JfyP CHl NGHIEN CLKU Y HQC
PHAT HIEN DOT BI^N GEN ATP7B TREN GIADiNH BENH NHAN WILSON
Phan T 6 n H o i n g \ T r i n VSn K h S n h \ H6 Ciim T u \ Nguyfin Vi§t T i 6 n ^ T? Thanh Van^
' Trwdng Oai hoc Y Hd Ndi. 'B0nh vien Phu sdn Trung wong Wilson Id b$nh di truydn l$n trdn nhidm sdc thd thwdng gdy ndn do ddt bidn gen ATP7B Gen ndy cd vai trd quen trong trong didu hda qud trinh hdp thu. phdn phdi vd thdi tru- ddng trong ca thd. Nghien cim dwoc thwc hi$n vdl muc tidu phdt hidn ddt bidn gen ATP7B trdn 6 bdnh nhdn dwac chdn dodn mdc b$nh Wilson trong cung mdt gia dinh. Gen ATP7B duvc khudch d$i bdng 21 c$p mdi ddc hidu cho 21 axon cua
*gen. sdn phdm PCR dwac gidi trinh tw gen dd xdc djnh ddt bidn Kdt qud cho thdy 6/6 benh nhdn dwoc phdt hidn cd 2 ddt bidn didm dang di hap ti> trdn gen ATP7B bao gdm: ins47-48CGGCG trdn exon 1 vd P.V456L trdn exon 3
Ti> kh6a: gen ATP7B, b^nh Wilson, d$t bifin gen
I. DAT VAN B^
B6nh Wilson, hay b0nh r6i loan chuyen hda ddng, \d m6t benh ly lien quan den ton thu'ong gen D^y la b6nh dt truydn ian tr&n nhi§m sac the (NST) thu'6'ng, dong tich tij 6"
mpt s6 ca quan trong co" the, dac biet la gan va nao, gay nhieu bi6n chirng Tr6n the gidi, tan SLiai mac b&nh dao dgng trong khoang 1/30.000 - 1/100 000 ngutf\ [1]. 0 Viet Nam, 8 tru'd'ng ho'p benh Wilson dau li6n du'o'c b^o cao bat Bill Quoc Hu'O'ng v^ cpng su lai hOi nght Than ktnh hoc quoc te lan Ihu' IX tgi My nam 1969 [2] Nhin chung, nghien ciru ve b6nh Wilson a nudc la chu'a nhieu, chi la nhO'ng nghien cCru ve dac diem lam sang va can lam sang [3, 4). NSm 2011, Trung tam Nghi6n ciru Gen- Protein thudc Tru'dng dgi hoc Y Y Ha NOI 3d lien hanh nghiSn ciru phat hi^n dot bien gen ATP7B gay b6nh Wilson [5]
V^ sinh bfenh hoc phan t i i , nguyfen nhan gay benh Wilson la do thieu hut enzyme
Dia chi lien /j# Ta Thdnh VSn - Trudng Bai hpc Y HA Ndi S6 1 Ton That Tung. Ddng Da, Ha NCi
Email tathanhvan@hmu edu vn Ngaynhan 03/03/2013 Ngdy duoc chip thudn 26/4/2013
ATPase typ P (P-ATPase), du'p'c mS hda bdt gen ATP7B trgn NST 13q14 3. Enzym P-ATPase dong vai Iro van chuyen dong qua mang te bao S i / thi^u hut ho$c giam hoat tinh ciJa enzym d i n den giam su bai tiel dong li> le bao gan vao mat va gdy ir dong dong lat gan Khi lu'P'ng ddng trong gan tang qua mu'C, ddng se theo h§ thong tuan hoan l d i cac ca quan nhu" nao, mat, Ih^n vd gay cac ton thu'O'ng cho cac Cff quan nay
Chan doan b0nh Wilson cd the du'a tren cac tneu chirng \dm sang cua gan, nao, vong Kayser - Fleischer d mat va cac xel nghietn hda sinh trong mau (n6ng do dong trong nu'dc tieu 24 gid, ham lu'ong ceruloplasmin huy^t thanh, n6ng do dong trong gan) [6]. Cac nghien ciru d mirc 66 sinh hoc phan t i i la vo cung can thiel de xac dinh chinh xac cac d<it bien gay benh va xac dtnh ngu-di ianh mang gen bgnh. Bdt vay de tat du'o'c thu'c hien nhSm muc t i e u : Phat hien dpi bi6n gen ATP7B tren benh nhan du'ac c h i n doan mSc b6nh Wilson.
II. 06\ TU'O'NG VA PHU'aNG PHAP
1. D 6 i tu'O'ng
TCNCYH 82 (2) - 2013
• TAP CHi NGHteN CU'U Y HQC Nh6m bpnh 6 b§nh nhan trong mot gia
fllnh, duac chan doan mSc b$nh Wilson du'a vSo trieu chirng lam sang va x6t nghi6m hda smh mau
Nhdm chirng 1 ngu'di binh thu'dng, ti^n sir gia dinh khdng cd ngu'di m^c b§nh di truydn NghiSn ciru du'p'c thyc hi§n tat trung tam Nghien ciru Gen - Protein, tru'dng Ogi hpc Y
2. Phipang phap
Mlu benh p h i m : 2 ml mau ngoai vi ch6ng dflng bang EDTA
Tcich DNA tlf mau ngoai vi va khudch d^i gen (PCR) DNA long so du'p'c tach chi^t lir mau toan phan bang phenol - chloroform - isopropanol (25 2 4 1 ) . Toan bo 21 exon cua gen ATP7B du'p'c khuech dai vdi cac cap moi 6dc hi&u
Giai trinh ti^ gen va phan tich dot bidn:
san pham PCR du'dc giai trinh tu- tru'C tiep trgn may ABl 3100 Genetic Analyzer Kel qua du'p'c thu thap va xir ly bang phan mem ABl PRISM^^ 3100 - Avant Data Collection, DNA Sequencing Analysis 5 2 va BLAST NCBl
III. K^TQUA
1. Kdt qua tach chidt DNA
Tat ca mau DNA du'p'c tach chiel cd nong d6 va 66 tinh sach cao vdi ty s6 mat d6 quang d bu'dc sdng 260 nm/280 nm la 1,8^1,9.
2. Kdt qua xdc djnh ddt bidn gen trdn nhom benh nhan
21 cap moi dac hieu du'ac sir dung de khuyech dai gen ATP7B, Kich thu'dc cua cac san pham PCR trong khoang 100 - 200 bp Hinh 1 mmh hoa san pham PCR ciia gen
6 M C(-i CM
300bp -
Hinh 1. Hinh anh san phSm PCR doan gen tip exon 3 cua gen ATP7B C, mdu ddt chO'ng, 1 - 6 mdu benh nhan Marker0174.
San pham PCR thu du-ac la dac hieu, rd n^tva dam bao cho phan irng sequencing liep theo de phal hien d6l bien diem Kdt qua cho thay cd 2 ddt bi4n diem di hap tir, gi6ng nhau ff 6 thanh vien trong gia dinh, ins47- 48CGGCG (tai exon 1) va p V456L (lai exon 3) (hinh 2)
Giai trinh tu gen ATP7B phat hi6n cung dang ddt bidn Iren 6 bfenh nhan (ma so W7 00 ddn W7-05) BOt bidn dt hap tir thdm
5 nucleotid CGGCG sau vi tri 47 tai exon 1 (NM - 000053, GeneBank) lam thay ddi khung dich ma Day la dpi bidn mdi chu'a duac cdng bo tai ngan hang dCr ligu ve benh Wilson (wllsondiseas med.ualberta ca) Dot bidn di hap tir thir hai la thay the G > C d vi tri 1523 (C.1523G > C, NM_000053, GeneBank) tai exon 3 iam cho b6 ba lai vi tri 456 ma hda Valine chuyen Ihanh Leucm (p,Val456Leu) (hinh 3)
TAP CHl NGHIEN CU'U Y HQC
Dot bien c 47-48insCGGCG (dOt b i i n mdi)
C.47 48insCGGC C.47G
h'^)'V'S: 'mi^mtA
B e n h n h a n ma s 6 W7.00 N g i r w l b i n h t h u - f f n g Dot bien missense: p Vai456Leu (NOV: non disease variant)
c. 1523G > CI455 p.Val456Leu •=• 1523G p Val456
J, *
l\ 'i ^'i'l' !\ >^.i\fi\f"\i\ l\f\' l\
[__ -- - > . - - 1 ' I - ;-. - i . •• ' - . - 1 - ^ : . , ^
B e n h n h a n ma s o W7.00 Ngu-di b i n h t h u ' d n g Hinh 2. Hinh anh giai t r i n h tu gen ATP7B exon 1 va exon 3 cua b e n h nhan ma s d W7.00
Mui ten thdng dung chi vi tn dot bidn. cac chCt so tren mui len chi vi tri nucleotid vd acid amin thay doi
O I D
W7 (H> W 7 n i VJ7 02 W7.05
Hinh 3. Pha he c u a g i a d i n h b e n h n h a n W i l s o n ma s o W 7 . 0 0
Chu- so tren pha he Ihd hien ndng dd Cerulapiasmin huydt thanh cua cac benh nhdn trong gia dinh (mg/m!) (^ NO: binh thuoncj \Z\Nam. binh thuung % NO: benh. ^ Nam. benh.
Ket qua phat hign d6t bien phu hap vdi kel qua xdt nghidm hda stnh mau, cac bdnh nhan ddu c6 chi s6 Ceruloplasmin huyet Ihanh thap han binh thu'dng
TCNCYH 82 (2) - 2013
IV. BAN LUAN
Cho den nay, khoang han 500 ddt bi^n da ijir^c tim thay trdn bdnh nhan mac bdnh Wilson [7, 8] Moi quoc gia, m6i chung tOc ngirdi khac nhau cd cac logi dot bien khac nhau DOt bi^n pHis1069Glu la dOl bien Ihu'trng gap nhat trong qu^n the ngudi da IrSng, chi^m khoang 37% den 63% t6ng so dot bien Bdnh nhan ngu'di Trung Qu6c, ddt bien p Arg778Leu chi^m 34 - 38% l6ng so dOt bien [9, 10] Protein ATP7B mang d^y du cac ij|c diem dac Iru'ng cho mOt protein thuOc hp protein P - APTase vdi 5 viing chirc nSng chinh Vung MBSs (vi tri bam cho cSc nguyen tu'dong), vung N (vung bam ATP), vung P (vung phosphor hda), vung A (vung khir phosphor) va vung xuydn mang ( n i m tren 8 exon exon 6 - exon 12 va exon 19, exon 20) [1]
OOI bien thdm nucleotid c47-48insCGGCG ndm trdn exon 1 cua gen ATP7B, day la vung nam ngay tru'dc vi tri bam cua d6ng Dot bien dl hgp tiK nay gSy Idch khung dich ma, do vay anh hudng den toan bo doan gen phia sau trang viec long hap protein P-ATPase 0 nhOng bdnh nhan mang dot bien nay, kdt hp'p vd'i cac dot bidn di hap tCr khac, la nguyen nhan anh hudng den nong dp ceruloplasmin huydt thanh Tuy vdy, day la bdnh di truydn trSn gen ian, ndn su' cd m^t cua 2 dOt bien di hop tu cOng tao ra su' "cpng hu'dng" anh hirong ldi su' hlnh thanh ciia protein P-ATPase
Dot bien lhay the, p Val456Leu da du'ac c6ng bo la dpi bien khdng gay benh (non disease variant). Tuy nhidn, ddt bien nay luon
>!uat hidn cung vdi cac ddt bidn khac, day cd the la yeu to iam tang thdm mirc dd nang cua b^nh, the hidn qua su' tut giam cua nong do ceruloplasmin huyel thanh DOt bi^n nay chi xuat hi0n d bdnh nhSn Wilson, khOng c6 d igu'fi'i binh thu'dng, dieu dd chirng td day la
T ^ P CHi NGHlfeN CtPU Y HQC bien dl hay SNP (single nucleotide polymorphism) dSc Iru'ng cua quan the ngu'di mSc bdnh Wilson.
Bdnh nhan Wilson cd the mang mdt gen bdnh cua ngu'di bo ho^c mOt gen bdnh cua ngu'di me hoac ca hai gen bdnh cua ngu'di bo hoac cua ngu'di me. Bo, mp cua bdnh nhan cd I h l mang gen ddng hap lan hoac gen di hap c a c anh chi em cua bdnh nhan c6 the mang' gen bdnh hoSc khdng Nhdng ngu'di mang gen bdnh deu c6 kha nang di truyen cho con chau Mpt diem khac bidt so vdi nhidu bdnh di truyen khac la ngirdi mang gen di hap tir cung bieu hidn benh va thu'dng kdm theo dgng dOl bidn khac [1, 11]
Theo nhan dmh tren, pha he gia dinh bdnh nhan Wilson cho thdy (hinh 3) de nhirng ngu'di con a thd he F IV mdc bdnh, thi chong cua benh nhan W7 00, W7 01 (thd he F llf) phai la nhirng ngirdi mang gen bdnh Tu-ang tu", cap vo chong a thd he F 11- phai la nhCpng ngu'di mang gen benh theo dung quy luat Menden Tuy nhien, nhom nghidn ciru co gia thuyel khac de ly giai linh di truydn Irong pha he gia dinh benh nhan s6 W7 00: do la du'a vao tinh chat ciia ca thd di hap (Heterozygosity) Khai mem nay doi khi da bi nhdm l l n vdi ngu'di ianh mang gen bdnh (earner) Ca the dj hap trong y smh hpc di truydn la hidn tuang d cac alien lan chira hai dot bidn di hap tir, nam d vi tri (locus) co kha nang gay bdnh ly di truydn 0 nhung ca the nay, hai dot bidn di hap tir cua ciing mOt gen nhu'ng thudc hai alien khac nhau (nghTa la hai alien ciing mang dot bidn nhu'ng n i m a vi tri khac nhau)[12] Dac diem nay cho t h l y tinh da dang vd di truydn cua cdc bdnh ly di truydn gen lan tren n h i l m s i c the Ihudng Bdnh hinh thanh sau nhidu lan trao doi cheo - hoan vi gen
So vdi cac cac the mang dot bidn dOng
TAP CHf NGHIEN CLPU Y HQC •
hp'p l^n, d cac ca t h ^ d| hp'p, bOnh x u l t hidn muOn hen va bi^u hi0n lam sdng cOng nh? han Khdi ni$m ndy giSi thich rO han s y x u l t hidn bdnh a th^ h$ thir III va IV cua phd hd gia dinh s6 W7 00 Cac bdnh nhan t h ^ hd F,IV cd th^ mang gen chira d6ng thdi hat dOl bien d| hdp tu' trdn ciing mOt alien (do trao d6i chdo - hoan v| gen) du'p'c nh^n tiJ' ngu'di mp ( I h l hd F 111) NhO'ng ngu'di mp nay nhdn 2 dot b i l n d| hp'p tir ngu'di mp cua minh ( t h i hd F 11) - la ngu'di c6 I h l mang dac d i l m ca the d|
hdp D i l u nay can du'p'c k i l m chirng ve iam sdng d nhirng ngudi IhuOc I h l thd F 11
V. K E T KUAN
Nghidn ciru da phat hidn du'dc 2 dang ddt b i l n diem di hap tir. ins47 - 48CGGCG trdn exon 1 vd p V456L trdn exon 3 d 6 bdnh nhan Wilson trong mot gia dinh
Kdt qua cd y nghTa trong vide sang Ipc ngu'di mang gen vd cac the di hdp tir Didu nay khong nhu'ng gdp p h i n chan doan va dieu tn sdm cho benh nhan Wilson (thu'dng x u l t hidn muon) ma cdn cd la lien de cho cac nghien ciru v l bdnh fy di t r u y i n gen l$n ndt chung vd benh Wilson ndi ndng trong lu'ang lai
Lo'i cam an
Nghidn ciru du'ac thu'c hien lai Trung ldm nghidn ciru Gen - Protein, Tru'dng Dai hgc Y Ha NOi trdn ca s d cua d l tai d p BO' "Nghidn CO'U U'ng dung cdc ky thudt sinh hoc phdn tt>
dd xdc dmh dpt bidn gen ATP7B gay bdnh Wilson d Vidt Nam", vdi s y tai trp' kmh phf tir ngan sach s y nghiep Khoa hpc cap Bp Y l l nam 2012
TAI LIEU THAM K H A O
1. Xin-Hua Li, Yi Lu, Yun U n g et al (2011). Clinical and molecular characterization of Wilson's disease in China: identification of
14 novel mutations, BMC Medical Genetics.
12,6.
2. Chu v a n T u ^ n g , Le Vdn Thi4ng, N g u y i n Thj Hi4n va c6ng sy- (1975 - 1 9 7 9 ) . B$nh Wilson d trd em (qua hai tru'dng hdp) Ky ydu cdng trinh nghidn ciru khoa hgc, Vidn bcio vd sire khoe tre e m
3. Nguydn Quach Thu Thuy (2006). Dac d i l m ldm sang va c | n iam sang b0nh Wilson d tre em lgi Vidn Nht Qu6c gia Ludn vdn Bdc sl chuydn khoa cdp II.
4. Ld Oi>c Hinh (1989 - 1990). MOt s6 ddc d i l m bdnh Wilson d Vidt Nam, Ky ydu cdng trinh nghidn cu-u khoa hpc, 3, 316-334
5. Ta Thanh Vdn, Hd C l m T u , Ta Minh H i l u , Tran Van Khanh (2011). Xay d y n g quy trinh phat hidn dOt b i l n gen ATP7B gay bdnh Wilson, Tap chl nghidn ciru Y hpc, 74 (3), 26 - 29.
6. Cumings JN (1951). The effects of B A L in hepatolenticular degeneration Brain 74(1), 1 0 ^ 2 2
7. Peter Ferenci (2006). Regional distnbu- tion of mutations of the ATP7B gene in pa- tients vi/ith Wilson disease impact on genetic testing, Hum Genet. 120, 151 - 1 5 9
8. Aftab Ala, Ann P Walker, Keyoumars Ashkan et al (2007). Wilson's disease. Lan- cet. 369, 397 - 408
9. Danadevil Kuppala, Jie Deng, George J . Brewer et al (2009). Wilson Disease Muta- tions in the American Population Identification of Five Novrl Mutation in ATP7B, The Open Hepatology Journal, 1 , 1 - 4
10. Lei Wan, Chang - Hai Tsai, Yuhsin Tsai et al (2006). Mutation analysis of Taiwan- ese Wilson disease patients Biochemical and Biophysical Research Communications, 345, 734 - 738.
11. G. Loudianos, V. Dessi, M. Lovlcu, A.
Angius, B et al (1999). Mutation analysis in
TCNCYH 82 (2)-2013
• TAP CHl NGHlfiN CtfU Y HQC patients of Mediterranean descent with Wilson (2000) Compound heterozygous tiemochro- disease, identification of 19 novel mutations, J matosis genotype predicts increased iron and Met) Genet 36(11), 833 - 836 erytlirocyte indices in women, Clinicai Chemis-
12. Rossi E, Olynyk JK, Cullen DJ et al try, 46 (2), 162 -166,
Summary
MUTATION ANALYSIS OF ATP7B GENE IN WILSON DISEASE FAMILY
Wilson disease an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene that encodes a P-type copper transporting ATPase, The aim of this study was to screen and detect mutations of the ATP7B gene in a Wilson disease famlily (n = 6), Ivlutattons were screened and detected by DNA sequencing in 21 exon of ATP7B gene The results showed tiiat 6/6 patients had 2 different heterozygous mutations including ins47_48CGGCG in exon 1 and P.V456L in exon 3 of ATP7B gene
Keywords:
XAC DjNH DOT BIEN GEN VA PHAT HIEN NGU-QI LANH MANG GEN BENH TANG SAN THLKCNG THAN BAM SINH
THE THIEU 21 - HYDROXYLASE
Ngo Thj Thu Hu'0'ng\ Tr^n Van K h a n h \ Nguydn Vi§t Ti#n', Nguydn Phii D a t \ Ta Thanh Van^
' Trwong Dai hgc Y Ha NQI. ^B$nh vien Phu sdn Trung wong Tang san thwong th$n bdm smh do thieu hut enzym 21- hydroxylase la bdnh di truyen lan nhidm sac the thirdng do dot bidn gen CYP21A2 Nghidn ciru ndy dwoc thwc hidn vdi muc tieu (1) xdc dmh ddt bien gen tr^n benh nhdn tang san thwang thdn bdm sinh the thidu hut enzym •21- hydroxylase. (2) phat hi^n ngwdi lanb mang gen benh cho cdc thanh vien gia dinh cd quan he huydt thdng vdi benh nhdn Nghidn ciru dupc tien hanh tren 33 b$nh nhdn va 78 thanh vien thudc 33 gia dmh cua nhCmg benh nhan tang san thwong thdn bam smh; Ky thudt giai trinh tw gen vd MLPA dwoc sw dung dd xac dinh dot bidn vd ngwdi Idnh mang gen ti^nh Ket qua cho thdy ddi vdt nhdm bdnh nhdn, 33/33 (100%) benh nhan da dwoc phat hien ddt bidn vdi 7 il9ng dot bien khdc nhau. tmng dd ddt bidn 656 /v'a > G (IVS2-13A/C > G) chiem ti 1$ cao nhat 13/33 (3S,5%}, tidp theo Id dqt bidn xda doan chiem ti le 7/33 (21,2%), ddi vdi nhdm ngudi Idnh mang gen benh.
32/33 ngwdi bd, 33/33 ngudi me vd 5/12 thdnh vien gta dinh khdc (anh, cht. em) dwdc xac djnh la ngudi lanh mang gen b$nh Ket qua thu dwac la co sd di truydn quan frong cho chan dodn trudc smh vd tw vdn di tmyen nhdm lam gidm ty 1$ mdc bdnh va cd phdc do didu trj sdm cho cdc thai nhi ngay tir nhung tudn ddu
Tfrkhoa: tang san thi/p-ng th^n b^m sinh, dot biln gen CYP21A2, ngu-o-i lanh mang gen benh
