DOT BI^N GEN HBx 6 BENH NHAN

- TAP CHI NGHllN CU'U Y HQC

DOT BI^N GEN HBx 6 BENH N H A N

• •

UNG THLf GAN NHI^M VIRUS VIEM GAN B

Trln Hd H i l u \ Le Hu'u Song^

^Bdnh vidn 175; ^Bdnh vidn Tmng wang Qudn ddi 108 Dpt biin gen HBx ctJa vimt vidm gan B (HBV) da dwac ehung minh Id cd lien quan din sw tiin triin thdnh ung thw gan (UTG). Nghidn cwu nhim khdo sdt ddt biin gen HBx trdn bdnh nhdn UTG nhiem HBV va bwdc diu tim hiiu mdi lien quan cua nd vdi mdt sd chl sd edn Idm sang khde. Kit qua nghidn cwu cho thiy cdc ddt biin tren gen HBx dwac tim thiy Id A1762T, G1764A. C1653T. T1753C/G va C1766T. Trong dd cdc dpt biin A1762T, G1764A vd C1653Tld thwdng gdp nhitvdityip twang ung Id 66%, 83% va 81%. Dpt biin gen HBx tpi cde diim A1762TvdG1764A cd ty Id cao han trdn nhdm bidt hda kdm so vdi nhdm bipt hda cao (100% so vdi 67%. p < 0,05). Ddt biin gen HBx khdng lien quan din ehdc nang gan cung nhw tinh trang mang khdng nguyen e. Ddt biin gen HBx c6ty Id twang ddi eao trdn bpnh nhdn UTG vd cd lien quan din dd bidt hda cua ti bdo gan.

Tir khoa: dpt biln gen, HBx, ung thu> gan

I. OAT VAN Dt

Ung thu- t l bdo gan nguyen phdt (UTG) hay la ung thu- bilu md t l bdo gan (Hepatocellular Carcinoma, HCC) la bfnh ly thudng gap dfrng hang thfr 5 tren the gidi, cd ty If tfr vong dfrng hang thfr 3 trong cdc tfr vong lien quan d i n ung thu- [1, 2], Nguyen nhdn gdy UTG da du-gc xdc djnh Id do virut vidm gan B (HBV), C (HCV) vd mft sd hda chit nhu' Aflatoxin. Trong dd HBV Id nguydn nhdn chii y l u , chilm han 90% cdc trudng hgp UTG. Tuy nhidn, khdng phai t i t ca cde benh nhdn nhilm HBV d i u tiln triln thdnh UTG. Nhiem HBV cd t h i gdy vidm gan c l p tinh tg hdi phue, hodc tiln triln thdnh vidm gan man tinh, xa gan, viem gan dc finh, hodc trd thanh ngu-dl mang HBV mgn tfnh khdng trifu chfrng [3]. Nguyen nhdn nao d i n d i n sg khde biet dd v i n cdn nhilu tranh ludn. Mft trong nhfrng v l n d l du-gc .nhilu ngu-di quan tdm dd Id sg khde biet v l kilu gen, dft biln gen eua HBV. Gen HBx eua HBV Id mft gene cd kich thu'dc nhd, vdi khoang 460 nucleofid, ma hda cho 154 acid amin. D i l m khdi dau eua gene ndy Id nucleotid 1374 vd dilm kit thuc la

nucleotid 1836. VI vdy gen HBx cd phan chdng lln Idn mft doan gene ma hda enzym polymerase (tfr nucleotid 1374 din nucleotid 1621) vd chdng lan len gen pre-Core mot doan tfr 1814 din 1836. Nhu vay, khi xudt hien mot diem dot biln tren 2 doan gen chdng lln ndy khdng chi anh hudng den mft gen ma cd the anh hudng din chfrc ndng cua ca 2 gen. Nhilu nghidn cfru dd chi ra dot biln gene HBx cd lien quan chat ehe vdi sg filn triln thdnh UTG tren bfnh nhdn nhilm HBV [4, 5, 6].

Vift Nam Id nudc cd ngu'di nhilm HBV dfrng hdng eao nhit t h i gidi, ty If HBsAg (+) d ngudi ldn khoe manh tfr 10 20% cd nai ldn tdi 26% [7]. Nhu- vdy, wdc tinh cd han 10 trifu ngu-di dang mang HBV man tinh d nudc ta vd nguy ca phdt sinh UTG d nhfrng ngudi nay Id r i t ldn. Tuy nhidn, sd lifu v l ty If vd mdi lidn quan gifra dft biln gen HBx vdi bfnh canh UTG d nudc ta v i n cdn hgn c h l . Do dd, ChQng tdi fien hdnh nghien cfru d l tdi ndy nhim muc fidu xdc djnh ty If dft biln gen HBx cua HBV d bfnh nhdn ung thu gan nhilm HBV va bu-dc d i u fim hilu mdi lidn quan gifra dft biln ndy vdi mft sd chi sd xdt nghifm khde.

TCNCYH 76 (5)-2011

•Jii'CHllc [UYHQC

II. 061 TUONG VA PHUONG PHAP

I.Ddl tu'gng

Tdng sd cd 94 bfnh nhdn UTG dilu trj tgi Khoa Tidu hda, bfnh vifn Trung uang Qudn dfi 108 dd du-gc du-a vdo nghidn "cfru. Tidu chuln Iga chpn bfnh nhdn ung thu gan:

Ldm sdng: bfnh nhdn mft mdi dn udng kdm, giy sut cdn nhanh. Cd t h i cd cdc trifu chfrng khde nhu gan to du-di bd su-dn, bd sic mft df chic, ed u cgc Idn nhdn ndi trdn b l mft, dau khi thdm khdm.

Xdt nghifm: ed t h i cd mft trong nhfrng diu hifu nhu AFP tdng cao, bilirubin trge tilp vd gidn filp, AST, ALT cd t h i tdng. Sidu dm phdt hifn khdi tdng dm khu tru hodc lan tda.

Chgp CT hodc MRI xdc djnh cd khdi u. Sinh thilt gan xdt'nghifm Idm t l bdo hpc hodc/vd md bfnh hpc eho k i t qua xdc djnh cd UTG vd mfre df bift hod kdm theo. Xdt nghiem huylt thanh cd HBsAg (+).

2. Phu'ang phdp

Nghidn cfru du'gc filn hdnh theo phuang phdp md t l elt ngang. Cdc xdt nghifm vk huylt hpc, sinh hda, miln djch, chin dodn hlnh anh dugc filn hdnh thu'dng quy tgi cdc Khoa xdt nghifm bfnh vifn TUQD 108. Chpc hut t l bdo gan du'gc tiln hdnh tai Khoa tidu hda, miu bfnh phim du'gc xfr ly vd xdt nghifm t l bdo hpc tgi Khoa Giai phiu bfnh ly, bfnh vifn Trung u-ang Qudn dfi 108. Xdt nghifm gill trinh tg gene HBx du-gc filn hdnh tgi Khoa Sinh hpc phdn tfr, bfnh vifn Trung uang Qudn dfi 108.

Phdn tich thdng kd

Cdc sd lifu dugc phdn tfch bing thudt todn non-parametric Mann-Whitney L/-test, y^ test, so sdnh khdng ddi xfrng T-test, so sdnh 2 ty If, 2 sd trung binh bdng cdc phin mim Statview, version 4.57 (www.statview.com) vd chuang trinh STATA (www.stata.eom) Stata.

KllT QUA

1. Ddc dilm chung v l benh nhan

Bang 1. Ddc dilm benh nhdn nghidn cii'u ChTsd

Gid trj Chl sd Gid trj

Uamlniy 78/16 Bilirubin (pMol/L) 35,4 ±15,1

Tudi 56,1 ± 12,2 Protein TP (g/L)

72 ±13

TC (G/L) 107 ±34 Albumin (g/L)

35 ± 3

AST (U/L) 110,8 ±107.4 Prothrombin (%)

72 ± 1 0

ALT (U/L) 100,3 ±91,9

HBeAg (+/-) 56/40 Tdng sd cd 94 bfnh nhdn, trong dd nam chilm ty If 82,97%. Cde chi sd nhu- tilu c l u , enzym AST, ALT, bilirubin fflu ed biln ddi. Cd 56/94 bfnh nhdn ed HBeAg (+), chilm 59,57%. 70.

2. Cac ddt biln thu'd'ng gdp tren gen HBx

Cd 3 dft biln dilm du-gc fim thiy vdi ty If tu-ang ddi cao Id C1653T, A1762T vd G1764A.

Ngodi ra cdn ed cdc dilm dft biln khde nhu' T1753C vd C1766T (hlnh 1, 2, 3).

3. So sdnh ty If dot biln gen HBx tren cdc nhdm theo mipc do bift hda t l bdo gan

Dft biln gen HBx cd ty If t,u-ang ddi cao trdn bfnh nhdn UTG, chilm tfr 66% d i n 83%. Dft biln gene HBX tai vj tri C1653T khdng ed sg khde bift gifra cde nhdm theo mfre dd bift hda t l bdo gan. D f t biln dilm ddi A1762T/G1764A xay ra d 100% bfnh nhdn ed bift hda kdm, giam

" T A P CHl NGHllN CCPU Y HQC

d i n d nhdm bift hda vfra vd eao, sg khde bift ed y nghTa thdng kd gifra nhdm bift hda cao vd kdm (xem bang 2).

250 260 C C C ii .a. a 0 T C T T .•s^ C .-••. T .», A 0 .i( C

270 * 280 C C C . ^ A O G T C T T A T A T A A O A

C1663T H94Y

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Hinh 1. Hinh dnh dft biln tai vj tri 1653.

[Nhfn xdt: Tgi vj tri 1653 binh thudng Id cytosine (A), khi dft biln ed 2 dgng Id ddng hgp tfr (C—T,

homozygous, hlnh B) vd di hgp tfr C—>C/T, heterozygous, hlnh C)]

BT

BT

1753

A1762T K130M

i l

/ W

- ' • • r ' " ' - — ^ '

DB

0 ^M

DB

A1762T j ei764A/G K130M* V131I

• 1753 1766

Hinh 2. Hinh anh dot biln tai dilm

1762. (Nhan xet: D f t biln tgi vj tri 1762 ^^'"*^ 3. Hinh anh dot biln nhilu dilm tren 1 bfnh (A-^T) diln hinh, khdng ed dft biln tgi vj "han. (Nhfn xdt: D f t biln tgi 2 dilm thudng gfp

^^'^ 1754) A l 762T/G1764A ddng hgp tfr vd dj hgp tfr. Ngodi ra cdn cd mft sd dft biln khde kit hgp tgi cdc vj tri

1753(T^C/G, 1766 (C-»T))

4. So sdnh mdt sd chi sd can lam sang khac glQ'a cdc benh nhan cd dot biln gene HBx va khdng dot biln gene HBx

Kit qua phdn tfch eho thiy ndng df AST, ALT, bilirubin, protein, albumin, ty If prothrombin, sd lu-gng tilu c l u , ty If HBeAg (+/-) ...gifra ddc bfnh nhdn cd gene HBx dft biln vd gene HBx binh thu-dng khdng ed sg khac bift ndo du-gc ghi nhdn (sd lifu khdng trinh bdy). Tu-ang tg nhu vfy, ndng d f HBV DNA cQng khdng ed sg bift gifra nhdm ed dft biln vd nhdm khdng dft biln tai cdc dilm C1653T, A1762T vd G1764A (hlnh 4).

TCNCYH 76J3)-?ai1

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Bdng 2. So sdnh ty If d f t b i l n gen HBx trdn cdc nhdm theo mii'c d f bift hda t l bdo gan

Cdc logi

dft biln

^{Axit amin}

Tdng n = 94

Mii'c d f b i f t hda cao (1)

n = 18

vira (2) n = 65

kdm (3) n = 11 C1653T n (%)

A1762T, n(%) G1764A, n (%)

H94Y K130M

V131I

62 (66) 78 (83) 76(81)

14 (78) 12 (67) 12 (67)

40 (62) 55 (85) 53(82)

8(73) 11 (100) 11 (100)

p > 0,05 P 1-3 < 0,05 p 1-3<0,05

9E8 8E8 7E81 6E8 5E81 4E8 3E8- 2E8- 1E8

0 A)

P>0,06

DB BT

- 1E9

I 9E8 o 8E8 8-7E8 i i 6 E 8 g 5E8 Q 4E8 m 3E8

^ 2E8 1E8i 0

B)

P>0,06

; ' I

DB BT

1E9 9E8 8E8- 7E8 6E8 5E8 3Ee 2E8- 1E8- 0

C)

P>0,06

Hinh 4. So sdnh ndng do HBV DNA giira cdc nhdm cd dot b i l n gene vd khdng d f t b i l n gen (tai cdc dilm C1653T (A), A1762T (B), G1764A (C).

Sg khde bift khdng cd y nghTa thdng kd)

IV. BAN LUAN

UTG cQng nhu tat ea cde bfnh ung thu' khde ndi chung Id hfu qua cua biln ddi nhilu gen, tuang tdc qua lai cua nhilu protein, DNA trong cde t l bdo. Do ddc dilm bf gen vd vdng ddi ciia HBV cd lidn quan chft che d i n sg hinh thdnh UTG ndn vifc fim ra cde d i u I n phdn tfr ciia HBV nhu dft biln gen, mfre df bilu hifn gen HBV vd gen ngu-di Id mft trong nhfrng hu-dng nghidn cfru dang r i t du-gc quan tdm [3]. Gen HBx Id mft dogn gen cd kieh thu'dc tuang ddi nhd, nhung ed vai trd quan trpng trong ea c h l nhdn Idn eiia HBV cQng nhu lidn quan d i n sg hlnh thdnh UTG [4, 5, 6].

Nghien cfru trudc ddy cho thiy dft biln gen HBx cua HBV thudng xay ra d ede chung virus cd ngudn gdc tfr md UTG, dilu ndy eho thiy cd sg lidn quan gifra dft biln gene HBx cua virus vdi qud trinh phdt sinh UTG. Dft biln dilm thudng gdp d vj tri 31 tfr Serin ddi

thdnh Alanin trdn gen HBx x i y ra ehu ylu d cdc bfnh nhdn UTG so vdi cdc nhdm bfnh gan khdng ung thu. D f c bift, dft biln mat dogn d phin cudi eua gen HBx chi xay ra a trdn nhdm UTG md khdng thiy d cdc nhdm bfnh gan khde do HBV Hdu qua ciia dft biln ndy gdy ra sg phdn bd b i t thudng cua protein HBx d mdng nhdn vd nhdn t l bdo, ddng thdi ldm biln ddi hogt hod ddng d i n truyin tin hifu STAT-3. Tdc dfng eiia protein HBx ciia HBV gdy rdi logn tdng cudng hogt hod STAT-3, ddy Id mft protein din-truyin tin hifu lidn quan dilu hod qud trinh tdng sinh vd chit theo chuang trinh eua t l bdo. ddy Id mft m i t xlch quan trpng trong bfnh sinh eiia UTG cua HBV. Ddng thdi cde dft biln m i t dogn xay ra trdn phin cudi eiia gen md hod HBx protein, ldm tdng cudng ehuyin dgng de tfnh t l bdo vd hogt hod ede gen sinh ung thu [10].

Trong nghidn cfru ndy ehung tdi khdo sdt trdn 94 bfnh nhdn UTG nhiem HBV dugc dilu

J'^AP CHl NGHllN CLPU Y HQC trj tgi bfnh vifn Trung uang Qudn dfi 108. Ty

If bfnh nhdn nam:nfr Id khoang 5:1. K i t qua ndy hodn todn phu hgp vdi cdc nghidn cfru trudc ddy. T i t ea bfnh nhdn d i u duge tiln hdnh xdt nghifm t l bdo hpc vd k i t qua eho thiy ed 18 bfnh nhdn ed bift hda cao, 65 benh nhdn cd bift hda vfra vd 11 bfnh nhdn cd bift hda kem. K i t qua ndy eho thiy ty If benh nhdn ung thu gan kdm bift hda thlp han so vdi nghien cfru cua Sumihito Tamura vd cfng sg, Trong nghidn cfru dd, ngudi ta thiy ed 28,3% bfnh nhdn ed bift hda kdm [9]. Tuy nhidn, do sd lifu cdn It, nhdm ddi tugng phdt hifn bfnh mft cdch ngiu nhidn, khdng phdi n i m trong chuang trinh sdng Ipc. Do dd, tinh khdng ddng nhit gifra 2 nghidn cfru Id cd t h i xay ra.

Trong nghidn cfru ndy chung tdi thay dft biln gen HBx d bfnh nhdn ung thu gan Id tuang ddi eao. Trong dd dft biln tgi dilm 1653 (Cytosin duegc thay t h i bing Thymin, C1653T) chilm 66%, tuy nhien, khdng cd khac biet gifra ede bfnh nhdn cd mfre df bift hda khac nhau. Dot biln gen tai dilm 1762 lam thay ddi nuclefide tfr Adenln thanh Thymin (A1762T) vd tgi dilm 1764 ldm thay ddi nucleofid tfr Guanin thdnh Adenin (G1764A) Id 83% vd 81%. Nhfrng d f t biln ndy cd sg khde bift gifra cdc nhdm phdn chia theo mfre df bift hda t l bdo. Tuy nhidn, vl sd lugng bfnh nhdn ed mfre d f bift hda cao vd kdm cdn it ndn k i t qua khdng thgc sg cd gid trj cao.

Nhung du sao chung tdi cQng thiy trong 11 bfnh nhdn cd bift hda kdm thi d i u cd dft biln gen tgi 2 dilm ndy. K i t q u i ndy mft lln nfra Idm tang tinh thuylt phgc v l nhfrng nhdn djnh trirdc ddy cho ring dot biln gen HBx se gdy ung thu nhilu han cd y nghTa so vdi nhdm khdng dft biln. Ddng thdi cQng ggi y ring dft biln gen HBx ed t h i lidn quan d i n mfre df ndng ciia ung thu gan. Trong nghien efru.dd ngudi ta filn hdnh khao sat tren 820 bfnh nhdn vidm gan B m.gn finh va theo ddi sg xult hifn ung thu gan trong thdi gian 76 thdng. Kit

qua cho thiy t i n suit phdt triln UTG sau 5 ndm vd 10 ndm lln lugt Id 4,4% vd 6,3%.

Phdn tfch thdng'kd bing phuang phdp hdi quy Cox cho thiy dft biln gen HBx Id mft trong nhfrng y l u td nguy ca dfc Ifp gdy UTG vdi (p = 0,007. RR 3,66), Qua dd ngudi ta finh dugc df nhgy vd df dfc hifu ciia vifc tidn lugng xult hifn UTG sau 5 ndm vd 10 ndm trdn nhfrng bfnh nhdn c6 dft biln ndy lln lugt Id > 84% vd > 76%, Ngudi ta cQng tinh dugc difn tich dudi dudng cong. (AUC) d l fidn lugng UTG sau 5 ndm vd 10 ndm Id 0,88 vd 0,89 [11],

Mft nghidn cfru g i n ddy (ndm 2009) phdn tieh hdi cfru cdc sd lifu cua cdc cdng trinh dufgc cdng bd trudc ddy dd cho nhfrng kit qua rit cd gid trj, Cg thi, sau khi sfr dgng cdc phuang phdp thdng kd chlnh xdc ngudi ta cd t h i dua ra dugc cdc gid trj v l df nhay vd df ddc hifu d l fidn lugng ung thu gan qua cdc dot bien gene. Qua dd ngudi ta thdy nlu cd dft bien gene tai dilm ddi A1762T/G1764A thi fien lugng bfnh nhdn se ung thu gan rat cao vdi df nhay han 70% va do dac hifu la han 60%. Nlu k i t hgp cdc dft bien gen A1762T/

G1764A vdi cdc dot biln khde nhu C1653T vd T1753V thi nguy ea ung thu g i n nhu la chic ehln (dd ddc hifu = 93,9%) [12]. Nhu vdy, bing nhfrng k i t q u i budc d i u chQng tdi da chfrng minh ring ty If dft biln gen HBx trdn nhdm bfnh nhdn ung thu gan Id tuang ddi cao. D f t biln gen HBx cd lidn quan d i n mfre df bift hda t l bdo gan md khdng lidn quan d i n chfrc ndng gan, finh trang mang khdng nguydn e.

V. K^T LUAN

Dft biln gen HBx dugc thiy tuang ddi phd biln trdn bfnh nhdn ung thu gan nhilm HBV.

Dft biln tgi dilm ddi A1762T/G1764A cd lien quan d i n mfre df biet hda t l bdo gan nhung khdng cd Ndn quan d i n chfrc ndng gan vd finh trgng mang khdng nguyen e.

TCNCYH 76jm^?ni1

TAP dWi NGl^ilN

CCPU Y HQC

TAI LIEU THAM

K H A O

1. Parkin, D.M et al (2000). Esfimafing the worid cancer burden: Globocan Int J Cancer, 2001.94(2): 153-156.

2. FeHay, J et al (2008). Esfimates of worldwide burden of cancer in GLOBOCAN.

Int 2008 J Cancer.

3. McClune, A.C. and M.J. Tong, Chronic hepatitis B and hepatocellular carcinoma, Clin Liver Dis, 14(3): 461-476.

4. Kim, C M et al (1991). HBx gene of hepatifis B virus induces liver cancer in transgenic mice Nature. 351 (6324): 317-320.

5. Lee, J.H et al. Impact of hepatifis B virus (HBV) X gene mutafions on hepatocellular carcinoma development in chronic HBV infection. Clin Vaccine Immunol. 18 (6): 914-21.

6. Wei, Y et al. Molecular biology of the hepatifis B virus and role of the X gene. Pathol Biol (Paris). 58 (4): 267-272.

7. Hipgrave, D.B et al (2003). Hepatifis B infection 4n rural Vietnam and the implications

for a nafional program of infant immunization' .V

Am J Trop Med Hyg,. 69 (3): 288-294.

8. Nguyen, V.T., M.G. Law, and G.J.

Dore, (2008), An enormous hepafitis B virus- related liver disease burden projected in Vietnam by 2025 Liver Int, 28(4): 525-531,

9. Tamura, S et al (2001) Impact of histological grade of hepatocellular carcinoma on the outcome of liver transplantation Arch Surg, 136 (1): 25-30; discussion 31,

10. Bock, C T et al (2008). Subcellular mislocalization of mutant hepafifis B X proteins contributes to modulation of STAT/SOCS signaling in hepatocellular carcinoma Intervirology. 51(6): 432-443.

11. Yuen, M.F et al (2009). Independent risk factors and predictive score for the development of hepatocellular carcinoma in chronic hepatifis B J Hepatol, 50(1): 80-88.

12. Liu, S et al (2009), Associations between hepatitis B virus mutations and the risk of hepatocellular carcinoma: a meta-analysis J Nafi Cancer Inst, 101(15): 1066-1082.

Summary

HBx GENE MUTATION IN HEPATOCELLULAR CARCINOMA PATIENTS INFECTED WITH HEPATITIS B VIRUS

HBx gene mutafion is associated with the progression of hepatocellular carcinoma (HCC). The study was conducted to investigate the HBx gene mutation in HCC patient infected with HBV and its relafionship with laboratory parameters. Five point mutafion were identified in HBx gene such as A1762T G1764A, C1653T, T1753C/G vd C1766T. .Among these, A1762T, G1764A and C1653T were found more frequent with 66%, 83% and 81%, respectively. A1762T and G1764A was more frequent in poor differentiated group compared to well dlfferenfiated group (100% vs 67%, p < 0.05). HBx gene mutafion is no associated with liver funefion as well as HBeAg status..

In conclusion, HBx gene mutation rate is relative high in HCC pafient infected with HBV and A1762T/G1764A mutation are associated with dlfferenfiated grade of liver cell.

Key words: mutation, HBx, HCC