B Y T

TRNG I HC DC H NI

H MINH TN

TNG QUAN V CC T BIN GEN

M HA CYP2D6

KHA LUN TT NGHIP DC S

H NI 2013

B Y T

TRNG I HC DC H NI

H MINH TN

TNG QUAN V CC T BIN GEN

M HA CYP2D6

KHA LUN TT NGHIP DC S

Ngi hng dn:

ThS. Nguyn Xun Bc

Ni thc hin:

B mn Ha Sinh

H NI 2013

LI CM N

u tin cho ti xin c gi ti Ths. Nguyn Xun Bc li bit n chn

thnh v su sc. Thy l ngi tn tnh ch bo, gip ti trong qu trnh

nghin cu v hon thnh kha lun.

Ti xin cm n cc thy c trong b mn Ha Sinh Trng i hc Dc H

Ni, cc dc s v bc s trong bnh vin Trung ng Qun i 108 gip

to iu kin thun li cho ti trong sut qu trnh thc hin ti.

Cui cng ti xin dnh nhng li tri n ti nhng ngi thn trong gia nh v

bn b lun lun ng vin, c v ti vt qua nhng kh khn m c lc tng

chng nh mun b cuc, gip ti hon thnh tt kha lun ca mnh.

H Ni, ngy 21/5/2013

H Minh Tn

MC LC

Trang

Danh mc k hiu, ch vit tt

Danh mc bng

Danh mc hnh

T VN 1

CHNG 1. I CNG V CYP2D6.............................................................2

1.1. CYTOCHROM P450 ..............................................................................2

1.1.1. Phn b................................................................................................3

1.1.2. Phn loi v danh php ........................................................................3

1.1.3. C ch xc tc ca CYP.......................................................................4

1.2. CYP2D6.....................................................................................................6

1.2.1. Cu trc ca CYP2D6..........................................................................7

1.2.2. C ch tc dng ca CYP2D6..............................................................8

1.2.3. C cht ca CYP2D6...........................................................................9

1.3. GEN M HA CYP2D6 .........................................................................15

CHNG 2. T BIN GEN M HA CYP2D6...........................................16

2.1. KHI NIM C BN V T BIN GEN...........................................16

2.1.1. Cc loi t bin ................................................................................17

2.1.2. Nguyn nhn t bin........................................................................20

2.2. NHNG T BIN GEN CYP2D6........................................................25

2.2.1. Nhng allen ca gen CYP2D6............................................................25

2.2.2.Kiu hnh v kiu gen CYP2D6 ca cc chng tc trn th gii .............37

2.3. HU QU LM SNG CA TNG TC KIU GEN KIU HNH

CA CYP2D6 ...................................................................................................38

CHNG 3. PHNG PHP PHT HIN T BIN GEN CYP2D6 .......43

3.1. K THUT ADN CHIP ..........................................................................43

3.1.1. Nguyn tc:........................................................................................43

3.1.2. u im:............................................................................................44

3.1.3. Nhc im:......................................................................................45

3.2. K THUT LONG-PCR.........................................................................45

3.2.1. Nguyn tc.........................................................................................45

3.2.2. u im.............................................................................................45

3.3.3. Nhc im .........................................................................................45

3.3.4. p dng xc nh s nhn i gen CYP2D6 .........................................46

3.3. K THUT REAL-TIME PCR ...............................................................47

3.3.1. Nguyn tc:........................................................................................47

3.3.2. u im.............................................................................................48

3.3.3. Nhc im .......................................................................................48

3.3.4. p dng xc nh t bin CYP2D6 ..................................................48

3.4. K THUT PCR-RFLP ..........................................................................49

3.4.1. Nguyn tc.........................................................................................49

3.4.2. u im.............................................................................................51

3.4.3. Nhc im:......................................................................................51

3.4.4. p dng trn CYP2D6 .......................................................................51

3.5. K THUT PCR PHC HP.................................................................52

3.5.1. Nguyn tc:........................................................................................52

3.5.2. u im.............................................................................................53

3.5.3. Nhc im .......................................................................................53

3.5.4. p dng trn CYP2D6 gip chn on allen *5 v allen*10 ..............53

3.6. MT S K THUT KHC GIP XC NH T BIN CYP2D6 ..56

CHNG 4. BN LUN ...................................................................................56

4.1. HIN TNG T BIN GEN CYP2D6..............................................56

4.2. CC NGHIN CU V A HNH CYP2D6 TRN TH GII.............57

4.3. XUT HNG NGHIN CU CYP2D6 VIT NAM .................58

CHNG 5. KT LUN ...................................................................................59

DANH MC K HIU, CH VIT TT

K HIU/CH TN TING ANH TN TING VIT (nu c)

ADN Deoxyribonucleic Acid

ADR Adverse Drug Reaction Phn ng bt li ca thuc

ARN Ribonucleotic Acid

CLint Clearance instrict thanh thi ni ti

gan

CYP Cytochrom P450

EM Extensive metabolism Chuyn ha nhanh

FADH2 Flavin Adenine Dinucleotide

FDA Food and Drug Administration Cc qun l thc phm v dc phm Hoa K

IM Intermediate metabolism Chuyn ha chm

kb Kilo base

MR Metabolic ratio T l chuyn ha

NADPH Nicotinamide Adenine Dinucleotide Phosphate

PCR Polymerase Chain Reaction Phn ng khuch i gen

PM Poor metabolizer Chuyn ha rt chm

SNP Single nucleotide polymorphism a hnh n nucleotid

UM Ultra metabolism Chuyn ha siu nhanh

DANH MC BNG

Trang

Bng 2.1. nh hng ca a hnh di truyn CYP2D6 ln mt s thuc iu tr 38

Bng 3.1. Cc mi v enzym ct tng ng tng allen [79] 50

DANH MC HNH

Trang

Hnh 1. T l chuyn ha thuc bi cc CYP [65] ..................................................4

Hnh 2: C ch xc tc ca CYP [1] ........................................................................5

Hnh 3. M hnh phn t CY2D6 [100] ...................................................................7

Hnh 4. Cu trc vng hot ng ca CYP2D6 [100] ..............................................7

Hnh 5. C ch xc tc ca CYP2D6 [68]................................................................9

Hnh 6. Chuyn ha ca dextromethorphan bi CYP2D6 [105].............................12

Hnh 7. V tr CYP2D6 trn nhim sc th [37].....................................................15

Hnh 8 M t cc loi t bin im [2]...............................................................19

Hnh 9 Gen EGFR trong ung th phi: p.T751_I759>N [2]..................................20

Hnh 10. Hnh thnh gen lai [33] .........................................................................29

Hnh 11. S lp li mt hay nhiu ln v xa ton b gen CYP2D6 [43]...............36

Hnh 12. Kiu gen CYP2D6 cc vng a l khc nhau [109] ............................37

Hnh 13. Kiu hnh ca CYP2D6 cc vng a l khc nhau [109] ....................38

Hnh 14. Phn loi kiu hnh da trn kiu gen v hu qu lm sng da vo kiu xc tc bi hin tng a hnh enzym [109] ..........................................................39

Hnh 15. S minh ha mt AND chip [118].....................................................44

Hnh 16. Sn phm Long-PCR [95] ...................................................................46

Hnh 17. Ph in di [95] .....................................................................................47

Hnh 18. Mi v trnh t mi [82].........................................................................49

Hnh 19. PCR-RFLP chn on t bin 360C>G trn gen m ha enzym deoxycytidin kinase [144]......................................................................................50

Hnh 20. Cc allen CYP2D6 [84] ........................................................................53

Hnh 21. Chn on kiu gen bng k thut PCR truyn thng v PCR phc hp [84]........................................................................................................................55

1

T VN

Vo cui nhng nm 50 ca th k 20, cc nh khoa hc pht hin v tp

trung nghin cu mt h thng enzym c vai tr v cng quan trng i vi con

ngi, l h thng cytochrom P450 hay cn c gi l CYP450. H thng

enzym ny l tc nhn xc tc chuyn ha cho cc cht ni sinh nh cc steroid,

cholesterol, cc acid mtcng nh cc cht ngoi sinh c cu trc khc nhau

trong c cc cht c, cht gy ung th v c bit l chuyn ha thuc. Hin

nay ngi ta pht hin c 57 isozym trn h thng CYP450 ngi v chia n

thnh cc h v phn h khc nhau.

Trong s nhng isozym ny, CYP2D6 ginh c s quan tm c bit. Bi

l, d ch ng th hai v mt s lng cc thuc chuyn ha nhng CYP2D6 li l

mt trong nhng enzym c hin tng a hnh ph bin nht. V nhng hu qu ca

hin tng ny nhiu khi rt nghim trng. Hn th na cc nhm thuc iu tr

c enzym ny chuyn ha nh tim mch, gim au, thn kinh, ung th l nhng

nhm thuc rt ph bin hin nay v c ca s iu tr hp. Do , vic hiu bit v

CYP2D6 trn cc phng din: cc t bin gen, nhng bin i kiu hnh, hu

qu lm sng km theo v cc phng php pht hin t bin l cn thit, gp phn

vo vic ti u ha iu tr.

Xut pht t thc t trn, chng ti tin hnh ti:

Tng quan v cc t bin gen m ha CYP2D6 vi nhng mc tiu sau:

1. Tm hiu cc t bin gen m ha CYP2D6 c pht hin.

2. Tm hiu v cc phng php pht hin t bin gen CYP2D6.

2

CHNG 1. I CNG V CYP2D6

1.1. CYTOCHROM P450

Cytochrom P450 (Cyt-P450) l enzym mi c pht hin cch nay khong

60 nm. y l mt enzym oxy ha cui cng ca h thng monooxygenase (phn

ng oxy ha s dng mt nguyn t oxy), tham gia phn ng oxy ha rt nhiu hp

cht khc nhau v c vai tr rt quan trng trong chuyn ha nhng cht c nhn

thm (gm c cht ni sinh a m nh steroid, acid bo, nhng vitamin tan trong

du m, prostaglandin, leukotrien, thromboxan v nhng cht ngoi sinh).

Cytochrom P450 thuc nhm b theo cch phn loi ca D. Keilin [53]. Enzym

ny c pht hin t nhng nm 1940 trong khi cc nh khoa hc ang tm

cch gii thch cho mt loi phn ng khng bnh thng c s tham gia ca phn

t oxy. Trong nhng phn ng ny, mt nguyn t oxy lin kt vi hai nguyn t

hydro (c cho t hai phn t NADPH) to thnh H2O, trong khi nguyn t oxy

th hai c gn vo c cht (k hiu l RH). Phn ng c th c biu din theo

cng thc sau:

2 NAD(P)H+ + O2 + RH 2 NAD(P)+ + H2O + ROH

Cytochrom P450 khc bit vi cc Cytochrom khc ch chng chuyn

thng in t n phn t oxy cn cytochrom khc chuyn in t n cc protein,

v c sng hp th cc i 450nm khi gn vi CO. S c mt ca mt cht mu

phn ng vi CO v i km vi cytochrom b5 trong microsom c pht hin

bi G.R.William vo nm 1955 v Garfinkel (1958) trong qu hc bng Johnson v

bi Klingenberg [59]. Cht mu ny c c trng bi mt bng hp th ca hp

cht vi CO 450 nm, v vy gi l P450. n gin v thng nht v mt thut

ng, ngi ta qui c dng ch CYP thay cho Cyt-P450. Sau , Sato v Omura

(1962,1964) xc nh bn cht hem ca nhm ngoi ca n. Loi protein cha

hem c mt trong microsom mi c pht hin ny c nhng thuc tnh ring bit

c xc nh l mt enzym oxy ha cui cng ca nhiu phn ng oxy ha

khc nhau tham gia vo mt s qu trnh chuyn ha quan trng c th ng vt:

3

sinh vt tng hp hormon steroid, acid mt t cholesterol v chuyn ha thuc [85,

86].

1.1.1. Phn b CYP l h enzym gn mng c hu ht cc m trong c th, trong tp

trung nng cao ch yu vi th trn li ni sinh cht nhn ca t bo gan v

mt lng t hn rut non, phi, thn v no.

ngi, CYP tp trung nhiu nht t bo gan. CYP cng c tm thy

trong ti th ca thng thn chut cng v ti th v thng thn b [56, 132].

Ngi ta cng pht hin ra rng CYP khng ch c vi th v ti th ca ng vt c

v m cn phn b rng ri cc dng tn ti khc nhau ca s sng bao gm:

ng vt v vi sinh vt. Nhng nghin cu v CYP tng ln c bit nhanh chng

trong na sau ca nhng nm 1960, khi nhng nghin cu c tp trung ch yu

vo chc nng ca CYP trong chuyn ha thuc vi th gan. Vai tr ca CYP trong

s hot ha cc cht gy ung th khc nhau (carcinogen) c khng nh vo

u nhng nm 1970 v nhiu nh khoa hc ang nghin cu cc ha cht gy ung

th chuyn sang nghin cu CYP, mang li s pht trin vt bc ln th hai trong

lnh vc nghin cu ny. S pht trin quan trng tip theo l nhng nghin cu

lm sch CYP t vi th v ti th v dn ti vic tinh ch v c trng c nhiu

dng khc nhau ca CYP ngun gc ng vt. Hu ht nhng dng tinh th ny

u tham gia vo s chuyn ha ca nhng cht ni sinh khc nhau, khng nh vai

tr thit yu ca CYP trong s chuyn ha bnh thng ng vt [77].

1.1.2. Phn loi v danh php

CYP l tn chung ca mt i gia nh cc enzym gm nhiu isozym c

trng lng phn t khong 45-60 kDa, trong c cha mt nhn Hem dng Fe

protoporphyirin IX v mt chui polypeptid. H gen ngi c 57 gen v 33

pseudogen m ha CYP. Cc isozym c sp xp li thnh h v trong h li c

chia thm thnh cc phn h theo s tng ng ca chui acid amin trong phn t.

Cc h c t nht 40% trnh t ging nhau, trong khi cc phn h t l ny t nht

4

l 55%. Mi h c k hiu bi mt ch s (v d CYP2), mi phn h c k

hiu bi mt ch ci vit hoa (v d CYP2D) v gen n hoc isozym c biu th

bi ch s th hai ( v d CYP2D6). Ngoi ra, ngi ta cn s dng ch s th ba (

c ngn cch vi ch s biu th isozym c th bi mt du hoa th *) biu

din cho v tr allen (dng bin i ca gen) [107].

V d:

CYP H Phn h Isozym Allen

Trong 30 h c phn loi, ch c 3 h CYP l CYP1, CYP2, CYP3 chu

trch nhim chuyn ha phn ln cc thuc, trong ring 6 enzym 1A2, 3A4,

2C9, 2C19, 2D6 v 2E1 m nhn chuyn ha hn 90% thuc cc thuc iu tr

[65, 94].

Hnh 1. T l chuyn ha thuc bi cc CYP [65]

1.1.3. C ch xc tc ca CYP C ch phn ng vng c xut cho phn ng oxygen ha bi CYP. Theo

quan im chung, mt chu trnh phn ng oxy ha c cht ca h thng CYP c th

chia thnh 6 bc chnh nh sau:

5

Hnh 2: C ch xc tc ca CYP [1]

Bc 1: C cht c gn vo CYP (dng oxy ha) to ra hn hp enzym-c

cht. S gn c cht ny lm bin i ng lng t ca CYP t trng thi spin

thp (low spin). S bin i th nng oxy ha c gy ra bng cch s to

thun li cho vic chuyn ca in t th nht.

Bc 2: L bc nhn in t th nht t NADPH di tc dng xc tc ca

enzym Cyt-P450-reductase (hoc t mt loi protein cha st- lu hunh (2Fe-2S)

gi l adrenodoxin trong c th vi sinh vt) to thnh hn hp enzym-c cht dng

kh.

6

Bc 3: Hn hp enzym-c cht trn gn vi mt phn t O2. Trong hn hp

ny mi c s chuyn in t ni phn t to thnh hn hp enzym-c cht,

trong st dng oxy ha gn vi O2-.

Bc 4: Hn hp va to thnh nhn in tip in t th 2 t phn t

NADPH th hai do NADPH-reductase xc tc hoc t NADH (xc tc bi NADH-

Cytochrom b5 reductase v Cytochrom b5), vi sinh vt bc ny do adrenoxin xc

tc. Hai in t nhn c kh phn t O2 trong hn hp enzym-c cht thnh hp

cht trung gian peroxo (RH) Fe3+-(O2--).

Bc 5: Oxy c hot ha s phn ng vi 2H+ (do 2 phn t NADHP+

cung cp) to thnh phn t nc tch ra khi hp cht trung gian trn, cn li hp

cht (RH) (Fe-O)3+.

Bc 6: L s oxy ha c cht bng nguyn t oxy cn li v gii phng c

cht dng oxy ha, enzym CYP tr v trng thi Fe+3 ban u (dng oxy ha),

hon thnh mt chu k phn ng. Sn phm to ra l c cht c oxy ha v mt

phn t nc [1, 77].

1.2. CYP2D6

CYP2D6 c pht hin bi Robert Smith trng i hc Y Saint Mary. Khi

tham gia tnh nguyn th thuc h p debrisoquin, Smith b nh hng ln ti

sc khe v iu gip ng pht hin ra mnh b thiu ht CYP2D6 [1].

CYP2D6 ng vai tr nh mt enzym oxy ha mt nguyn t oxy (mono-

oxygenase) thng c tm thy trong cc vi th gan. V n chuyn ha ti 30

% thuc. Cc nhm thuc quan trng c enzym ny chuyn ha l : thuc iu tr

trm cm, thuc chn Beta v thuc gim au. c im ca cc thuc ny l

nhng cht thn du vi mt nhm amin c proton ha v mt vng thm

[13]. S sng t v cu trc tinh th ca CYP2D6 v vic FDA ph duyt vic th

nghim kim tra gen a vic nghin cu v enzym ny ln v tr mi nhn [28,

119].

7

1.2.1. Cu trc ca CYP2D6

Phn t CYP2D6 l mt protein gm 479 acid amin, cha mt nhm heme, ID

trong d liu Ngn hng protein: 2f9q [100]. (hnh 3). CYP2D6 c mt v tr hot

ng, nm pha trn nhm hem. (hnh 4)

Hnh 3. M hnh phn t CY2D6 [100]

Hnh 4. Cu trc vng hot ng ca CYP2D6 [100]

8

Cc acid amin xon li vng tip nhn v gn c cht l Asp301 (D301), Glu216

(E216), Phe483 (F483) v Phe120 (F120). (hnh 4). Vai tr rt quan trng ca

D301, E216, F120 v F483 l nhn bit v gn c cht. Trong D301 v E216

gip gn kt c cht. Cn F483 v F120 cn li iu khin s lin kt phn t c

cht v tr hot ha [91].

CYP2D6 c pht trin sau khi tr c sinh ra. CYP2D6 hoc hot tnh ca

n khng c pht hin cc t bo gan ca thai nhi mc d ARN CYP2D6 tng

ln cng vi s pht trin ca bo thai. Lng CYP2D6 cng vi hot tnh ca n

khng lin quan n tui ca thai nhi v tng ln cng vi s pht trin ca tr sau

khi ra i. Sau khi sinh 7 ngy, hot tnh enzym ny xp x bng 25% so vi ngi

trng thnh. V khi mt tui hot tnh CYP2D6 gan bng vi mc ngi

trng thnh [80].

1.2.2. C ch tc dng ca CYP2D6 Chc nng chnh ca CYP2D6 l oxy ha thuc thng qua cung cp electron

u tin t vic tng tc vi phc hp hem-oxy. (Hnh 5) Phn ng xc tc lin

quan n s chn mt nguyn t oxy vo phn t cht nn v nguyn t oxy th

hai th c chuyn thnh nc.

Ngun electron c cung cp t phc hp kh NADPH - cytochrom P450,

y l mt flavoenzym ci cha 1 phn t ca mi coenzym, flavin adenin

dinucleotid (FAD) v flavin mononucleotid ( FMN) [55]. FAD nhn electron t

NADPH v gim th, FADH chuyn electron ti FMN,[87]. Sau electron c

chuyn t FMNH- ti phc hp hem, lm gim ha tr ca st t +3 xung +2.

Vng lin kt ca CYP dng kh ny c tm thy u C tn ca CYP2D6.

Vng ny ch yu cha nhng v tr c bn, kt ni CYP dng kh thng qua

cc cu mui vi Arg-440 ng mt vai tr kt ni rt quan trng [6]. Phn ng oxy

ha c khi ng bi s hot ha mt phn t oxy thng qua ion st 2. Phn ng

hydroxyl ha ny xy ra vng 5-7 A so vi nguyn t Ni t trong phn t cht

nn. iu ny to ra cp st oxy hot ha, chnh phc hp ny phn ng li vi

cc phn t thuc thng qua nhiu c ch khc nhau [41].

9

Hai phc hp phn ng st oxy chnh lin quan ti phn ng li gii l phc

hp i in t st- hydroperoxo v st oxo [104]. Loi st hydroperoxo

c hnh thnh bi phc hp dioxygen- Fe2+, st- peroxo.( Nh hnh 5). Phc

hp st oxo lm tch lin kt O O. C ch phn ng i hi s gii phng hp

cht trung gian, mc d iu ny vn cn nhiu s tranh ci [68].

Hnh 5. C ch xc tc ca CYP2D6 [68]

1.2.3. C cht ca CYP2D6

1.2.3.1. C cht mu ca CYP2D6 C cht mu ca CYP2D6 thng l nhng thuc c dng iu tr t rt

lu (ti nay c th khng cn dng iu tr na) v c chuyn ha thng qua

CYP2D6. S thay i trn CYP2D6 s nh hng rt ln ti tc dng iu tr ca

cc thuc. Khi cc nh khoa hc bit c iu ny cng vi s ra i ca cc

phng php php hin t bin trn gen, h tm cch a ra mi quan h v

kiu gen v kiu hnh CYP2D6. Do m cc thuc ny c la chn lm cht

phn loi cc kiu hnh chuyn ha nhanh (EM), chuyn ha chm (IM), chuyn

10

ha siu nhanh (UM) v chuyn ha rt chm (PM). S phn bit ny s c trnh

by c th mc 2.3.

a. Spartein Spartein v debrisoquin l hai c cht u tin c nghin cu v n cng

c s dng rng ri xc nh kiu hnh chuyn ha chm (PM) ca CYP2D6

[138]. Nhng nghin cu sm ch ra rng spartein chuyn ha ch yu bi mt h

thng cc CYP thnh mt N-oxid. N-oxid tip tc phn ng, v b mt nc

tr thnh 2-dehydrospartein (v d: 2,3-didehydrospartein) v 5- dehydrospartein (v

d: 5,6 didehydrospartein). So snh s to thnh 2-dehydrospartein vi th gan

ngi t hai kiu hnh EM (MR < 2) v PM (MR > 20) ch ra hn s hn km

nhau 32 ln i vi gi tr ca hng s thi tr Km gia EM v PM (58.3 v 1880

micromol/L ) [89].

Vi t l chuyn ha ca spartein (MR) = spartein thi tr qua nc tiu (

2-dehydrospartein v 5- dehydrospartein thi tr qua nc tiu).

kiu hnh PM, s thi tr spartein ph thuc hon ton vo s thi tr ca

thn vi t l thi tr 99,9% liu dng qua thn dng khng thay i. kiu hnh

EM c s tng ln 30% cht chuyn ha ca spartein sau khi dng rifampicin liu

php iu tr 600 hoc 1200 mg trong 7 ngy, trong khi rifampicin khng nh

hng ti thi tr spartein kiu hnh PM [22]. Mc d spartein chuyn ha ch

yu bi CYP2D6 (>85%) [21, 23, 69], n cng b chuyn ha bi nhng CYP

khng c xc nh. C th nhng CYP ny b gim tc dng bi rifampicin. iu

ny c th gii thch c s tng thi tr ton phn spartein kiu hnh EM. C

EM v PM u khng b nh hng bi liu php iu tr bng pentobarbital

(phenobarbi-ton) mt tc nhn gy cm ng mnh ti CYP2B6 [66]. Tc ng

khng ng k ca nhng enzym cm ng in hnh i vi s chuyn ha spartein

ch ra rng nhng enzym ny chu tc ng ch yu do gen. Nhng ca nghin cu

xa hn na xc nh rng s khim khuyt chuyn ha spartein l do nhng t

bin gen CYP2D6.

11

b. Debrisoquin Debrisoquin l thuc iu tr tng huyt p v c chuyn ha ch yu bi

CYP2D6 (CYP2D6 cn c gi l tc nhn hydroxyl ha debrisoquin). Cc cht

chuyn ha ca n l: 4-hydroxy-debrisoquin, 3-hydroxydebrisoquin v 1-

hydroxydebrisoquin [24]. Mahgoub v cng s bo co mt trng hp thi tr

60.8% liu ban u di dng nguyn vn v ch 5.4% di dng 4-

hydrodebrisoquin, ch s MR l 11.3 [67]. Mt chui cc nghin cu ch ra rng

trng hp ny l mt kiu hnh PM, trong khi 3 trng hp khc trong ca

nghin cu ny ch c MR mc 0.5 1.4 in hnh ca kiu hnh EM. T l

chuyn ha (MR) ca debrisoquin (lng debrisoquin / lng 4-

hydroxydebrisoquin trong nc tiu thu thp trong 8h) dng xc nh kiu hnh

ca CYP2D6. qun th ngi da trng mc MR >12.6 c xc nh l kiu

hnh PM, cn mc MR < 12.6 c xc nh l kiu hnh EM. C t hn 1% ngi

chu c phn loi l PM nu s dng MR ca debrisoquin = 12.6 l mc

nh gi [8]. Phn b t l chuyn ha debrisoquin thay i cao hn nhng ngi

chu c kiu hnh EM so vi ngi da trng c kiu hnh EM. Cc th nghim

kim tra kiu hnh s dng spartein hoc debrisoquin c gi r v ng tin cy tuy

nhin ngy nay khng cn dng na.

c. Dextromethorphan Dextromethorphan mt cht tng hp tng t thuc gim au gy nghin

c s dng nh mt thuc gim ho khng cn k n. ngi n c thi tr

12

ch yu dng nguyn vn v dextrorphan c tc dng dc l ( hnh 6).

Hnh 6. Chuyn ha ca dextromethorphan bi CYP2D6 [105]

S hnh thnh dextrorphan t dextromethorphan ch yu do CYP2D6 thng

qua vic ct b nhm methyl qua cu ni oxy [18, 49]. Do dextromethorphan

ngy nay c s dng nh l mt c cht mu xc nh hot tnh CYP2D6 c

in vivo v in vitro. Ty thuc qun th, chng tc m biu tn s phn b, gi tr

phn nh m c cc gi tr MR tiu chun (antimode) khc nhau. Gi tr MR nc

tiu = 0.3 khi s dng dextromethorphan lm thuc th phn loi EM v PM

ngi da trng, gi tr ny cng c p dng cho cc qun th khc [105]. Cn i

vi MR huyt thanh gi tr s dng l 0.126. Tuy nhin c s chng cho gia kiu

hnh IM v EM khi s dng gi tr antimode bng 0.03 [60].

d. Bufuralol Bufuralol l mt thuc chn rt hay c s dng lm cht mu trong cc

th nghim kim tra CYP2D6 in vitro. N c chuyn ha thnh 3 cht: 1-

hydroxybufuralol, 1-oxobufuralol v 1,2-ethenylbufuralol. Nng 1-

hydroxybufuralol - cht chuyn ha chnh ca bufuralol, thng c nh lng

13

nh l mt ch s nh gi hot tnh hoc mc ca CYP2D6 v lng 1-

hydroxybufuralol to thnh t bufuralol kiu hnh PM ca CYP2D6 l rt thp.

Tuy nhin, chnh bufuralol ch khng phi spartein hay debrisoquin c chuyn

ha rt mnh bi CYP2C19 v mt phn bi CYP1A2, iu ny c th nh hng

n s c trng ca n nh mt c cht mu ca CYP2D6 [133]. Ngoi ra, S-

metoprolol cng c s dng lm cht mi cho CYP2D6 nhng qui m nh

hn.

e. Tramadol Tramadol l mt thuc gim au gy nghin kiu aminocyclo-hexanol, c hai

trung tm bt i xng cng c s dng lm c cht mu cho CYP2D6. (-)-

tramadol c chuyn ha ch yu bi CYP2D6 thnh O-desmethyltramadol (M1)

trong khi (+)-tramadol th c chuyn ha t hn. Mt cht chuyn ha khc ca

tramadol l N-desmethyl-tramadol (M2). Nhng ca nghin cu in vitro ch ra

rng tramadol ch yu chuyn ha thnh M1 bi CYP2D6 v thnh M2 bi

CYP2B6 v 3A4 [90, 117].

Gia 5 loi c cht mu trn ca CYP2D6 th dextromethorphan v bufuralol

l hai cht c s dng rng ri trong iu kin in vitro vi t l s dng bufuralol

l 60% v dextromethorphan l 30% [136].

1.2.3.2. Thuc iu tr l c cht ca CYP2D6

a. Cc thuc tc dng trn h thn kinh trung ng CYP2D6 c th chuyn ha mt s lng ln thuc c ch tc dng l h

thn kinh trung ng v trong chng c nhiu thuc vi tr s iu tr hp [36, 141,

142]. Nhng thuc ny bao gm: thuc iu tr trm cm ba vng (nh: amitriptylin

[124], clomipramin [83], imipramin [123], doxepin [42], thuc c ch chn lc thu

hi serotonin (SSRI; nh l: fluoxetin, fluvoxamin, paroxetin v sertralin), nhng

thuc iu tr trm cm khng vng khc (nh l: atomoxetin, maprotilin, mianserin

v venlafaxin ), thuc iu tr ri lon tm thn (nh : chlorpromazin, perphenazin,

thioridazin, zotepin, zuclopenthixol, risperidon v haloperidol ), nhm opioid (nh

l: codein, dihydrocodein v tramadol ). Thuc chng nn nh l tropisetron,

14

ondansetron, dolasetron v metoclopramid cng c CYP2D6 chuyn ha. Tuy

nhin nhng CYP khc nhau th m nhn vai tr khc nhau, chng c th m

nhn hon ton hoc ch mt phn. Chng hn nh CYP1A2 v 3A4 cng lin quan

n chuyn ha ca hu ht thuc iu tr ri lon tm thn trong khi CYP2C19,

2C9 v 3A4 li c vai tr rt ln i vi chuyn ha ca phn ln thuc c ch thu

hi chn lc serotonin [141]. Do m nh hng ca hin tng a hnh di truyn

ca CYP2D6 ln s chuyn ha hon ton nhng thuc ny l rt a dng v ph

thuc rt nhiu vo vai tr ca CYP2D6 trong s chuyn ha ca chng.

CYP2D6 ng mt vai tr khng ln trong s chuyn ha ca citalopram.

Trong khi s chuyn ha qua CYP3A4 v CYP2C19 ln lt chim t l 70% v

7%. Tuy nhin CYP2D6 li c vai tr rt quan trng i vi chuyn ha

escitalopram- ng phn S ca citalopram. S phn b tng quan gia CYP2D6,

2C9 v 3A4 ti thanh thi ni ti ca gan (CLint) ca escitalopram c c tnh

theo th t l 28%, 37% v 35% [125]. Gepiron l mt thuc chng trm cm th

h mi c chuyn ha ch yu bi CYP2D6, nhng nng thp th vai tr ca

CYP3A4 li ng vai tr chnh [39]. Mt thuc chng trm cm mi hn,

duloxetin, c chuyn ha chnh bi CYP2D6 v 1A2 [110]. Methadon l mt

thuc ch vn trn receptor , c chuyn ha ch yu bi c CYP2D6 v 3A4

[127].

Atomoxetin, mt thuc iu tr chng tng ng, gim tp trung hay

nicergolin mt dn xut ca ca la mch iu tr mt tr nh cng c

chuyn ha ch yu bi CYP2D6 [10, 99]. Selegilin, mt thuc s dng iu tr giai

on u ca bnh Parkinson, trm cm, chng mt tr nh c chuyn ha mt

phn bi CYP2D6, nhng CYP2B6 v 2C19 mi l nhng enzym chnh trong s

chuyn ha thuc ny [44]. Almotryptan mt cht ch vn chn lc receptor 5-

HT1B/1D c pht trin iu tr triu chng ca bnh au na u cp l mt

c cht ca CYP2D6 [38].

15

b. Thuc tim mch CYP2D6 ng mt vai tr quan trng trong chuyn ha cc thuc iu tr bnh

tim mch. CYP2D6 chuyn ha cho rt nhiu thuc iu tr lon nhp tim (nh l:

spartein, propafenon, encainid, flecainid, cibenzolin, aprindin, lidocain,

procainamid v mexiletin) v thuc chn (nh l: atenolol, bufuralol, carvedilol,

metoprolol, bisoprolol, propranolol, bunitrolol, bupranolol, timolol andalprenol)

[36, 141].

c. Nhng thuc khc Tamoxifen c chuyn ha bi CYP2D6 thnh endoxifen, mt cht chuyn

ha c tc dng dc l thng qua hai qu trnh ct b nhm methyl v gn vo

nhm hydroxyl (N-demethylation v 4-hydroxylation) lin tc [9, 19]. Cc

thuc khng histamin, chng d ng hay khng cholinergic cng c CYP2D6

chuyn ha.

1.3. GEN M HA CYP2D6

CYP2D6 chim 1 vng 4.2 kb trn nhim sc th 22 v tr q13.1 v cha 9

on exon v 8 on intron. N bao gm 1491 cp base m ha cho 497 acid amin.

Nm cng trn nhim sc th 22 cn c 2 pseudogen l CYP2D8P v CYP2D7P

[37].

Hnh 7. V tr CYP2D6 trn nhim sc th [37]

16

CHNG 2. T BIN GEN M HA CYP2D6

2.1. KHI NIM C BN V T BIN GEN

t bin theo ngha rng ch cc bin i di truyn xy ra t ngt. Mt t

bin gen l mt s thay i trong trnh t nucleotid trn gen, dn n kt qu l to

ra mt protein t bin vi trnh t acid amin thay i hay tc ng n qu trnh

iu khin s tng hp ca cc sn phm gen. Nu protein t bin c chc nng

khc vi dng t nhin th s to ra mt s thay i tng ng nhng c tnh c

th quan st c v to ra th t bin. S khc bit c th do hot tnh hay tnh n

nh ca enzym. Mt s t bin c th tn ti trong qun th bn cnh cc dng t

nhin lm thnh mt th a hnh [4].

t bin xy ra cc t bo khng sinh sn c gi l t bin sinh dng

v to ra cc thay i c tnh cc b, khng di truyn c nh cc u sc t ngoi

da ngi to ra nt rui. Nhng t bin sinh dng c th l nguyn nhn gy ra

ung th v c th l nguyn nhn ca s lo ha .

t bin xy ra trong giao t c gi l t bin dng mm v c truyn

cho i sau. Khi nghin cu cc t bin giao t v o t l t bin sinh vt a

bo ni chung v ngi ni ring cn xt n tnh lng bi. Hu ht cc t bin

gen l t bin ln v s khng c pht hin nu hp t khng c hai bn ca

allen t bin. Do vic pht hin v o tn s t bin phi da vo quan st cc

t bin tri ang xy ra trn nhim sc th thng, cc t bin ln v tri trn

nhim sc th X.

V d: chng lon sn sn xy ra ri rc l bi cc t bin mi trong gen ca

th th yu t tng trng nguyn bo si. Trong 1 nm c 7 tr s sinh b lon sn

sn trn tng s 242257 tr s sinh , t l l 7/242257 1/2 ( 2allen/ hp t) =1,4

10^-5.

Trong t nhin, d trong iu kin no tt c cc gen u c t bin c gi

l t bin t nhin hay t bin t pht. Cc t bin t nhin thng xut hin rt

t. Cc gen khc nhau ca cng mt sinh vt c th c tn s t bin khc nhau

nhng tn s t bin t nhin i vi mi gen l n nh.

17

Tn s t bin c nh gi cn c trn mt ln sao chp, mt ln phn bo

hay trn mt giao t v trn mt t bo trong mt th h. Tuy tn s t bin ca

tng gen rt thp nhng tng cc t bin ca nhiu gen li l mt s ng k, c

ngha quan trng trong tin ha. Cc kt qu nghin cu in vivo v in vitro trn t

bo ngi khong 10-6/gen/th h. c tnh cc thay i xy ra trong mi th h

da vo t l t bin ngi nh sau: 10-6 t bin/gen*5.104 gen/ b gen n bi

bng 5.10-2 t bin /giao t (1/20) . Mi hp t c (1/20). 2=1/10 c hi xy ra t

bin gen . Con s ny c v rt cao nhng cn nh rng hu ht cc t bin l ln

v nh vy s khng c biu hin trong iu kin d hp t. S thay i cc base

c th xy ra trong vng khng m ha ca phn t ADN v cc acid amin b thay

i cng c th khng phi lc no cng lm thay i c tnh protein. Do vy ,thay

i trnh t nucleotid c th khng biu hin thnh s khc bit tnh trng v do

khng phi l i tng ca chn lc. Cc t bin ln tch t trong b gen.

Hu ht cc t bin l bt li v do cc c ch c hiu thng c dng

gim thiu cc bin i trong chui ADN hoc hon phc trnh t gc bng cch

sa cha t bin.

2.1.1. Cc loi t bin a. t bin im

L kt qu ca s thay i mt base trong chui ADN gy bi tc nhn gy

t bin hoc sai st trong sao chp ADN.

Thay th cp nucleotid

Ph bin nht l chuyn v, pyrimidin c thay th bi pyrimidin khc hay

purin bi purin khc ( AG , C T ). S chuyn v, bt cp sai c th gy ra bi

cc acid nitro hoc cc ng ng base nh 5-bromo-2-deoxyuridin ( BrdU). Cc

t bin im t ph bin hn l o chuyn, pyrimidin c thay th bi purin hay

18

purin bi pyrimidin (C/T A/G). t bin im c khuynh hng c trng l t

bin im c khuynh hng c trng l t bin li (hi bin), ngha l chuyn li

dng nguyn thy. iu ny xy ra v ln t bin tip theo ti cng mt im c

mt phn ba c hi quay li chui ban u. C ba loi t bin im ty theo codon

mang t bin m ha:

bin ln hay cn gi l t bin ng ngha: m ha cho cng acid amin,

khng nh hng n protein cui. V d, codon GCA hoc GCG trong mARN u

mang ngha l arginin ( Loi ny thng ng vi t bin chuyn v tr th 3-

base linh hot ca codon ).

t bin sai ngha : M ha cho cc acid amin khc, c th lm protein

khng c chc nng. V d, trong chui protein -globulin gy bnh thiu mu hng

cu hnh lim, GAG m ha cho glutamat, nu thay bng GUG s m ha cho valin.

t bin lch ngha c th gy hu qu rt nghim trng nh thiu mu t bo li

lim, hoc nh nh hemoglobin C ( acid amin v tr 6 ca -globulin b thay th bi

acid amin khc) hoc khng c kiu hnh.

t bin v ngha: M ha cho codon kt thc, c th lm ct protein. Tc

ng ca cc t bin v ngha ty thuc protein b ct bao nhiu v mc cn

cho hot ng ca protein.

t bin thay th base c th xy ra trong promoter hoc cc vng iu ha 5

ca gen hoc trong intron v c th nh hng ti s phin m , dch m hoc mc

xon bn ca ADN. Nhiu -thalassemia l hu qu ca cc t bin khng cu trc

ny nh hng mc biu hin cc gen globin. Mc d nh hng ty thuc

nucleotid b thay th v v tr ca n trong ADN [2].

19

Hnh 8 M t cc loi t bin im [2]

((A) ng ngha (silent); (B, C)Sai ngha (missense); (D) V ngha (nonesense))

t bin lch khung

Cc t bin ny do chn hoc mt mt hoc nhiu nucleotid trong vng m

ha ca gen. iu ny lm thay i khung c, v cc codon l cc nhm ba

nucleotid nn c th c ba khung c nhng ch mt ci c dng. t bin loi

ny thay i tt c cc acid amin pha sau t bin v rt d to sn phm khng

chc nng v khc hn vi cc protein thng. Ngoi ra, cc khung c sai thng

cha cc codon stop, s lm qu trnh tng hp protein dng sm.

Vic thm hay bt mt nucleotid vo khung c s lm thay i cc acid amin

k t tr i. Nu t bin thm hoc bt cng xy ra trn mt phn t ADN th

do trao i cho gia cc base tng ng m t bin m c th sa cha li

khung c sai do t bin dng gy ra. Nh ch c acid amin no nm gia hai

im t bin b sai m thi. Nh vy t bin m tc ng nh mt t bin

km hm ngoi gen i vi t bin dng hoc ngc li.

b. t bin a im Cc t bin a im l nhng thay i tc ng n hn mt base, thay i t

hai n hng ngn base, nhng thng nht l ch vi base. Hu qu thng l xa

b mt phn ca trnh t m ha. t bin a im khng c tnh hi bin, ngha l

t bin c n nh.

S xa mt on ln s lm thay i hon ton protein to ra, thm ch xa b

on ngn cng gy hu qu nghim trng nu n lm thay i khung c.

Hnh..m t s mt mt on gen ln bnh nhn ung th phi.

20

Hnh 9 Gen EGFR trong ung th phi: p.T751_I759>N [2]

S chn a im c th xy ra do gen nhy hoc li khi sao chp ca cc yu

t lp li ( v d cc lp li AT). Hu ht khi gen b chn s b lch khung hoc thay

i kt ni trong mARN, c hai trng hp ny u lm thay i sn phm ca gen.

Gen nhy di ti vi ngn base (kb) v v vy , n lm ngng qu trnh sao m v

dch m ca bt k gen no chng xen vo. Gen nhy c hai loi, retrotransposon v

transposon. Cc transposon c th di chuyn trc tip t v tr ny sang v tr khc

ca b gen, retrotransposon trc ht c phin m thnh ARN v sau li thnh

ADN bi enzym phin m ngc v chn li vo b gen. Transposon rt c ch cho

cc nh nghin cu lm cng c bin i ADN trong c th sng.

2.1.2. Nguyn nhn t bin C hai loi t bin l t bin t nhin ( xy ra mt cch t pht )v t bin

cm ng( gy ra bi cc tc nhn t bin)

a. t bin t nhin Xy ra trong t nhin mt cch ngu nhin vi tn s nht nh v khng xc

nh c ngun gc. Bt k mt tin trnh no dn n s gn sai base trong qu

trnh sao chp ADN s to ra mt t bin im. Tn sut sai st t nhin vo

khong 10-10 base. T l thp ny t c nh kh nng sa li ca cc ADN

polymerase ch yu. Cc enzym ny ko di ADN theo hng 5 3 nhng c

hot tnh exonuclear 3 5 . Nu li nm gia base c chn cui cng vi si

khun c pht hin, enzym loi b base sai v thay th base ng vo . Chui m

ca mt protein trung bnh 300 acid amin l khong 1000 base chiu di. T l t

21

bin t pht l mt trong 107 bn m sao ca gen s c mt t bin im, ngha l

10-7 t bo s c t bin v gen .

t bin t nhin mc phn t gm c hn bin, kh amin, chuyn v, o

chuyn, t bin lch khung, oxy ha ph hy.

Hn bin: 4 loi base trong ADN c th tn ti dng hn bin, tc l c

kh nng tn ti hon chuyn gia hai dng ( Keto Enol v Amino Imino).

Base pyrimidin bnh thng c dng keto (C = O) , nhng i khi chng cng c

dng enol (C OH). dng enol, thymin c th bt cp vi guanin v enol cytosin

c kh nng bt cp vi adenin, tng t base purin bnh thng tn ti di dng

amino (NH2) nhng cng c dng hn bin imino (=NH) him gp, c th dn n

s bt cp nhm. Nu trong khi ADN sao chp, G dng enol, polymerase s thm

T vo thay v bnh thng l C v qui lut bt cp b thay i ( khng phi do li

ca polymerase). Kt qu l s chuyn v G C thnh A T; s hn bin ch gy

cc t bin chuyn v.

Kh amin: Cc phn ng kh amin thng thng l phn ng thy phn

cytosin thnh uracil, qu trnh ny phng thch ammonia v kh amin 5-

methylcytosin thnh thymin v ammonia. Trong ADN, s kh amin cytosin t pht

c chnh li bng cch loi b uracil (v uracil khng c trong ADN) v thay th

bng cytosin, cn s kh amin 5- methylcytosin khng c chnh v c ch sa

cha khng nhn thymin l li. Trong ADN b gen cytosin ti cc trnh t CG (

CpG) thng b methyl ha, iu ny l u CpG xy ra trong gen, n l im

nng cho t bin. Mi y ngi ta tm thy gen lin quan n lon sn sn c

nhng im nng nh vy.

t bin lch khung (chn hoc mt trn mt si), thng l li ca

polymerase khi sao chp cc on lp li ca mt nucleotid. Mi polymerse c

22

chnh xc khc nhau. Yu t chnh nh hng n chnh xc ca polymerse l c

hot tnh exonuclease c sa 3 5, lm loi b cc base bt cp khng ng chn

vo bi polymerase.

Oxy ha ph hy do cc gc oxy. Cc gc oxy tng trong t bo do chuyn

ha oxy ha (v cng c to bi cc tc nhn vt l nh tia phng x ). Sn phm

oxy ha quan trng l 8 hydroxy guanin bt cp sai vi A, to o chuyn G C

thnh T A.

b. t bin cm ng Cc tc nhn l tng tn s t bin cao hn mc t nhin c gi l tc

nhn gy t bin. Cc tc nhn ny c th lm thay i trnh t ADN. Cc tc nhn

t bin cm ng c chia thnh cc tc nhn ha hc v cc tc nhn vt l [2].

Tc nhn ha hc gy t bin:

C th c chia ra lm 4 nhm:

Nhm base ng ng

- Bromouracil (BU) l hp cht nhn to dng rng ri trong nghin cu .

Ging vi thymin ( c nguyn t Br thay cho nhm methyl) v s gn vo ADN v

bt cp vi adenin ging nh thymin. C nhiu kh nng h bin thnh dng enol(

BU)v c kh nng bt cp vi guanin. Nh vy, BU c th xm nhp v bt cp

b sung vi adenin v thay cho thymin to A BU v vng sao chp tip theo BU

hn bin thnh BU v bt cp vi guanin to BU - G, do A T c thay th

23

thnh G C. Trong khi BU li c th xm nhp sai, khi gn thay ch cho

Cytosin to G - BU, sau BU li hn bin thnh BU v tip theo bt cp vi

adenin to A BU v vng sao chp tip theo adenin bt cp vi thymin thnh A

T.

- Aminopurin l ng ng ca adenin, c th bt cp vi thymin hoc vi

cytosin, gy chuyn v A T thnh G C hoc G C thnh A T. Cc ng ng

base gy chuyn v nh gy hn bin t pht.

Cc cht ha hc thay i cu trc v c tnh bt cp ca cc base, gm:

Cc cht kh amin v cc cht alkyl ha.

- Cc cht kh amin. V d in hnh : cc acid nitr to bi s phn gii cc

nitrit (cht bo qun) trong thc phm lm cytosin thnh uracil, methylcytosin thnh

thymin, v adenin thnh hypoxanthin kh amin, hypoxanthin trong ADN bt cp

vi C gy chuyn v. Methylcytosin c mt trong cc gen ca vi sinh vt bc cao c

th nh hng n hot tnh phin m. Cc base ngoi nh xanthin, hypoxanthin v

uracil c th khi ng c ch sa cha. Thymin, dn xut t methylcytocin ni

sinh v nhiu s chuyn v c th xy ra theo cch ny. Cc gc 5 methylcytosin,

sau tr thnh im nng ca s t bin. Hydroxylamin (NH2OH) ch kh

amin ca cytosin, do tc ng rt gii hn:

Cytosin NH2OH 4-N- Hydroxycytosin, bt cp vi adenin

- Cht alkyl ha. V d in hnh: Nitrosoguanin (NTG), methyl methan

sulfonat (MMS), ethyl methan sulfonat (EMS ) l cc tc nhn ha hc gy t bin

24

phn ng vi cc base v thm nhm ethyl hoc methyl gi l cc tc nhn alkyl

ha . Ty yu t b tc ng, chng c th gy t bin t nht bng 3 cch:

Thm nhm methyl (-CH3) hay ethyl (-C2H5) vo guanin to base

ng ng ca adenin dn ti s bt cp b sung sai.

Mt purin do guanin b alkyl ha to l hng trn ADN , khi sao

chp c th lm t mch.

Lin kt cho gia cc mch ca mt hoc cc phn t ADN khc

nhau lm mt nucleotid.

Nhm chn vo ADN (tc nhn lm lch khung) .

Nhm ny gm cc cht proflavin, cam Acridin, ethidiumbromide dng trong

phng th nghim lm cht nhum. Tt c u l cc phn t a vng phng tng

tc vi cc base ca ADN v chn vo gia chng. S chn ny lm dn si i

ADN v ADN polymerase b nh la chn base vo i din nhiu hn bnh

thng, lm lch khung ADN to thnh.

Cc tc nhn thay i cu trc ADN

Cc tc nhn ny c th l:

- Cc phn t ln (knh cng) gn vo base trong ADN v lm chng tr

thnh khng m ha. V d, N-acetoxy-2-acetylaminofluorene( NAAAF).

- Cc tc nhn gy lin kt cho trong v gia cc si. V d cc psoralen c

trong mt s rau v dng trong iu tr mt s bnh v da.

- Cc cht ha hc gy t ADN v d nh cc peroxid.

- Cc hydrocarbon a vng. V d cc benzypyrene c trong xng.

Tc nhn vt l gy t bin

Cc bc x c bc sng khc nhau nh l tia X, tia gamma, ht phng x ,

gy ra t bin khc nhau. Nhng nh hng ca chng ln ADN thng qua cc

gc t do v tc ng trc tip:

t mt hoc c hai si.

Ph hy cc base.

Lin kt cho trong ADN.

25

2.2. NHNG T BIN GEN CYP2D6

2.2.1. Nhng allen ca gen CYP2D6 C t nht 74 t bin allen khc nhau ca CYP2D6 ( *2 ti *75 ) v mt lot

cc t bin nh c tm ra v miu t bi hip hi Human Cytochrome P450

Allele Nomen-clature. V c 196 a hnh n nucleotid ca CYP2D6 c miu

t c s d liu National Center for Biotechnology Information SNP vi t nht 52

a hnh n nucleotid khng ng ngha c bo co c th c tc ng ln

protein [128]. Xt theo chc nng, nhng allen ca CYP2D6 c th c phn loi

thnh 3 nhm: nhng allen gy tng hot tnh, nhng allen gy ra mt hoc gim

chc nng v nhng allen c chc nng bnh thng. Nhng bin i ny c hnh

thnh t nhng kiu t bin quan trng nh: mt hoc thm, sp xp li gen, xa

gen, lp li mt hoc nhiu ln gen CYP2D6. S phn b ca nhng allen ny vo

cc qun th khc nhau l rt khc nhau. *1A c xem nh l allen bnh thng

dng i chiu vi cc allen khc.

a. Nhng allen CYP2D6 v ngha Nhng allen v ngha khng to ra cc protein hoc c to ra protein nhng

khng tm thy vng c hot tnh enzym. Nhng allen *3, *4, *5, *6, *7, *8, *11,

*13, *14, *15, *16, *18, *19, *20, *21, *38, *40, *42, *44, *56, *62 m ha ra cc

protein khng c hot tnh enzym. Chng gy ra kiu hnh PM khi kiu hnh

ng hp t hoc d hp t. Nhng allen ny l gy ra nhng hu qu lm sng r

rt nh thay i thanh thi ca thuc v p ng thuc. Trong nhng allen v

ngha th *3, *4, *5, *6 chim t l gn 97% tt c nhng allen gy ra kiu hnh PM

ngi da trng [101].

C ch ca vic cc allen v ngha ny lm mt ton b chc nng ca enzym l :

t bin mt base hoc chn / xa mt on nh ci ph v khung c hoc

ngn cn vic ghp ni ng, dn ti sm tng hp protein cui cng / stop codon (

V d: CYP2D6*3, *4, *6, *7, *8, *11, *15, *19, *20, *38, *40, *42, *44, *56 v

*62 [52].

26

Mt chc nng ton b chiu di allen m ha ( V d: CYP2D6 *12, *14 v

*18) [25].

Xa ton b mt on gen hoc xa mt chui rt ln ( V d: CYP2D6 *5 ).

Hnh thnh mt gen lai ( V d: *13 v *16 c s mt mt on chui ln l

hu qu ca gen lai CYP2D7- 2D6) [30].

Allen v ngha do t bin mt base hoc chn / xa mt on nh

CYP2D6*3A c khm ph nhng ngi da trng c kiu hnh PM i

vi debrisoquin [52, 111]. t bin allen *3 dn ti xa base A v tr 2549 trn

exon 5, gy ra s ph v khung c, v s gii thch c s mt chc nng v mt

CYP2D6 kiu hnh PM do sm tng hp protein kt thc nhng c th d hp

t. CYP2D63B cha thm mt t bin 1749 A>G gy ra mt trng hp N166D

(Asparagin c thay bng Asparatic v tr 166 trn protein) [72]. nh hng

chc nng ca allen ny vn cha c bit n.

t bin allen *4 xy ra vi tn sut 22% v chim nhiu hn 75% cc t

bin ngi Thy in da trng [14]. Tuy nhin allen ny xy ra tn sut thp

ngi Trung Quc (~ 1%) [129] v ngi chu Phi ( 3.9%) v y l l do ca vic

t l thp ca kiu hnh PM ngi Trung Quc (~1%) v chu Phi (0-5%) [74,

108], trong khi t l ny ngi da trng l 5-10%. Gaedigk v cng s xc nh

mt trng hp allen CYP2D6*4 khng c a hnh n nucleotid 100C>T v

4180G>C [31]. Th a hnh di truyn 4180G>C c pht hin tt c cc allen

CYP2D6*4 ngoi tr CYP2D6*4J. Trng hp ny l mt kiu hnh PM vi kiu

27

gen CYP2D64/4, ngi c xc nhn c b gen d hp t c t bin

100C>T.

CYP2D6*7 c s bin i v tr 2935A>C trn exon 6 ca gen CYP2D6

gy ra s thay th H324P [26]. Nhng t bin ny lin quan ti kiu hnh PM i

vi spartein v xy ra ngi da trng vi tn sut gn 1%. Khi s thay i H324P

c hnh thnh t cDNA bi v tr t bin gene trc tip, khng c ph CYP no

c kh nng pht hin c to thnh v khng c hot tnh chuyn ha bufuralol

v spartein c pht hin. Hn na, tri vi dng protein t nhin, protein t bin

hu nh ch nm vng cht ly gii khng tan ca mnh v t bo. Nhng kt qu

ny khng nh rng s thay th H324P chu trch nhim v kiu hnh PM trong c

th sng c lin quan n allen CYP2D6*7 bng cch ngn cn s gp np bnh

thng ca protein v s st nhp vi nhn hem.

CYP2D6*11 c xc nh t qun th ngi Php cha cc t bin

883G>C, 1661G>C, 2850C>T v 4180G>C [73]. t bin 883G>C lm ph hy

vng tip ni, gy hu qu thiu ht enzym. S chuyn v 2850C>T v 4180G>C

gy ra s thay i R296C v S486T.

CYP3D6*19 vi s xo 4 base t 2539 n 2542 (AACT), c xc nh t

qun th ngi da trng [72]. S xa ny lm ph v cu trc v tr 255 trn

protein v to ra mt enzym mt hot tnh. CYP2D6*20 l mt allen him vi s

chn G vo gia v tr 1973 v 1974 gy ph v cu trc v tr 211 trn protein.

t bin ny dn ti kiu hnh PM [70].

CYP2D6*56 c xc nh c s thay i 3201C>T trn exon 7. Theo qui

lut n s to ra Arg344 (CGA), nhng by gi c thay th bi mt stop codon

(TGA) v gy ra mt CYP2D6 thiu 153 amino acid cui [64]. Hu qu l enzym

b mt hot tnh.

28

CYP2D6*62 l allen t bin trong xy ra s thay th 4044C>T v gy ra

s bin i R441C trn protein, lm mt s gn kt vi nhn hem. Cc c th c t

bin ny c chng minh gim hot tnh oxy ha spartein [58].

Nhng allen m ha khng cn chc nng y

CYP2D6*12 l allen c pht hin t qun th ngi Php, cha cc t

bin 124G>A, 1661G>C, 2850C>T v 4180G>C [71]. S chuyn v 124G>A,

2850C>T v 4180G>C theo th t gy ra cc bin i G42R, R296C v S486T

trn protein. Protein b t bin b mt hon ton hot tnh chuyn ha spratein, dn

ti kiu hnh PM.

CYP2D6*14A l allen cha cc t bin 100C>T, 1758G>A, 2850C>T v

4180G>C, c tm thy u tin ngi Trung Quc v sau l qun th ngi

chu [61, 130]. C 4 acid amin b thay i CYP2D6.14 l: P34S, G169R,

R296C, v S486T, vi G169R l bin i duy nht ch gp protein t bin ny

[16, 130]. Hai s thay i P34S v S486T c s trng hp vi s thay i

CYP2D6.10. Mt ngi Trung Quc c kiu gen CYP2D6*5/*14 c ch ra l

c kiu hnh PM vi t l chuyn ha MR >12 i vi debrisoquin [130]. Mt

nghin cu chc nng gn y cho thy khng pht hin c hot tnh ca

CYP2D6.14A khi chuyn ha bufuralol v dextromethorphan [102]. CYP2D6*14B

cng cha intron 1 hon i vi CYP2D7 (base nucleotid t 215- 245) ngoi 4 SNP

trong CYP2D6 pha trn. Allen ny c tm thy ngi Trung Quc vi tn

sut 2.0% . Allen *14B khc vi nhng allen 14 khc l s thiu chuyn v

188C>T thay vo 1749G>C c thm vo. Tri vi allen *14A, allen *14B c

hot tnh enzym ng k, y c th l do s vng mt ca s thay th P34S [50].

Allen CYP2D6*18 l allen c 9 base c chn vo ( GTGCCCACT) t

4125 ti 4133 trn exon 9 v c tm thy u tin qun th ngi Nht. Allen

29

ny lin qua n kiu hnh PM. Hng s Km i vi s hydroxyl ha bufuralol thc

hin bi CYP2D6.18 cao gp 236 ln so vi mc bnh thng (990 so vi

4.2mol/L ) [102, 134].

S xa ton b on gen

CYP2D6*5 l t bin xa ton b on gen v c tn sut tng t nhau

cc qun th khc nhau ( 4 7% ) [30, 116]. y l t bin bt hot allen ph bin

th hai ngi Anh v tn sut ca n qun th ngi da trng l 4% [81]. i

vi ngi Trung Quc tn s xut hin t bin ny l 7.2% [50]. Gaedigk v cng

s xc nh s xa ton b on gen di 11.5kb trn gen trong c cha

CYP2D6 v khng tm thy CYP2D6 trong gan [30].

Hnh thnh gen lai

CYP2D6*13 l allen lai cha exon 1 ca CYP2D7 v exon 2 9 ca gen

CYP2D6 c xc nh u tin qun th ngi Php, gy ra kiu hnh PM [92].

Allen CYP2D6*16 cng l mt gen lai cha exon 1 7 ca CYP2D7 v

exon 8 - 9 ca CYP2D6 [17].

C hai gen lai ny u to ra s xa mt on ln gen do s trao i cho bt

thng hoc c ch mt s mc ni.

Hnh 10. Hnh thnh gen lai [33]

b. Allen mt chc nng mt phn Nhng allen CYP2D6*10, *14, *17, *18, *36, *41, *47, *49, *50, *51, *54,

*55, *57 gy ra s gim ng k hot tnh enzym. Nguyn nhn l do s gim tnh

n nh ca protein, ph v cu trc vng gn c cht, hoc gim i lc c cht

enzym. S thay i hot tnh enzym c th ph thuc c cht i vi vi allen nh

*17. Cc trng hp c th c th gy ra kiu hnh PM hoc IM.

Allen *10 xy ra vi tn s 33 43 % i vi ngi chu bao gm ngi

Nht, Hn Quc v Trung Quc [11, 48, 50]. Tuy nhin tn s ny ngi da trng

30

v ngi M gc Phi v nhng qun th khc (v d nh n ) l 2 5% [63].

Allen *10A cha a hnh n nucleotid 100C>T gy ra s thay i P34S vng

giu Prolin (PPGP) gn nhm NH2 cui cng ca protein v lin quan ti t l

thanh thi thp spartein (MR > 1.5) trong cc th nghim in vivo [135]. Nhng c

th ng hp t *10/*10 yu cu mc liu metoprolol v nortriptylin thp hn c

th c kiu gen *1/*1 to ra hiu qu lm sng tng t [45, 137]. V tr P34 c

th hot ng nh bn l gia vng mng k nc v vng lin kt vi nhn hem

ca enzym. a hnh n nucleotid 100C>T to ra mt protein khng n nh c

hot tnh enzym gim i vi chuyn ha debrisoquin, bufuralol v

dextromethorphan. Allen ny cng cha cc t bin 1661G>C v 4180G>C gy ra

t bin S486T trn protein [102]. Shen v cng s khm ph ra hot tnh xc

tc ca CYP2D6.10 i vi mt lot cc c cht. ng tm ra rng gi tr CLint

ca s hydroxyl ha bufuralol ti v tr carbon 1 (bufuralol 1 hydroxylation), ct

nhm methyl khi dextromethorphan qua cu ni oxy (dextromethorphan O-

demethylation), hydroxyl ha debrisoquin v tr carbon 4 (debrisoquin 4-

hydroxylation), hydroxyl ha atomoxetin carbon 4 (atomoxetin 4- hydroxylation),

ct nhm methyl khi S-fluoxetin qua cu ni ni t (S-fluoxetin N-demethylation),

hydroxyl ha nortriptylin carbon 10 (nortriptylin 10-hydroxylation), ct nhm

methyl khi tramadol qua cu ni oxy (tramadol O-demethylation) v ct nhm

methyl khi codein qua cu ni oxy (codein O-demethylation) bi CYP2D6.10

ln lt gim xung cn 3.65%, 5.29%, 11,84%, 8.58%, 7.54%, 1.32%, 6.90% v

27.86% so vi protein bnh thng.

S nhn i allen CYP2D6*10 (*10 2 ) c tm thy u tin qun th

ngi Trung Quc nhng hot tnh ca enzym li gim [35, 50]. Tn s ca allen

ny ngi Nht Bn kh thp (0.6%) [78].

Allen *17 xy ra vi tn s cao ngi Chu Phi v ngi M gc Phi

nhng li hu nh khng xut hin ngi da trng [11]. Tn s ca allen ny

qun th ngi Zimbabwe l 34% [75], ngi Tanzania l 17% [131], ngi Ghana

l 28% [40]. Hn 10% ngi Zimbabwe c kiu gen ng hp t vi allen ny.

31

Nhng bin i ny gii thch ti sao ngi Chu Phi c ch s MR trung bnh

cao hn ngi da trng [63].

Allen CYP2D6*17 cha 4 SNP l : 1023C>T trn exon 2; 1661G>C trn exon

3 (mt SNP ng ngha); 2850C>T trn exon 6 v 4180G>C trn exon 9. Kt qu

khi dch m ln lt gy ra cc s thay th: T107I, R296C v S486T [75]. So snh

vi CYP2D6*2 th CYPD6*17 c thm 1 s thay i T107I. S thay i ny xy ra

vng helix v v tr 107 c th lin quan n vng nhn c cht. Mt th

nghim in vivo ch ra rng allen ny gy ra s gim hot tnh phn ng hydroxyl

ha debrisoquin [75]. S biu hin ca cADN trong COS-1 cell tit l enzym

CYP2D6.17 th hin ch 20% hot tnh so vi dng t nhin, trong khi s thay i

mt mnh T107I khng c nh hng ng k n chc nng enzym [88]. Enzym

cha c t bin T107I v R296C c ch s Km i vi bufuralol cao hn 5 ln so

kiu hnh bnh thng, trong khi t bin S486T khng gy ra nh hng. Tri

li khi codein c dng lm c cht, t bin T107I mt mnh n cng gy ra

s tng hng s Km biu kin, iu ny ch ra rng nh hng khc bit ca cc t

bin ny ph thuc vo c cht ca CYP2D6 c s dng. CYP2D6.17 c hng

s Km biu kin i vi codein cao gp 5-10 ln so vi enzym bnh thng. Shen

v cng s khm ph ra hot tnh xc tc ca CYP2D6.17 i vi mt lot c

cht. ng ch ra rng gi tr CLint ca s hydroxyl ha bufuralol, ct nhm

methyl khi dextromethorphan, hydroxyl ha debrisoquin, hydroxyl ha

atomoxetin, ct nhm methyl khi S-fluoxetin, hydroxyl ha nortriptylin, ct nhm

methyl khi tramadol v ct nhm methyl khi codein thc hin bi CYP2D6.17

gim ln lt 22.69%, 16.69%, 64.25%, 21.89%, 8.17%, 7.33%, 35.70% v

80.35% so vi enzym bnh thng. Allen *17 c th to ra mt protein vi c ch

xc tc khc thng bi s tng tc c cht enzym c th thay i rt khc nhau

ty thuc c cht [106].

CYP2D6*36 thng c gi *10C lin quan vi kiu hnh PM. Allen ny

cha rt nhiu t bin a dng nh : 1426C>T, -1235A>G, -1000G>A, 100C>T,

310G>T, 843T>G, 1039C>T, 1661G>C, 2097A>G, 3384A>C, 3582A>G v gen

32

hon i vi CYP2D7 exon 9 [31]. Cc protein t bin bao gm: P34S, P469A,

T470A, H478S, G479A, F481V, A482S v S486T. S bin i 100C>T (P34S)

CYP2D636 l nguyn nhn chnh to ra s mt n nh protein v 6 acid amin

khc exon 9 khng c nh hng ng k n hot tnh enzym. C CYP2D6.10

v CYP2D6.36 u c khng nh s gim thanh thi ca venlafaxin in

vivo do s gim to thnh protein v tng hng s Km ca c cht [29]. Gi tr Km

ca CYP2D6.1, CYP2D6.10A v CYP2D6.10C ln lt l 1.7, 8.5 v 49.7 mol/L

i vi s hydroxyl ha bufuralol; v ln lt l 9.0, 51,9 v 117.4 mol/L i vi

s ct nhm methyl khi venlafaxin. Nhng kt qu trn ch ra rng s gim

CLint do CYP2D6.10 v CYP2D6.36 c gy ra khng ch bi hot tnh protein

thp m cn bi s tng gi tr Km do gim i lc i vi enzym. Khi c miu t

trong COS-7 cells, CYP2D6.36 th hin hot tnh ngho nn i vi chuyn

ha bufuralol v dextromethorphan [102].

Allen CYP2D6*41 l mt bin i ca allen CYP2D6*2 vi thm mt t

bin 1548G>C, c hai allen ny u gy ra hai s thay i acid amin ( R296C v

S486T). Hai ca nghin cu gn y ch ra rng 1584G>C gy ra s thiu kt ni ca

phn t mARN [121]. Tuy nhin, nhng c th ng hp t CYP2D6*41 c kiu

hnh tng t IM do s khim khuyt allen CYP2D6 [97]. CYP2D6*2 v

CYP2D6*41 l hai trng hp thng c s lp li mt hoc nhiu ln gen

CYP2D6, n c xem nh l s bin i c chn lc thng xy ra vng bc Phi

[46].

CYP2D6*47, *51 v *57 l nhng allen m ha cho nhng enzym t bin

vi hot tnh cn li b hn 5% so vi thng thng [102]. Allen CYP2D6*47 ln

u tin c xc nh qun th ngi Nht, n bao gm cc thay i R25W,

P34S v S486. Ngi ta cho rng nhng c th c kiu gen *47 s gy ra kiu hnh

33

PM [114]. Allen *51 c bo co bi Soyama v cng s. N lin quan n s

thay i R296C, E334A, S486. Glu334 nm helix J v c trng trong cc CYP,

gi ti vai tr c bit quan trng ca n trong chuyn ha [100]. Allen *57 c

xc nh bi Soyama v n cha cc bin i P34S, R62W, P469A, T470A, H478S,

G479A, F481V, A482S v S486T [115]. S mt gn ht hot tnh ca CYP2D6.51

l do s xut hin ca P34S, iu ny cng c th hin nhng protein c m

ha bi gen *47, *36. C s lin h mt thit gia CYP2D6.10 v CYP2D6.57 v v

vy chng thng c so snh vi nhau. nh hng chc nng ca *51, *57 vn

cha c r rng nhng ngi ta cho rng n gy ra kiu hnh PM.

Allen CYP2D6*10, *18,*49, *50, *54 v *55 c xc nh ln u tin

qun th ngi Nht, n m ha cc protein ch cn 7 36% hot tnh so vi

protein bnh thng. Nhng allen ny c cho l lin quan n kiu hnh PM v

nhiu kh nng hn na l IM. CYP2D6.50 cha E156A, CYP2D6.54 cha P34S,

T216I v S486I; CYP2D6.55 cha cc t bin R296C, K404Q v S486T.

CYP2D6.2 v CYP2D6.10, CYP2D6.14, CYP2D6.18 v CYP2D6.55 c miu t

trong COS-7 cells ch ra s tng ng k gi tr biu kin Km, trong khi

CYP2D6.49, CYP2D6.50 v CYP2D6.53 li c gi tr Km biu kin thp hn so vi

dng protein bnh thng. Gi tr Vmax ca CYP2D6.10, CYP2D6.49, CYP2D6.54

ln lt l 20%, 17% v 6% so vi dng bnh thng [102].

c. Nhng allen gy tng nh hot tnh Nhng nghin cu chc nng khng chng minh c s bin i ng k gia

hot tnh enzym v mt s allen CYP2D6, bao gm c *2A, *172, *27, *35, *39,

*412 v *48. Marez v cng s nghin cu ngi chu u v khng tm thy

nh hng chc nng ca nhng allen ny, hay ni cch khc th hot tnh enzym

m n m ha tng t vi loi bnh thng. H cng xc nh c cc allen

CYP2D6*2B, *2C, *2D, *2E, *2F, *2G, *2H v *2K [72]. Gaedigk v cng s xc

nhn thm c 2 allen na trong chui *2 l :*2L v *2M . Hai allen ny cha

nhiu t bin a dng (v d: 310G>T, 746C>G v 843T>G). Nhng t bin -

1584C>G, -1235A>G, -740C>T v -678G>A c th tm thy ph bin cc nhm

34

t bin thuc nhm CYP2D6*2 nh *2A ti *2H, *2J ti *2N v *2J = *59.

Nhng nh hng ti chc nng ca cc allen ny cha r rng nhng ngi ta cho

rng n c tc ng nh ti hot tnh enzym [96].

Allen CYP2D6*24, *26, *27 l nhng allen him c tm thy ngi da

trng vi tn sut 0.1- 0,3%. CYP2D6.24 (I297L) v CYP2D6.26 (I369T) th hin

hot tnh ng k vi chuyn ha codein v dextromethorphan. CYP2D6.24 to ra

mc CLint cao gp 6 ln i vi s chuyn ha dextromethorphan nhng li gim

50% s chuyn ha codein so vi CYP2D6.1 do tng Km ln 3 ln. CYP2D6.26 to

ta mc CLint thp hn 37.7% i vi s s chuyn ha dextromethorphan nhng li

cao hn 42.6% i vi s chuyn ha codein so vi dng t nhin. CYP2D6.27 to

ra mc CLint cao hn 45.9% vi s chuyn ha codein nhng thp hn 64.8% vi s

s chuyn ha dextromethorphan khi so snh vi dng t nhin [139].

Allen CYP2D6*27, *39, *48 m ha to ra nhng protein c hot tnh

chuyn ha kh mnh so vi dng t nhin [102]. CYP2D6.27 (E410K) ,

CYP2D6.39 (S486T) v CYP2D6.48 (A90V) c miu t trong COS-7 cells lm

tng nh ch s CLint so vi dng t nhin. A90, E410, v S486 nm -sheet 3,

gia cc helix K v K v trn helix B. iu cho thy nhng v tr ny l khng

quan trng i vi chc nng ca CYP2D6 [100].

Mt pht hin th v khi CYP2D6.53 c miu t trong COS-7 cells, n

cho thy s gim 73% tr s Km biu kin i vi s chuyn ha bufuralol (2.46 so

vi 9.2mol/L). Kt qu l lm tng 4 ln tr s CLint so vi dng t nhin [102].

CYP2D6.53 cha t bin F120I, A122S v c s thay i phn cn li c nm

trn mt vng nhn c cht ca CYP2D6. V tr F120 ng mt vai tr quan trng

trong s kt ni c cht v nh hng [27]. Khi F120 c thay i thnh Ala, tr

s Km i vi s chuyn ha coumarin (7-methoxy-4-(aminomethyl)-coumarin)

gim 50% cng vi i lc enzym c tng ln [54]. Mt iu ng ngc nhin

khc l protein t bin mang t bin F120A c th chuyn ha quinidin thng qua

vic thm vo nhm hydroxyl v ct i nhm methyl, dng t nhin khng th lm

c iu ny [76]. t bin F120I (358T>A; rs1135822) c th c tm thy mt

35

t l nh ngi ng Nam . Nhng c th c kiu gen *53 c th to ra kiu

hnh UM [112].

d. S lp li mt hoc nhiu ln gen CYP2D6

Gen CYP2D6 c cha nhiu t bin lm tng s lng bn sao gen. mt

gia nh ngi da trng c ngi cha v 2 ngi con c kiu hnh UM i vi

debrisoquin (MRT trn exon

6 v 4180G>C trn exon 9. t bin 2850C>T dn n s thay i R296C. t

bin 4180G>C dn n s thay th S486T [51]. Oscarson v cng s tm ra rng

CYP2D6.2 lm tng Km v Vmax i vi s chuyn ha codein thnh morphin so vi

CYP2D6.1. Kt qu ny c lm r hn khi m cu trc tinh th ca CYP2D6

c tit l, theo th vng 296 ca helix I gn Asp301 v vng 486 -sheet 4

ng vai tr l vng nhn c cht ca CYP2D6 [100].

Lc u, nhng allen vi 0,1,2,3,4,5 v 13 bn sao gen c bo co bi

Johanson v Aklillu. Trong mt gia nh ngi Thy in (cha, con gi v con trai)

c khong 13 bn sao ca allen CYP2D6 c xc nh. Nhng ngi c kiu

gen CYP2D62N (N = 2,3,4,5 hoc 13) vi hot tnh CYP2D6 rt cao c xc

nh ngi Thy in, mt qun th thuc chng tc Ethiopian. S lp li mt

hay nhiu ln ton b on gen l do s bt thng trong qu trnh trao i cho v

do nhng c ch khc. Vic lp li mt hay nhiu ln gen CYP2D6 c th gy ra

nhng enzym c chc nng bnh thng, c chc nng mt phn hoc khng c

chc nng [5, 51]. Gaedigk v cng s tm ra nhng gen nhn i l *1, *2, *4,

*6, *10, *17, *29, *35, *43 v *45. S nhn i xy ra vi tn s ln lt l 1.3%,

36

5.75% v 2% ngi da trng, ngi chu M gc Phi v nhng chng tc khc.

Hu ht nhng t bin nhn i tr *35N c tm thy ngi Chu M gc

Phi [34].

Hnh 11. S lp li mt hay nhiu ln v xa ton b gen CYP2D6 [43]

Tn s xut hin ca s lp li gen CYP2D6 nhng chng tc khc nhau th

khc nhau. Vi allen CYP2D6*2N, tn sut xy ra ngi chu Phi (28 56%)

thng cao hn so vi ngi chu v ngi da trng. t bin *4N to ra mt

allen khng c chc nng c tm thy vi tn s 37% ngi ng nhng

him gp ngi chu Phi v ngi da trng. Tn s ca CYP2D6*102 lm gim

ng k hot tnh enzym ngi Trung Quc v Nht Bn l thp (0.5-1%) v hu

nh bng 0 ngi da trng [35, 78]. Kiu hnh UM c phn b chnh ng

Bc Phi ( ch yu *2N) v chu i Dng( ch yu *1N) [109]. Tri vi kiu

hnh PM c mc MR ca debrisoquin c chun ha mc 12.6 th kiu hnh

UM khng c nh ngha r rng m ch c tiu ch c nhiu hn 2 bn sao gen. C

s chng cho khi xc nh kiu hnh UM v EM. kiu hnh UM, lng cht

37

chuyn ha c th tng cao gp 10 ti 30 ln so vi bnh thng v do c tnh

tim n ca nhng cht chuyn ha phi c theo di cht ch [15].

2.2.2.Kiu hnh v kiu gen CYP2D6 ca cc chng tc trn th gii

Trong mt nghin cu ca i hc Helsinki Phn Lan, ngi ta tng hp

hng trm bi bo t nm 1991 n 2007 vi 1064 c th thuc 52 qun th trn

ton cu c chn on kiu gen cng nh kiu hnh a ra c bc tranh

ton cnh v a hnh CYP2D6 v gen CYP2D6 [109]. Kt qu c khi qut ha

qua biu 2.5 v 2.6.

Cc allen gi nguyn chc nng CYP2D6*1, CYP2D6*2 l nhng t bin

ph bin, c phn b khc nhau cc vng a l. Cc allen lm gim chc nng

CYP2D6*10, CYP2D6*17 ln lt rt ph bin qun th chu v chu Phi.

Trong khi CYP2D6*41 ph bin nht Trung ng v Trung/Nam . Allen

khng c chc nng rt ph bin - CYP2D6*4 c tn sut cao nht qun th ngi

chu u. Allen gim chc nng CYP2D6*2N t tn s cao Bc Phi (28.3%).

Hnh 12. Kiu gen CYP2D6 cc vng a l khc nhau [109]

38

Hnh 13. Kiu hnh ca CYP2D6 cc vng a l khc nhau [109]

(Hai ng song song ch s nhn i gen. Lin quan hot tnh ca enzym vi kiu

gen: tng(), gim() mt chc nng();UM : kiu hnh chuyn ha siu nhanh,

EM : kiu hnh chuyn ha nhanh, IM: kiu hnh chuyn ha chm, PM: kiu hnh

chuyn ha rt chm hoc khng chuyn ha).

Kt qu bt ng l qun th c kiu hnh UM cao nht thuc v ngi Bc Phi

vi t l ln ti 40%. Tip theo sau ln lt l ngi chu c vi t l 25.6%, ngi

Trung ng 12.2% v ngi chu M 8.3%. Trong khi kiu hnh PM li rt ph

bin ngi chu u vi t l 7.6%. i vi kiu hnh IM, qun th ng , Chu

Phi, Trung ng chim t l cao do cc qun th ny allen gim chc nng

CYP2D6*10, CYP2D6*17 v CYP2D6*41 xut hin vi tn s cao [109].

2.3. HU QU LM SNG CA TNG TC KIU GEN KIU HNH

CA CYP2D6

Gen CYP2D6 l gen c tnh a hnh cao gia cc qun th ngi, dn n s a

dng v kiu hnh tng ng. i vi cc gen m ha CYP, cc trng hp a hnh

v kiu gen c th dn ti 4 loi kiu hnh tng ng:

39

Kiu gen bnh thng tng ng vi kiu hnh enzym chuyn ha nhanh

(extensive metabolism EM)

Kiu gen t bin dn ti kiu hnh chuyn ha siu nhanh (ultra metabolism

UM)

Kiu gen t bin dn ti kiu hnh chuyn ha chm (intermediate

metabolism IM)

Kiu gen t bin dn ti kiu hnh chuyn ha rt chm hoc khng chuyn

ha (poor metabolism PM)

Cc trng hp thay i v kiu hnh enzym chuyn ha thuc ny c th dn

n nhng khc bit ng k v p ng iu tr, bao gm nhng s thay i v

hiu qu iu tr v tc dng khng mong mun ca thuc. Hnh 14 miu t s phn

loi kiu hnh da trn kiu gen v hu qu lm sng ca n trn chuyn ha thuc.

Bt hot Tht bi tc dng nhy cm ADR

iu tr mong mun ADR

Hot ha nhy cm tc dng gim tht bi

ADR mong mun tc dng iu tr

Hnh 14. Phn loi kiu hnh da trn kiu gen v hu qu lm sng da vo

kiu xc tc bi hin tng a hnh enzym [109]

(t bin mt chc nng:mu en; gim chc nng:mu xm;cn nguyn hot

tnh: mu trng; ng t nt: xa ton b gen; mu :phn t thuc c hot

40

tnh; mu xanh: phn t thuc bt hot; UM: chuyn ha siu nhanh, EM: chuyn

ha nhanh, IM: chuyn ha chm, PM: chuyn ha rt chm hoc khng chuyn

ha).

Bng 2.1. nh hng ca a hnh di truyn CYP2D6 ln mt s thuc iu tr

[140].

S

th

t

Thuc Nhm

thuc

Chnh lch v nng

/ AUC (gia PM

v EM)

p

ng/c

tnh

1 Atomoxetin

Thuc

iu tr hi

chng tng

ng

8 ln

Nhiu tc

dng ph

kiu hnh

PM

2 Codein Opioid

Liu n v liu lp

li:Khng c s

khc bit v nng

; nng rt thp

hoc khng pht

hin c kiu

hnh PM

Gim tc

dng gim

au, t gy

ph thuc

thuc

3 Chlorpromazin Thuc an

thn

Liu n:khng c

s tng ng k

Cha xc

nhn

4 Flecainid

Thuc

chng lon

nhp

Liu n: 1.7, 2.4,

3.6 ln i vi ln

lt hn hp, ng

phn S v R; Liu

lp li khng khc

bit

Khng c

s khc

bit thi

gian ko

di sng

QRS, nhiu

tc dng

ph kiu

41

hnh PM

4 Doxepin Trm cm

ba vng

Liu n: 2.9 ln

cao hn cc cht

chuyn ha c hot

tnh kiu hnh PM

t bng

chng v

tc dng

ph

6 Imipramin Trm cm

ba vng

Liu n: 9.1 ln i

vi desipramin; liu

lp li: 2.2, 6.8, 4.8

ln i vi

iminpramin,

desipramin v c hai

H huyt

p

7 Metoprolol Chn Beta Liu n: 4 ti 8

ln(PM)

Cha c

nhiu bng

chng v

cc tc

dng ph

8 Mexiletin Chng

lon nhp

Liu n: 2.0 n

2.3 ln Cha r

9 Mianserin Chng

trm cm

Liu n: 1.8 v 1.5

ln i vi ln lt

mianserin v

desmethylmianserin

Hi tng

p ng

10 Paroxetin SSRI Liu n: 7.1 ln

Ri lon

tnh dc

nhiu hn

kiu hnh

EM

11 Perhexilin Chng au

tht ngc Liu n: 6 ln

Nhiu tc

dng nguy

42

him PM

12 Perphenazin An thn Liu n: 4.1 ln ;

liu lp li : 2 ln

Tng phn

ng c hi

kiu hnh

PM

13 Tamoxifen iu tr

ung th

Mc endoxifen v 4-

OH-tamoxifen trong

mu thp hn kiu

hnh PM

T l ti

pht ung

th v cao

kiu hnh

PM

14 Tolterodin

i khng

th th

muscarinic

Liu lp li: 10 ln

kiu hnh PM

Cc tc

dng i

khng

muscarinic

khng i

15 Tropisetron Chng nn Liu n: 6.8 ln

Gim tc

dng chng

nn

Ngi ta c tnh rng CYP2D6 chuyn ha t 20 n 25% cc thuc thng

dng [47, 120]. Nhng bnh nhn a bi ha gen CYP2D6 thng c kiu hnh

chuyn ha nhanh (UM) v do h s khng t c nng thuc iu tr nu

ch s dng liu thng thng. Mt khc nhng bnh nhn thiu ht chc nng

enzym CYP2D6 l nhng ngi c kiu hnh chuyn ha chm (PM). Khi xc nh

c kiu gen CYP2D6 s gip d on c thanh thi ca cc thuc chng

trm cm nh l desipramin, fluvoxamin, mexiletin, mianserin, nortriptylin v

paroxetin, hay thuc an thn nh perphenazin v paroxetin, hoc l thuc i khng

receptor muscarin nh tolterodin [57].Ngi ta thng k trn hn 100 bnh nhn

tm thn, chi ph iu tr cho nhng bnh nhn c kiu hnh PM hoc UM s tn

43

hn cc bnh nhn thng thng t 4000 n 6000 la [12]. Thi gian iu tr cho

bnh nhn nhm PM l lu nht. Mt khc trong cc bnh nhn khng p ng iu

tr th c mt lng ln cc bnh nhn c kiu hnh UM. Ngi ta c tnh rng s

lng ngi chu u a b ha gen CYP2D6 c th l 40 n 60 triu ngi. iu

ny l gii v sao thiu p ng iu tr li thng xuyn xy ra vi cc bnh nhn

trm cm ngi da trng [46]. Vai tr chuyn ha sinh hc ca CYP2D6 i vi cc

thuc chng au tht ngc cng c lu . S gn mt nhm OH

(monohydroxylation) vo perhexilin c xc tc ch yu bi cc isoenzym v

ngi c kiu hnh PM s chuyn ha yu hn EM gn 100 ln. Kt qu l tn s

xy ra cc triu chng ph ca perhexilin bnh nhn c kiu hnh PM gp 5 ln

nhng bnh nhn EM [7, 113].

i vi mt s thuc hin nay nh tamoxifen vic c th ha iu tr ang

c thc hin cht ch. Bnh nhn trc tin c lm xt nghim chn on kiu

gen CYP2D6 sau d on kiu hnh chuyn ha thuc. Vic cung cp liu dng

thuc c da trn kt qu chn on ny. Nhng bnh nhn c kiu hnh EM s

nhn c mc liu tamoxifen l 20mg/ngy, trong khi cc bnh nhn c kiu

hnh IM v PM nhn mc liu t 20 n 40mg/ngy [126].

CHNG 3. PHNG PHP PHT HIN T BIN GEN CYP2D6

3.1. K THUT ADN CHIP

3.1.1. Nguyn tc: ADN Chip hoc ADN microarray l mt k thut mi pht hin cc t

bin c hiu vi tc rt nhanh, chnh xc da trn c s phn tch t bin bng

vi tnh. i vi CYP2D6 phng php ny cho php pht hin ng thi 33 t

bin [98].

sn xut cc ADN chip , cc oligonucleotid ng vai tr ca cc on d

(probe) c my ci ln mt phin knh nh. Mi phin knh nh vy vi din tch

khong 1cm2 c th cha t hng trm n hng ngn loi oligonucleotid khc nhau.

44

Cc oligonucleotid ny gm cc ADN c trnh t bnh thng v cc ADN c trnh

t mang cc t bin gy bnh bit. ADN ca mt i tng no c gn

hunh quang v cho lai vi cc oligonucleotid trn phin knh xc nh xem

chng lai vi oligonucleotid bnh thng hay t bin. Mu lai c phn tch trn

my vi tnh xc nh ADN ca i tng l bnh thng hay mang loi t bin

c th no [118]. Hnh 15 l minh ha ca mt AND chip.

Hnh 15. S minh ha mt AND chip [118]

(Cc oligonucleotid c t trn con chip, sau cho tip xc vi ADN c gn

hunh quang ca mt i tng. Qu trnh lai xy ra nu c mt oligonucleotid c

trnh t nucleotid b sung vi trnh t ADN ca i tng. V tr pht hunh quang

trn chip s nh du v tr ca on oligonucleotid trn chip).

3.1.2. u im: - Phng php ny c u im l thi gian tin hnh nhanh.

- C mu p dng ln.

- Thao tc nhanh gn, d lm.

- chnh xc cao.

45

3.1.3. Nhc im: Yu cu thit b t tin, khng lm gim gnh nng ti chnh i vi cc

trng hp ADR lin quan n CYP2D6. Ngoi ra, mt iu ng lu l cng vi

s gia tng ca cc t bin mi c pht hin, t l chnh xc ca phng php

ny ngy cng gim. Nm 2005 l ln u tin FDA ph duyt cho mt phng

php xc nh t bin CYP2D6 v CYP2C19 bng ADN chip ca hng Roche. Khi

phng php ny c cc nh khoa hc khuyn co l ng tin cy, chnh xc

nhng ti nm 2007 cng chnh FDA a ra khuyn co v s km tin cy ca n.

Theo nghin cu thc hin ngi Nam Phi, t l thnh cng trong vic xc nh

kiu gen CYP2D6 ca phng php ny ch l 75.9% [20]. Cn theo mt nghin

cu khc th ADN chip khng c kh nng pht hin c t bin CYP2D6*9 do

vy lm sai lch kt qu nghin cu [32].

3.2. K THUT LONG-PCR

3.2.1. Nguyn tc K thut long-PCR l mt bin th ca phng php PCR, dng khuch i

cc on ADN di hn 5kb. K thut ny khc bit vi PCR truyn thng v

khong thi gian ca vic bin tnh v thi gian ko di chui. Hn th na n s

dng nng mi, nng dNTP cng nh nng Mg2+ cao hn. k thut

long-PCR thnh cng, vic phi hp hai enzym polymerase l rt quan trng.

Enzym th nht c hot tnh 3-5 proofreading exonuclease v enzym th hai ch

l mt enzym ch tng hp theo chiu 5-3 [95].

3.2.2. u im - y l k thut n gin, d thc hin.

- Khng yu cu thit b phc tp, t tin.

3.3.3. Nhc im

- Ging nh cc k thut PCR khc, long-PCR rt d nhim tp ADN do

yu cu thao tc phi rt cn trng.

- C mu b.

46

3.3.4. p dng xc nh s nhn i gen CYP2D6

Phng php ny c dng pht hin s sao chp gen CYP2D6, cc mi

c s dng l: Mi xui 5-TCCCCCACTGACCCAACTCT-3 v mi ngc

5-CACGTGCAGGGCCTA GAT-3 [95]. Chng to ra mt sn phm 5.2kb mi

mu v mt sn phm 3.6 kb nhng bnh nhn c s lp li gen CYP2D6. Sn

phm 5.2 kb c ngun gc t vng ni gia gen CYP2D6 v pseudogen CYP2D7,

trong khi phn phm th hai c ngun gc t vng ni gia nhng bn sao gen

CYP2D6. Hnh 16 miu t cc sn phm long-PCR.

Hnh 16. Sn phm Long-PCR [95]

(5.2kb vng tip gip, 3.6kb: vng gen CYP2D6 nhn i)

Sn phm long-PCR c phn tch trn gen agarose 2% (Quiagen, Germany)

khi cho chy in di (BioRad, USA) . S kim tra gel v hnh nh phn tch thu

c bng cch s dng h thng d liu v my chp nh mu trn gel (Kodak,

USA). Hnh 17 l nh in di trn gel.

47

Hnh 17. Ph in di [95]

((1) C th cha s nhn i gen CYP2D6; (2) C th khng c s nhn ln ca

gen CYP2D6)

3.3. K THUT REAL-TIME PCR

3.3.1. Nguyn tc: Real-time PCR l k thut PCR m kt qu khuch i ADN c hin th

ngay sau mi chu k nhit phn ng, chnh v vy nn c gi l real-time. V do

c im ny nn ngi lm th nghim PCR khng cn thit phi lm tip cc th

nghim c v phn tch kt qu xc nh c sn phm khuch i ch hay

khng v kt qu cui cng ca phn ng khuch i cng c hin th ngay sau

khi hon tt phn ng khuch i. Nh vy c th ni real-time PCR l k thut

nhn bn ADN ch trong ng nghim thnh hng t bn sao da vo cc chu k

nhit v kt qu khuch i trong cc ng phn ng c hin th cng lc vi phn

ng khuch i xy ra ngi lm th nghim c th thy c. Thit b quan

trng ca k thut ny l my real-time PCR. Thit b ny cng ging cc my PCR

thng thng nhng c trang b thm mt b phn c gi l thit b real-time.

Kt cu ca thit b ny gm hai b phn chnh: (1) ngun sng pht ra tia sng kch

thch c bc sng xc nh ln cc ng phn ng, (2) Camera hay cm bin quang

48

ghi nhn c c nh sng hunh quang pht ra t cc ng phn ng sau khi cc

ng ny c kch thch bi nh sng do ngun sng pha trn. c c tn hiu

hunh quang ngi ta c th c nhiu cch thit k:

- Cht pht hunh quang l mt loi mu hunh quang chn vo si i ADN.

Cht hay dng l SYBR I. Tuy nhin cht ny c tn hiu hunh quang pht ra

khng cao v c th c ch qu trnh PCR [62].

- S dng probe l cht pht hunh quang

Probe l nhng on nucleotid si n c trnh t c th bt cp b sung vi mt

trnh t c hiu trn ADN ch (trong PCR th chnh l sn phm khuch i

c hiu t ADN ch). Khi nhng probe c nh du hunh quang ny bt

cp vi ADN ch n s pht ra tn hiu hunh quang. C th c nhiu cch to ra

probe, thm ch c th dng primer lm probe. Cc probe hay dng l Taqman

probe, Beacon probe, probe lai [93].

S lng bn sao s c c thng qua biu khuch i, biu chun v

cc h thng phn tch kt qu. Trong biu khuch i m t tng quan gia

cng hunh quang ghi nhn c tng ng vi s chu k PCR. Biu chun

m t tng quan s lng bn sao vi chu k ngng [3].

3.3.2. u im

Phng php ny nhanh cho kt qu, chnh xc cao, ng tin cy, cho php

sng lc c cc a hnh di truyn him gp.

3.3.3. Nhc im Yu cu thit b chuyn dng t tin.

3.3.4. p dng xc nh t bin CYP2D6

Theo mt th nghim p dng phng php real-time PCR vi mu d

TaqMan, cc tc gi thc hin xc nh kiu gen CYP2D6 cho 617 trng hp.

Tt c cc trng hp ny c xc nh kiu gen trc bng k thut PCR

truyn thng. Trong 617 trng hp ny c 33 trng hp gen CYP2D6 b xa,

49

551 trng hp c gen c lp li mt ln v 33 trng hp c lp li nhiu hn

mt ln. Trnh t cc loi mi v mu d c s dng trong th nghim ny l :

Hnh 18. Mi v trnh t mi [82]

Phn tch s b kt qu cho thy, vi mi CYP2D6 exon 9 c s dng cho

t l kt qu tng ng vi cc phng php trc ln ti 99.5%. Trong khi ,

mi CYP2D6 intron 6 c s dng s tng ng l 95.8% [82].

3.4. K THUT PCR-RFLP

RFLP c tn y l Restriction Fragment Length Polymorphism l k

thut a hnh chiu di on ct gii hn. N c kt hp cng k thut PCR

to nn k thut PCR-RFLP.

3.4.1. Nguyn tc Nhm khc phc hin tng to ra qu nhiu bng ADN khi phn ct genom

bng enzym gii hn v s kh khn khi thit k probe ph hp trong vic phn tch

a hnh di truyn trong k thut RFLP, ngi ta kt hp k thut PCR v RFLP

to ra k thut PCR-RFLP. Trong phng php ny mt on ADN s c

khuch i chn lc bng k thut PCR trc. Sau on ADN ny s c ct

bng enzym gii hn thch hp. S khc nhau ca cc bng ADN sau khi chy in

di v nhum ethium bromid s cho ta bit v tr cc t bin. C th n gm 3 bc

c bn:

50

- Tin hnh PCR vi cp mi thch hp phn lp on ADN cn phn tch

s a hnh di truyn.

- Tin hnh phn ct on ADN ny vi mt enzym thch hp.

- Chy in di xc nh s khc nhau ca cc bng ADN v a ra kt lun

[144].

c th hiu r hn nguyn tc ca phng php ny, ngi vit xin trnh

by mt v d v vic p dng phng php PCR-RFLP pht hin t bin gen m

ha enzym deoxycytidin kinase. Ti v tr t bin 360 trn gen khi C c thay

bng G s lm trn bng in di xut hin 2 bng in 490kb v 93kb thay v 3 bng

214kb, 276kb v 93kb nh bnh thng.

Hnh 19. PCR-RFLP chn on t bin 360C>G trn gen m ha enzym

deoxycytidin kinase [144]

51

3.4.2. u im

- Khng t.

- D thit k th nghim.

- C th p dng pht hin a hnh n nucleotid.

- Khng yu cu dng c t tin.

- Khng yu cu s o to chuyn su i vi i ng nhn vin.

3.4.3. Nhc im:

- Yu cu phi bit r trnh t on DNA v v tr enzym ct gii hn.

- Mt nhiu thi gian.

- Khng p dng c cho mu ln [103].

3.4.4. p dng trn CYP2D6

Mt nhm tc gi thc hin mt nghin cu nhm xc nh hin tng a

hnh di truyn CYP2D6 Nam n [79]. H s dng phng php PCR-RFLP

nhm xc nh cc t bin CYP2D6 *2, *3, *4,*5, *10, *14, v *17 trn 447 i

tng .Cc mi v enzym gii hn c trnh by nh bng di. Kt thc th

nghim, tn s xut hin cao nht l alen *2 vi 34.8%, tip theo l *10 vi 10.2%.

V tn s cc allen *4, *5 ln lt l 7.3% v 1.9%. Cc t bin *3, *14, *17

khng c pht hin trong th nghim trn.

Bng 3.1. Cc mi v enzym ct tng ng tng allen [79]

Allen Mi xui v mi ngc (theo th t t trn xung) Enzym ct

*2 5GCTGGGGCCTGAGACTT 3

5GGCTATCACCAGGTGCTGGTGCT 3 HhaI

*3 5GGCTGGGTCCCAGGTCATAC3

5GCTGGGGCCTGAGACTT3 BsaI

*4 5TGCCGCCTTCGCCAACCACT3

5TCGCCCTGCAGAGACTCCTC3 BstNI

*10 5GTGCTGAGAGTGTCCTGCC3

5 CACCCACCATCCATGTTTGC3 HphI

52

*17 5TTTTGCACTGTGGGTCCTCGG3

5 CCCGGGTCCCACGGAAATCT3 BstF5I

Mt bi bo khc cng s dng phng php PCR-RFLP xc nh tn

s xy ra t bin CYP2D6*10 bn qun th ngi Hn, Uighur, Hui v ngi

Trung Quc . Kt qu thu c ln lt l 57.4%, 22.4%, 39.7%, v 46.5% [143].

3.5. K THUT PCR PHC HP

S xa ton b on gen thng c pht hin bi k thut Southern blot

hoc s dng cc mu d TaqMan. Nhng phng php ny thng t, phc tp,

tn thi gian. V mt trong nhng kh khn trong vic chun on kiu gen

CYP2D6 l do n c mi lin h ng ng vi pseudogen CYP2D7P. Th nghim

long-PCR c bit c thc hin phn tch nhng mu ADN ca ngi da

trng to ra nhng sn phm c th t nhng vng bin d gy ra bi s xa ton b

gen [84]. Mc d k thut ny kh hu ch nhng gn y ngi ta pht hin ra rng

nhiu mu ADN mang gen CYP2D6 b chn on nhm l kiu gen CYP2D65

qun th ngi Nht. Do vy k thut PCR phc hp c thit k kim tra

s hin din ca gen CYP2D6 s dng. Nhng phn tch ch ra nhiu kh nng l

k thut PCR trc y chn on nhm t bin CYP2D6*10 thnh CYP2D6*5.

3.5.1. Nguyn tc: Phng php ny da trn k thut PCR c bn, trong c nhiu ci tin v

thit k cc mi, iu kin phn ng. Trong vic thit k mi l rt quan trng.

53

3.5.2. u im - Gi thnh thp.

- n gin.

- Tit kim thi gian.

- Rt hiu qu trong vic chn on chnh xc cc t bin.

3.5.3. Nhc im - Ging nh cc k thut PCR khc, k thut PCR phc hp rt d nhim tp

ADN do yu cu thao tc phi rt cn trng.

3.5.4. p dng trn CYP2D6 gip chn on allen *5 v allen*10

Hnh 20. Cc allen CYP2D6 [84]

((A) Gen CYP2D6 bnh thng, (B) Gen CYP2D6*5, (C) Gen CYP2D6*10D; V tr

cc mi v vng gen c khuch i, REP : vng lp li; : Cc mi khng

bt cp v khuch i; F: mi xui, R: mi ngc; mu xm v na xm ln lt

l gen CYP2D6 v nhng vng c ngun gc t CYP2D7).

Nhng mu ADN c cung cp t mu ADN ca ngi Nht cn li

c thu thp t nhng nghin cu trc . Vic chn on kiu gen CYP2D6 ca

54

nhng mu ny c thc hin t trc bng cch gii trnh t trc tip. Nng

ADN ca mu l 50ng/L.

- Cc th nghim PCR trc y: m PCR, 1mM MgSO4, 0.2 mM

dNTPs,1M primer F1, 1M primer R2, 0.4 n v KOD-Plus-DNA polymerase

(Toyobo, Osaka, Japan) v 1.5 L ADN trong tng 20L. Cc iu kin PCR

94 trong 1 pht, theo sau l 30 chu k 98 trong 20s v 68 trong 6 pht,

72 trong 10 pht v sau l 4. Sn phm PCR c phn tch bi in di gel

agarose 0.7% .

- Th nghim PCR trong th nghim ny: m LA-PCR, 2.5mM MgCl2, 0.3

mM dNTPs, 0.1M primer F1, 0.1 m primer R2, 1M F3, 1 n v LA Taq DNA

polymerase (TaKaRa Bio, Shiga, Japan) v 1.6L ADN trong tng 20L. Cc iu

kin phn ng 94 trong 1 pht, theo sau bi 25 chu k 98 trong 20s v 70

trong 6 pht, 70 trong 6 pht, cui cng l 4. Sn phm PCR c in di trn

gel agarose 0.7%.

55

- Trnh t cc mi dng l :

F1: 5-ACCGGGCACCTGTACTCCTCA-3

R2: 5-GCATGAGCTAAGGCACCCAGAC-3

F3: 5-AAGGAGTGTCAGGGCCGGA-3

- Kt qu bng in di. Hnh 21.

Hnh 21. Chn on kiu gen bng k thut PCR truyn thng v PCR phc

hp [84]

(Hnh nh ph in di: (A) PCR truyn thng, (B) PCR phc hp, Ging 1:

CYP2D6*1/*1, ging 2: CYP2D6*1/*5, ging 3: CYP2D6*5/*5, ging 4:

CYP2D6*1/*10D)

Hnh nh v ph in di gel t cc k thut PCR truyn thng i vi t bin

gen CYP2D6 c th hin hnh A: mu ADN 1 khng to ra bng no c chn

on kiu gen CYP2D6*1/*1, mu AND 2 4 to ra mnh c chiu di 3.5kb nn

c chn on l kiu gen CYP2D6*5 . Trong khi k thut PCR phc hp (hnh

B) cho thy mu ADN 4 mang t bin CYP2D610D bi v ngoi to ra bng

3.5kb n cn to ra bng 6.2kb. Nguyn nhn ca s nhm ln ny l do

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CYP2D6*10D cng c s xut hin ca vng lp li c t tn l REP7/6 (hnh

20 C) tng t nh ca CYP2D6*5 v cc mi truyn thng F1/R2 c thit k

pht hin vng ny. Bng 6.2kb c ngun gc t s bt cp mi F3/R2. Do iu

ny c th kt lun l t bin CYP2D6*10D c th b chn on nhm nh l

CYP2D6*5 trong nhng phn ng PCR truyn thng ch s dng mi F1/R2 [84].

3.6. MT S K THUT KHC GIP XC NH T BIN CYP2D6

Ngoi cc phng php trnh by trn, ti u ha vic pht hin cc

t bin CYP2D6 bit cc nh khoa hc c th s dng thm nhiu phng php

PCR ci tin khc nh nh AS-PCR, long multiplex PCRCn i vi nhng t

bin mi, vic s dng phng php gii trnh t Pyrosequencing, gii trnh t t

ng rt c a chung v c chnh xc rt cao v nhanh gn.

CHNG 4. BN LUN

4.1. HIN TNG T BIN GEN CYP2D6

CYP2D6 l mt trong nhng thnh vin rt quan trng trong gia nh CYP.

Mc d n khng phi l enzym chuyn ha nhiu loi thuc nht nhng n li c

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hin tng a hnh ph bin nht. C s ca hin tng trn l do mi lin quan

cht ch gia kiu gen v kiu hnh. Cc bin i kiu gen c th dn n dn n

nhng thay i kiu hnh. Hai kiu hnh quan trng c bit c ch l kiu

hnh chuyn ha siu nhanh v kiu hnh chuyn ha rt chm hoc khng chuyn

ha vi cc thuc c th.

Trong cc thuc c chuyn ha thng qua CYP2D6 c rt nhiu thuc quan

trng c gii hn iu tr hp nh: tim mch, gim au, ung th. Hin tng a hnh

ny c th khng nh hng nhiu ti vic iu tr nhng cng c th gy ra nhng

hu qu nghim trng nh gia tng cc phn ng bt li ca thuc, gy tht bi iu

tr, thm ch e da tnh mng bnh nhn. Bn cnh nhng bin i cc th a

hnh c th di truyn cho cc th h sau. Do vy, vic hiu r cu trc, chc nng,

cc t bin CYP2D6 v hu qu lm sng ca n l rt cn thit v s gip:

La chn phc ph hp cho tng bnh nhn. T nng cao c

hiu qu, tc dng, gim chi ph iu tr.

Ch ng phng nga cc bin c bt li ca thuc, c cc bin php

can thip kp thi, gii thch c cc tht bi iu tr.

4.2. CC NGHIN CU V A HNH CYP2D6 TRN TH GII

Do CYP2D6 c nhng bin i v kiu gen rt a dng m hnh thnh nn mt

th a hnh rt phc tp. Mc d vy cc nh khoa hc trn th gii nghin cu

rt y v CYP2D6 c in vivo v in vitro trn ng vt v ngi. H tm ra

cc t bin quan trng xy ra trn CYP2D6, hu qu lm sng cng nh cc

phng php pht hin t bin . Xt mt cch tng th v h thng enzym

Cyt-P450, cc nghin cu v a hnh CYP2D6 s l hnh mu cc nh khoa hc

p dng nghin cu cc CYP quan trng khc cng xy ra hin tng a hnh nh

l CYP2C19.

Tuy nhin s ra i ca nhiu hot cht mi v hin tng chn on nhm

kiu gen xy yu cu cc nh khoa hc khng ngng nghin cu v CYP2D6.

nh hng nghin cu v cc t bin gen m ha CYP2D6 ang c p dng

trn th gii bao gm:

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Tm ra cc t bin mi, nh gi tc ng ca n trn lm sng

tm cu tr li cho nhng tht bi iu tr, hng ti vic c th ha iu tr.

Tip tc pht minh, ci tin cc k thut pht hin t bin gen m

ha CYP2D6 a ra nhng k thut n gin, nhanh chng, hiu qu, chi ph

thp.

4.3. XUT HNG NGHIN CU CYP2D6 VIT NAM

Vic nghin cu v t bin gen m ha CYP2D6 trn th gii c tin

hnh t lu. Song song vi nhng t bin c pht hin, cc nh khoa hc trn

th gii nh gi c tn sut xy ra, nhng c cht ca CYP2D6 v nh hng

ca hin tng t bin trn lm sng. Hin ti Vit Nam cha c nhiu nghin

cu v hin tng t bin ca enzym ny. Theo mt nghin cu c thc hin

bi vin k sinh trng st rt quc gia v cc t chc quc t, 78 ngi Vit Nam

(75 ngi Kinh v 3 ngi Thi) khe mnh c nghin cu chn on t

bin cc gen m mt s CYP trong c CYP2D6 bng phng php PCR-RFLP.

Kt qu pht hin c allen CYP2D6*4, CYP2D6*5, CYP2D6*10 vi tn sut

ln lt l 1%, 8% v 44%. Bi bo cng ch ra rng kt qu ny l tng ng vi

nhng qun th ngi chu khc [122]. Tuy nhin s lng ngi nghin cu

cha nhiu, li phn ln l ngi Kinh. V vy, nhm gp phn vo vic ti u ha

iu tr thng qua nhng hiu bit v t bin gen m ha CYP2D6, chng ti xin

a ra mt s xut v hng nghin cu lin quan n CYP2D6 c th p dng

trong iu kin Vit Nam nh sau:

Tip tc tin hnh nhng nghin cu xc nh cc t bin gen

CYP2D6 ngi Vit Nam vi s mu ln v a dng hn nh gi c tn s,

phn b