con bj bpnh SMA;
DNA dwQfc tich chiit tir miu vi ti bio oi ciia cic thanh vien gia dinh va thai phij; Sir dung ky thuit MLPA, STR, RFLP. Kit qua: 48% cae thanh vien gia dinh ngwdi bpnh SMA dwp/c phit hipn la ngwdi mang gen bpnh; 02 thai nhi dwac chin doan khdng mac bpnh SMA. Kit luan: Buoc diu da thinh edng trong vipc phit hipn ngudi linh mang gen bpnh va ehin doan trude sinh benh thoii hoa ca tiiy a Vipt Nam.
TCr khoa: benh thoai hda co" tuy, nguo'i lanh mang gen benh, chin doan trLrac sinh
I. DAT
VANDt
Benh thodi hda ea tuy hay benh SMA thodi hda tuin tiln t l bdo thin kinh sirng trudc (Spinal Muscular Atrophy-SMA) Id mdt trong tuy song din d i n suy y l u vd thodi hda doi nhirng benh ly thdn kinh ea di truyin hay gap xu'ng gde ehi [2,3]. Tren Idm sdng, bdnh nhi nhit vdi t i n suit benh khd cao (1/10.000- giam van ddng tiln triln vdi cde ddc trung Id 1/25.000) chl dii'ng sau bdnh loan dudng ea nhugc ea doi xirng goc chi, truang lyc ca Duchenne. Thodi hda ca tuy Id benh di truyin giam, phan xa gdn xuang giam hodc mit, ludi lan trdn nhiim sde the so 5. Ddt biln gen Idm rung, biln dang long nguc vd cirng khdp. Dae
