TCNCYH Ph trng 91 (5) - 2014

XC NH T BIN LP ON GEN DYSTROPHIN BNG

K THUT MULTIPLEX LIGATION - DEPENDENT PROBE AMPLIFICATION
Phm L Anh Tun, Trn Huy Thnh, T Thnh Vn, Trn Vn Khnh
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T kha: t bin lp on, gen dystrophin, MLPA

I. T VN
K thut Multiplex Ligation - dependent Probe
Amplification (MLPA) hay k thut khuch i u d
a mi da vo phn ng ni - l mt bin th ca phn
ng PCR a mi (Multiplex Polymerase Chain Reaction)
cho php khuch i nhiu ch ch vi mt cp mi duy
nht [1, 2]. Mi u d bao gm hai oligonucleotide, mi
on u cha mt trnh t mi cho phn ng PCR v
mt trnh t b sung vi trnh t ch (c gi l trnh
t lai). Khi cc u d c lai chnh xc vo trnh t
ch, chng s c ni li bi enzym ligase bn nhit.
Phn ng PCR s ch khuch i cc u d c
ni li hon chnh. V mi u d u c nh du
hunh quang v c kch thc ring bit, nn c th
thu c tn hiu ca mi u d c phn tch
v xc nh trn h thng in di mao qun. K thut
MLPA c th nh lng s bn sao ca nhiu trnh
t trong mt gen vi nng sut ln ti 40 trnh t ch
trong mt phn ng [3; 4]. MLPA l mt k thut c
chnh xc cao c ng dng pht hin t bin
xa on v lp on gen dystrophin cho bnh nhn
DMD/BMD. Tuy nhin, gi thnh cao hn so vi mt s
k thut thng thng khc. Nghin cu ny la chn
nhng bnh nhn c xc nh t bin xa on
bng k thut mPCR v xc nh cc t bin lp on
gen dystrophin bng k thut MLPA. Gen dystrophin c
chiu di hn 3000 Kb nm trn NST X, m ha 14 - kb
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n h T Th nh
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Ng nh n 2
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Ng
h th n 1 11 2014
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nh tr ng

mRNA, bao gm 79 exon; quy nh tng hp protein

dystrophin, l mt protein nm trn mng t bo c, c
vai tr quan trng trong vic bo v c khi hot ng.
t bin gen dystrophin l nguyn nhn gy bnh DMD
do s mt ton vn ca protein dystrophin dn n t
bo c b tn thng. DMD/BMD l mt trong nhng
bnh l v c do di truyn thng gp nht c tn sut
mc bnh vo khong 1/3.500 tr trai, biu hin lm
sng nng vi triu chng yu c mang tnh cht tun
tin, tr b teo c, mt kh nng i li v cht trc tui
trng thnh do suy tim v ri lon h hp. t bin
to m kt thc sm hoc gy lch khung dch m s
gy nn th nng DMD trong khi t bin khng gy
lnh khung dch m s gy nn th nh BMD. Xc nh
t bin gen dystrophin c vai tr rt quan trng trong
t vn di truyn v chn on trc sinh, c bit gip
cho liu php iu tr gen [5; 6]. C nhiu dng t bin
trn gen dystrophin bao gm t bin xa on chim
50 - 65%, t bin im 20 - 25% v t bin lp on
chim 5 - 10% [6]. Nghin cu c tin hnh vi mc
tiu: ng dng k thut MLPA xc nh t bin lp
on gen dystrophin trn bnh nhn lon dng c
Duchenne / Becker.

II. I TNG V PHNG PHP

1. i tng
30 bnh nhn c chn on l DMD/BMD da
vo cc du hiu lm sng v cn lm sng in hnh.
Bnh nhn c du hiu yu c, i li hay vp ng, ph
i c cng chn, du hiu Gower dng tnh. Xt
nghim enzym Creatine Kinase (CK) tng cao (bnh
thng 200 UI/l). Bnh nhn c sng lc khng
mang t bin xa on trn gen dystrophin bng k
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TCNCYH Ph trng 91 (5) - 2014

thut mPCR [7]. 02 ngi bnh thng c la chn
lm mu i chng i km vi mu bnh nhn trong
mi phn ng MLPA.
2. Phng php
2.1. Quy trnh ly mu: Bnh nhn s c ly 2ml
mu tnh mch chng ng trong EDTA. Quy trnh m
bo tuyt i v trng.
2.2. Quy trnh tch chit DNA tng s: Quy trnh
tch chit DNA tng s c tin hnh theo quy trnh
phenol/chloroform [7]. Nng v tinh sch DNA
c kim tra nh phng php quang ph (OD 260
/ 280).
2.3. Quy trnh MLPA xc nh t bin lp on
Nguyn l: S dng 79 u d (probe) DNA lai c
hiu vi 79 exon ca gen dystrophin. Mi probe gm
hai chui oligonucleotide, mt chui ngn v mt chui
di. Mi chui c 2 trnh t quan trng:
- V tr lin kt c hiu v lin k nhau trn trnh t exon
ch tng ng, to iu kin cho enzym ligase gn 2
chui li thnh probe hon chnh. Nu c t bin lp
on trong mu DNA.
- V tr gn mi phn ng PCR khuch i probe, c
trnh t ging ht nhau, nn ch cn mt cp mi duy
nht khuch i tt c cc probe.
Quy trnh: S dng b kit MLPA P034 - A3 v P035 - A3
ca MRC - Holland.Chun b mu DNA, a v nng
30ng/l.Bin tnh DNA; Cho vo ng 0,2ml; 2l DNA +
3 l DW; Bin tnh ti 98C trong 5 pht, gi 25C.
Lai u d: Thm 1,5l MLPA buffer + 1,5 l ProbeMix

(P034/P035) vo ng mu. 95C trong 1 pht, sau

gi 60C trong 16 gi. Phn ng ni: Gi 54C.
Thm vo: 3 l Ligase - 65 buffer A + 3 l Ligase - 65
buffer B + 1 l Ligase - 65 + 25 l DW. 54C trong
15 pht, sau l 98C trong 5 pht v gi 15C.
Phn ng PCR: Thm vo: 7,5 l DW + 2 l Cy5 Primer
Mix + 0,5 l Salsa Polymerase. Chy chu trnh nhit : 35
x [95C/30 giy - 60C/30 giy - 72C/60 giy] - 72C/20
pht Gi 15C [4]. Chy trn my Beckman Coulter
CEQ600, phn tch trn phn mm GeneMarker. Tnh
ton theo cng thc: RPA(Relative Peak Area) = [As/
As] / [Ac/Ac]. X 1.5: t bin lp on.
3. o c nghin cu
Bnh nhn v ngi nh hon ton t nguyn tham
gia vo nghin cu. Bnh nhn hon ton c quyn rt
lui khi nghin cu khi khng ng tip tc tham gia
vo nghin cu. Bnh nhn v ngi nh bnh nhn
s c thng bo v kt qu xt nghim gen gip
cho cc bc s t vn di truyn hoc la chn phc
iu tr ph hp. Cc thng tin c nhn s c m
bo b mt.

III. KT QU
1. Kt qu xc nh t bin lp on gen bng
k thut MLPA
30 bnh nhn DMD/BMD c tin hnh xc
nh t bin lp on gen dystrophin, kt qu pht hin
2/30 bnh nhn c t bin lp on gen dystrophin.

Hnh 1. Kt qu phn tch t bin lp on gen dystrophin bnh nhn MS12 bng k thut MLPA (mi tn
ch exon t bin). A: Kt qu t bin lp on exon 14 - 17. B: T l RPA ca tng exon bnh nhn MS 12
so vi mu chng nam
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TCNCYH Ph trng 91 (5) - 2014

T l RPA ca exon 14 - 17 1,5, trong khi t l RPA ca cc exon cn li tng ng vi 1, chng t bnh nhn
c t bin lp on exon 14 - 17 gen dystrophin.

Hnh 2. Kt qu phn tch t bin lp on gen dystrophin bnh nhn MS 21 bng k thut MLPA
(
t n h
n t
n)
A: Kt qu t bin lp on exon 5-7. B: T l RPA ca tng exon bnh nhn MS 21 so vi mu chng nam
T l RPA ca exon 5 - 7 1,5, trong khi t l RPA ca cc exon cn li tng ng vi 1, chng t bnh nhn
c t bin lp on exon 5 - 7 gen dystrophin.
2. T l t bin lp on gen dystrophin
Bng 1. T l t bin lp on gen dystrophin
STT

Bnh nhn

t bin

Bnh nhn 12

Lp on Exon 14-17

Bnh nhn 21

Lp on Exon 5-7

Tng

Bnh nhn c t bin lp on

2/30

Bnh nhn khng c t bin lp on

28/30
30

2/30 bnh nhn c t bin lp on gen dystrophin chim t l khong 6,5%.

IV. BN LUN
Lon dng c Duchenne l mt trong nhng bnh
l di truyn c ph bin nht, cc t bin trn gen dystrophin c chng minh in vitro lm mt hoc gim
chc nng protein dystrophin - mt protein quan trng

trong qu trnh co c, t gy bnh lon dng c

Duchenne. K thut MLPA l mt k thut c chnh
xc cao c ng dng pht hin t bin xa on
v lp on gen dystrophin cho bnh nhn DMD/BMD.
Tuy nhin, k thut ny gi thnh cao hn so vi mt s
k thut thng thng khc. gim gi thnh, chng
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TCNCYH Ph trng 91 (5) - 2014

ti la chn bnh nhn c xc nh t bin
xa on bng k thut mPCR, k thut ny r hn so
vi k thut MLPA, ch nhng bnh nhn no khng
c t bin xa on mi c chng ti la chn
xc nh t bin lp on. t bin lp on trn gen
dystrophin chim t l t 5 - 10% [3; 4; 6]. Tuy nhin,
t bin lp on khng xc nh c bng nhng k
thut sinh hc phn t thng thng (PCR, gii trnh t
gen). Nhm nghin cu p dng k thut MLPA
gii quyt kh khn ny. K thut MLPA l mt k thut
sinh hc phn t mi, c nhy v chnh xc cao,
tit kim thi gian, c bit l trn mt gen di 79 exon
nh gen dystrophin [1; 2; 3; 4; 5]. K thut MLPA c
tin cy cao nh nhng mi ni chun c thit k sn
kim tra cht lng mu, c c hiu cao nh
thit k u d hai u hai trnh t c bit, khuch i
da vo phn ng ni. Kt qu nghin cu cho thy
u im ca phng php MLPA trong phn tch t
bin lp on nhanh chng, chnh xc, gip ng dng
trong chn on trc sinh bnh DMD v cc bnh di
truyn, lm rt ngn thi gian v cng sc [8].
Trong nghin cu ny, 30 bnh nhn c chn
on mc bnh DMD/BMD v c sng lc khng
mang t bin xa on bng k thut mPCR. 2/30 bnh
nhn (6,7%) c xc nh mang t bin lp on gen
dystrophin kt qu ny, tng ng vi nhng nghin
cu khc Vit Nam v trn th gii [4; 5; 6]. Tuy c
mu cha c ln, nhng y l nghin cu u tin
p dng k thut MLPA vo xc nh t bin lp on
gen dystrophin Vit Nam.

V. KT LUN
S dng k thut MLPA, nghin cu xc nh
c t bin lp on gen dystrophin 2/30 bnh
nhn c chn on xc nh l DMD/BMD.

Li cm n
Nghin cu c thc hin vi s h tr kinh ph t
ti cp nh nc KC.04.08/11-15 Nghin cu xy
dng quy trnh iu tr gen cho bnh lon dng c
Duchenne thc hin t nm 2013 - 2015.

TI LIU THAM KHO

1. Schouten JP, McElgunn CJ, Waaijer R et al (2002).
Relative quantification of 40 nucleic acid sequences by
multiplex ligation-dependent probe amplification. N 30 (12).
2. Murugan S, Chandramohan A, Lakshmi BR (2010).
Use of multiplex ligation-dependent probe amplification
(MLPA) for Duchenne muscular dystrophy (DMD) gene
mutation analysis. n n
132, 303 - 311.
3. Kent K.S. Lai, Ivan F.M. Lo et al, (2006).Detecting
exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification
(MLPA). C n
h
tr 39, 367 72.
4. Marzese D.M., Mampel A., Gomez L.C., (2008).
Detection of deletions and duplications in the Duchenne
muscular dystrophy gene by the molecular method
MLPA in the first Argentine affected families.
n t
n
r
r h 7, 223 - 233.
5. Mendell R.J., Griggs C.R. (1991). Muscular dystrophin. H rr n r n
nt rn
n 12th edition, 2112 - 2118.
6. Takeshima Y, Yagi M, Okizuka Y et al (2010). Mutation spectrum of the dystrophin gene in 442 Duchenne/
Becker muscular dystrophy cases from one Japanese
referral center. H
n t 55(6), 379 - 388.
7. Van Khanh Tran, Van Thanh Ta, Dung Chi Vu, et al
(2013). Exon Deletion Patterns of the Dystrophin Gene
in 82 Vietnamese Duchenne/Becker Muscular Dystrophy Patients. N r g n t
27(4), 170 - 175.
8. Minh-Hieu Ta, Thinh Huy Tran, Ngoc-Hai Do, et al
(2013). Rapid method for targeted prenatal diagnosis
of Duchenne muscular dystrophy in Vietnam.T
n
rn
t tr
n
g 52, 534 - 539.

Summary
MULTIPLEX LIGATION - DEPENDENT PROBE AMPLIFICATION FOR
DETECTION OF DUPLICATION IN DYSTROPHIN GENE
The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for
deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/
BMD). The purpose of this study is to detect duplication in dystrophin gene using MLPA technique. 30 unrelated male
patients with DMD/BMD were selected for this study. These patients had already been screened by multiplex PCR
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TCNCYH Ph trng 91 (5) - 2014

(mPCR). We detected two duplications that had been missed by mPCR, in one DMD patient showing a duplication
from exon 5-7 and other one showing a duplication from exon 14-17. The results of our study confirmed MLPA to be
the method of choice for detecting DMD duplication in DMD/BMD patients.
Keywords: Duplication, dystrophin gene, MLPA

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