NG DNG K THUT MLPA

(MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION)

TRONG CHN ON T BIN MT ON GY
HI CHNG WILLIAMS-BEUREN
Phm Quc An1, Nguyn Th Bng Sng1, Th Thanh Thy1,Nguyn Hong nh2, L Minh Khi2
1

Trung tm Y Sinh hc Phn T, i hc Y Dc Thnh ph H Ch Minh

Khoa Phu thut Tim Mch, Bnh vin i hc Y Dc Thnh ph H Ch Minh

Tm tt: Hi chng Williams-Beuren (WBS) c chng minh l do t bin mt on d hp t locus

7q11.23 trn trn nhim sc th s 7 m ho cho protein elastin vi biu hin tn thng nhiu c quan,
c bit l h tim mch. Chn on xc nh bnh c ngha quan trng trong tin lng cc d tt cng
nh nh hng iu tr cho bnh nhn. Vit Nam, cha c nghin cu no s dng phng php sinh
hc phn t trong chn on xc nh WBS. Nhng bnh nhn c chn on Williams-Beuren trn lm
sng vi v mt in hnh v cc tn thng tim mch c hiu c a vo nghin cu. Bc u chng
ti thu nhn c 5 bnh nhn c chn on mc WBS trn lm sng. Chng ti s dng phng php
MLPA (multiplex ligation-dependent probe amplification) xc nh t bin vi mt on locus 7q11.23.
Tt c cc bnh nhn ny u c mt on in hnh (khong trn 1,55 Mb). Nh vy k thut MLPA l
phng php sinh hc phn t hin i c th ng dng trong chn on xc nh WBS Vit Nam.
T kha: 7q11.23, MLPA, vi mt on, Williams-Beuren.
---------------------------------------------------Tc gi lin h: TS.BS. Nguyn Th Bng Sng, email: suongnguyenmd@gmail.com, T: 0914007038
I.GII THIU
Hi chng Williams-Beuren (WBS, Williams-Beuren syndrome) l mt dng ri lon nhiu chc nng h
thng vi nguyn nhn thng l do s mt on nhim sc th, khong t 1,5 n 1,8Mb vi khong t 26
n 28 gene. Tn sut mc bnh ca tr trn th gii c tnh 1/10000 ca sinh vi s mt on in hnh trn
1,55Mb chim phn ln. Trong , gen Elastin, nm gia vng mt on, c cho rng c vai tr rt quan
trng trong chc nng ca c th. S t bin lm mt i gene ny gy ra cc d tt c trng nht ca hi
chng Williams-Beuren nh hp trn van ng mch ch, hp ng mch phi ngoi vi hay bt thng ca
ng mch nui c. [13,2,12]
Trn th gii, c nhiu nghin cu v t bin gene gy nn hi chng ny v pht trin cc phng php
chn on sinh hc phn t khc nhau. Do nhng kho st trn qun th ngi ti y c trin khai
nhm a ra nhng khuyn co ph hp [6,10,12]. Vit Nam, cha thy c nhng nghin cu chuyn su,
cng nh trin khai rng ri cc phng php xt nghim chn on bng phng php sinh hc phn t
trong y khoa ni chung v trn WBS ni ring. a s cc trung tm phu thut tim mch v bnh vin u
chn on hi chng ny da trn c im lm sng. Chnh v vy rt d b st cc trng hp khng in
hnh. c bit ngi mc hi chng ny thng khng bit mnh mang bnh, do vn hot ng th lc nh
ngi bnh thng, nhng c th b t t hay vn kt hn, sinh con v c xc sut di truyn cho th h sau
[11]. V vy, chn on sinh hc phn t nh bng phng php MLPA khng nhng gip nng cao kh
nng pht hin bnh, thit lp c s cho vic theo di lu di m cn h tr thng tin cho bc s nhm t vn
chnh xc cho bnh nhn. V vy, chng ti tin hnh ti ng dng k thut MLPA (multiplex ligationdependent probe amplification) trong chn on t bin mt on gy hi chng Williams-Beuren.

II.VT LIU V PHNG PHP

* Nhm chng : 10 nam v 10 n bnh thng, tin s gia nh khng c ai mc bnh di truyn.
* Nhm nghin cu: 5 bnh nhn (2 nam v 3 n) c chn on mc phi hi chng Williams-Beuren da
vo cc triu chng lm sng c trng nh khun mt, hp trn van ng mch ch [9]; b v m ca
mt bnh nhn c kt qu dng tnh vi hi chng Williams-Beuren.
* Thu mu v ly trch DNA: Thu 1-2ml mu tnh mch, chng ng bng EDTA, c tch DNA trong
vng 24 gi. Quy trnh ly trch DNA c thc hin theo kit ly trch QIAamp @ DNA mini kit (QiAgene).
tinh sch ca DNA sau ly trch (OD 260/280) phi t trong khong 1,7 n 2. Nng DNA s dng
nm trong khong 20-30ng/l, nu ln hn cn phi pha long bng nc ct.
* Thc hin phn ng MLPA: Phn ng MLPA c thc hin vi kit MLPA (SALSA kit P029 - MRCHolland, Amsterdam, The Netherlands) bao gm 3 giai on chnh (1) Bin tnh DNA v lai vi probe (chu
trnh nhit: 98oC 5 pht, 25 oC tm dng, 95 oC 1 pht, 60 oC 16-20 gi (2) Ni cc probe (chu trnh nhit: 54
o
C tm dng, 54 oC 15 pht 98 oC 5 pht, 4 oC tm dng ) (3) Khuch i cc probe ni (Chu trnh nhit:
60 oC 1 pht; 33 chu k vi 95 oC 30 giy, 60 oC 30 giy, 72 oC 1 pht; 72 oC 20 pht; 4oC dng).
* Thc hin chy in di mao qun trn my CEQ 8800 (Beckman Coulter, Fullerton, CA) v c kt qu
bng phn mm GeneMarker 1.6 (Softgenetics, State College, PA, USA)
III. KT QU
* Nhm chng thc hin phn ng MLPA ng thi vi mu bnh nhn nhm so snh. Trong nhm
chng s cho kt qu t l cc nh sng l 1. i vi ngi bnh thng, t l nh sng so vi nhm chng
s l 10.3.
Kt qu phn ng MLPA ca 5 bnh nhn di dng hnh nh c trnh by hnh 1, bng 1. Kt qu phn
ng MLPA di dng hnh nh ca b v m mt bnh nhn dng tnh vi Williams-Beuren c trnh
by hnh 2, bng 2.

Hnh 1: Kt qu MLPA i vi bnh nhn (bnh nhn s 3). Mt s nh sng ca bnh nhn ch bng
khong mt na so vi nh sng ca ngi bnh thng, kt lun bnh nhn b t bin mt on trn vng
kho st.

Hnh 2: Kt qu MLPA i vi b v m bnh nhn (bnh nhn s 4). A: Kt qu t b; B: Kt qu t m.

Cc nh ca ngi cn kho st cao tng ng vi cc nh ca ngi bnh thng chng t khng b
t bin mt on trong vng cn kho st.

Tn probe
Rp_3q12
FKBP6_7q11.23

Kch
thc
156.2
163.1

Bnh
nhn 1
0.947
0.480

Bnh
nhn 2
1.000
0.484

Bnh
nhn 3
0.832
0.510

Bnh
Nhn 4
0.796
0.609

Bnh
nhn 5
0.795
0.535

b bnh
nhn 4
1.200
1.111

m bnh
nhn 4
1.114
1.009

Rp_7q15

172.8

1.128

1.120

1.047

0.975

0.716

0.915

1.059

Rp_4q34
Rp_7q21

182.7
192.0

0.850
1.007

0.877
0.903

0.820
0.917

0.849
0.950

0.768
0.817

0.979
0.912

1.119
0.872

Rp_8q21
Rp_10q22
CLIP2_7q11.23
Rp_7q21

201.8
218.5
229.7
238.2

1.132
0.759
0.574
0.945

1.022
1.001
0.589
1.009

1.008
0.845
0.460
0.922

0.740
0.940
0.444
0.946

0.781
0.893
0.427
0.761

0.979
1.096
1.019
1.155

0.909
1.014
1.006
1.042

Rp_11q13
FZ_7q11.23
Rp_7q22

246.2
256.2
266.0

0.727
0.476
0.928

1.100
0.644
1.126

0.726
0.437
0.862

1.228
0.649
0.986

0.990
0.496
0.819

0.979
1.007
1.063

0.885
0.935
1.010

TBL2_7q11.23
Rp_17q13
STX1A_7q11.23
Rp_10q21
ELN_EX1_7q11.23
Rp_22q11
ELN_EX6_7q11.23
Rp_5q35
ELN_EX33_7q11.23

273.5
281.7
290.1
301.5
310.6
317.9
327.8
336.6
345.8

0.580
0.954
0.553
0.895
0.554
0.903
0.512
0.858
0.609

0.567
0.968
0.577
1.012
0.556
0.979
0.640
1.002
0.629

0.477
0.932
0.528
0.823
0.522
0.894
0.524
0.832
0.543

0.558
0.947
0.543
1.246
0.662
1.047
0.486
1.169
0.540

0.431
0.774
0.426
0.830
0.453
0.794
0.416
0.837
0.410

1.051
0.982
1.346
1.167
1.308
1.091
1.039
1.166
1.106

1.069
1.026
1.202
1.179
1.181
1.006
1.081
1.125
1.064

Rp_8q22
ELN_EX20_7q11.23
Rp_9q21
LIMK1_7q11.23
Rp_3q26

354.8
363.6
374.5
383.9
391.6

0.979
0.467
1.017
0.527
0.922

1.227
0.674
1.000
0.543
0.924

0.916
0.500
0.911
0.477
0.853

1.165
0.690
0.944
0.572
1.086

0.864
0.528
0.762
0.432
0.843

1.011
1.072
1.002
1.093
0.942

0.968
1.019
1.046
1.027
0.918

RFC2_7q11.23
Rp_2q14
CYLN2_7Q11.23

400.4
409.3
417.9

0.573
0.876
0.522

0.602
1.024
0.528

0.511
0.804
0.493

0.532
1.325
0.571

0.417
0.875
0.470

1.069
1.001
0.987

1.032
1.013
0.984

Rp_7q35
Rp_17q13
Rp_2q16

426.6
433.4
444.1

1.029
0.943
1.091

0.902
0.878
0.867

0.904
0.850
0.984

1.018
1.111
1.029

0.836
0.830
0.709

0.976
1.097
1.024

0.929
1.000
0.941

Bng 1: T l din tch cc sng kt qu MLPA gia bnh nhn v mu chng. Nhng s liu c bi en
cho thy cc nh sng ca ngi cn kho st thp hn khong mt na so vi ngi bnh thng. T y
c th kt lun nhng ngi ny b t bin mt on trogn vng kho st.

IV. BN LUN
T khi MLPA ra i, k thut ny c ng dng cho cc chn on cc bnh lin quan n ri lon nhim
sc th. So snh vi cc phng php ng dng k thut sinh hc phn t khc nh FISH (fluorescence in
situ hybridization), real-time PCR, microsatellite, chng ti nhn thy MLPA c nhng u im ph hp bi
cnh cc nc ang pht trin nh Vit Nam.
Trc y, FISH c s dng rt rng ri trong cc xt nghim lin quan n h hng trn nhim sc th
v hin nay mt s phng th nghim vn coi l tiu chun vng cho mt s loi bnh. Tuy nhin FISH
li l mt phng php tn nhiu cng lao ng, i hi thi gian v s t m, khng th t ng ha c.
Thm vo , cc ha cht s dng cho FISH cng rt c hi. V mt hiu qu ca xt nghim, FISH c
nhy thp hn so vi MLPA, k thut FISH cng khng xc nh c mt on ngn hay di c bit l
trong nhng trng hp nh WBS ny [6,10].
Mt k thut khc cng c pht trin chun on WBS l real-time PCR (hay QPCR, PCR nh
lng). y cng c coi l mt chin lng hay v nguyn tc khng qu phc tp, thao tc cng n gin
c th t ng ha [14,7]. Tuy nhin, thc hin k thut ny cn thit b hin i, qu trnh chun ha v
thit k gp nhiu kh khn v k thut c bit l trnh t c trng. Bn cnh s lng phn ng cn
thit cho mt xt nghim (thng phi s dng nhiu cp mi khuch i nhiu vng mc tiu trn vng
Williams-Beuren)
lm cho gi thnh ca phng php ny c th tng cao.
Phng php s dng microsatellite cho nhy kh cao cng nh c th phn on c kch thc ca
trnh t b mt v gi thnh r [5]. Tuy nhin phng php ny li c th cho kt qu m tnh gi do ch kim
tra gin tip s tn ti ca vng gene mc tiu. V vy c th b st mt s trng hp.
Nhng li th quan trng ca MLPA lm cho k thut ny c pht trin ng dng rng ri hn. MLPA
tng i n gin v c nghin cu v cho ra thnh phm thng mi, chi ph thp v quay vng
nhanh (ch mt 2 ngy), c cc probe kim tra chnh xc ngay trong phn ng cng nh so snh d dng
mu i chng (thng l ngi bnh thng). N mang c u im ca kh nng phn tch s lng bn
sao v l tim nng ln cho nhng nghin cu ng dng khc nh pht hin s Methyl ha hay SNP.
nhy ca phng php MLPA l kh cao, 99,8%, mt s nghin cu c quy m ln [4,8].
Trong nghin cu, chng ti ng dng thnh cng phn ng MLPA trong chn on WBS.
Kt qu ca chng ti thu c cho thy cc bnh nhn c biu hin lm sng rt c trng u cho kt qu
dng tnh. Cc nh ca cc probe hin th cho cc gene nm trong vng kho st u gim mt na so vi
mu chng (hnh 1 v bng 1). S gim mt na ch khng mt hon ton c gii thch do ch b t bin
mt on trn mt nhim sc th, nhim sc th ch em cn li khng b mt on trn vng kho st. Nhng
gi tr ca cc probe s dng l chng ni cng nm trong gii hn cho php, tc tng ng vi cc nh
ca ngi bnh thng. T y chng ti t tin nh gi kt qu ca chn on WBS bng phng php
MLPA cho tin cy cao.
C 5 bnh nhn tham gia vo nghin cu u cho kt qu ging nhau vi mt on trn 1.55Mb, y l dng
mt on in hnh. iu ny ph hp vi cc nghin cu khc. Nhng dng mt on ngn hn cha c

pht hin trong nghin cu ca chng ti c th l do c mu cn nh trong khi xc sut nhng t bin
dng ny sut hin trong cc qun th l rt thp [1,4].
Kt qu MLPA i vi b v m ca mt bnh nhn trong s 5 bnh nhn c kho st cho m tnh (hnh
2, bng 1). iu ny cng hon ton hp l vi c ch gy ra WBS, do s sai hng trong qu trnh ti t hp
tng ng qu trnh gim phn to giao t [3,12]. Tuy nhin xc sut nhng ngi mc phi hi chng
ny ln ln, lp gia nh v di truyn cho con l c th xy ra [11].
V. KT LUN V NGH
T nhng bn lun trn chng ti kt lun rng MLPA l mt phng php c nhiu u im tt, ph hp
vi iu kin ca Vit Nam trong chn on cc bnh di truyn ni chung v bnh Williams-Beuren ni
ring.
V nhng nghin cu WBS trn qun th ngi Vit Nam l cha rng ri nhng li rt cn thit, chng ti
ngh cn c nhng nghin cu ln hn, trong s dng phng php MLPA kho st l rt ph hp.

APPLICATION OF MLPA
(MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION)
TECHNIQUE FOR DETECTING THE MICRODELETION CAUSING WILLIAMSBEUREN SYNDROME
Phm Quc An1, Nguyn Th Bng Sng1, Th Thanh Thy1,Nguyn Hong nh2, L Minh Khi2
1

Center for Molecular Biomedicine, University of Medicine and Pharmacy, Ho Chi Minh City
2

Department of Cardiovascular Surgery, University Medical Center, Ho Chi Minh City

Abstract
Williams-Beuren syndrome, the heterozygous microdeletions of chromosomal sub-band 7q11.23 on the
chromosome 7 encoding

for elastin, is proved to cause the with manifestations in multiple organs,

especially in cardiovascular system. Genetic diagnosis of WBS plays an important role in predicting
outcome as well as guiding the longterm management of patients with WBS. In Vietnam, until present, no
investigation using genetic technique has been conducted in patients with WBS. The patients who presented
clinical WBS based on facial dysmorphologic features and specific cardiovascular defects were recruited
into the present study. In this initial study, we recruted 5 patients with clinical WBS. We realized the MLPA
(multiplex ligation-dependent probe amplification) technique for detecting the microdeletions of sub-band
7q11.23 on the chromosome 7. All of them showed typical deletions (axproximately >1,55 Mb) upon MLPA
technique. Thus, MLPA technique is a modern biomolecular method that might be of high feasibility for
detecting patients with WBS in Vietnam.
Key words: 7q11.23, MLPA, Microdeletion, Williams-Beuren syndrome.

TI LIU THAM KHO

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