" TAP CHI NGHIEN CU'U Y HOC
PHAT HIEN NGirai LANH MANG GEN BENH LOAN DI/QNG CO DUCHENNE BANG KY THUAT MULTIPLEX LIGATION -
DEPENDENT PROBE AMPLIFICATION
TrSn Van Khanh, TrSn Huy T h i n h , Ta Thanh Van Trung tam Nghien CLFU Gen - Protein. Truong Dai hoc Y Ha Noi Loan dw&ng cc Duchenne la mot h$nh gay nen do dot bi§n gen dystrophin, trong do dot bien mit doan chiem khoang 60 - 65% Khoang 1/3 benh ntian duvc phat hien la do di truyen tu nguoi me Ky thuat Multiplex Ligation - Dependent Probe Amplification (MLPA) duuc cac nha khoa hoc ung dung rong rai trong xac djnh dot bi4n mSt doan. lap doan gen va phat hien ngwoi lanh mang gen benh. Nghien cuu nay su dung ky thuat MLPA de xac dinh ty le mang gen cua cac thanh vien nu-- trong gia dinh benh nhan m§c benh loan du&ng ca Duchenne KSt qua cho thiy 36/68 ngu&i mang gen di hop td. chiSm ty le 55,8%
Tif khoa: loan du'd'ng ccy Duchenne, ngu'd'i mang gen, MLPA I. DAT VAN DE
Loan du'ang ca Duchenne (Duchenne Muscular Dystrophy DMD) la mot trong nhCrng benh Iy v^ ca do di truy&n thuang gap nhat, c6 t&n su^t mSc benh v^o khoang 1/3,500 tre trai Benh gay nen do dot bi6n gen dystrophin nSm tren n h i i m s i c the X [1] Dang dot bien thu'ang gap n h l t cua gen dystrophin la dot bi^n mat doan gen, chiem ty le khoang 60 - 65% [ 1 . 2] Mpt s6 nghien cu'u tru-ac cho thly khoang 2/3 benh nhan loan du'ang co Duchenne nhan gen dot bien tu' ngu'd'i me, chi 1/3 la do dot bien moi phat smh trong qua trinh tao giao ti> [4, 10] Loan du'ang ca Duchenne la benh ca r l t nang vai bieu hien lam sang mang tinh c h i t t u l n ti^n, tre bi teo ca, m l t kha nang di lai va c h i t tru'ac tu6i tru-ang thanh do suy tim va r6i loan ho h i p Hien nay, chu-a CO phuang phap d i l u tn dac hieu, phat hien ngud'i l^nh mang gen benh la bu'O'c d l u tien quan trong d l c h i n doan tru'ac sinh, tu- v l n di
Dia ctii lien h$ Trail Van Khanh Trung lam Nghien cuv Gen • Prolein. Truong Dai hoc Y Ha Noi Email vankhanh73md@yahoo com NgiynhSn 13/1/2014 Ngdy duQC chip Ihuan 28/4/2014
truydn nham giam ti le mac benh trong cong d6ng [3, 4, 5] Co n h i l u ky thuat d l xac dmh ngu-ai lanh mang gen benh loan duang ca Duchenne, trong do MLPA la ky thuat du'ae li'ng dung r l t rong rai trong phat hien dot b i l n mat doan, lap doan va phat hien ngaai lanh mang gen benh Day la ky thugt co do nhay cao, cho ket qua nhanh chong, chinh xac [5, 6, 7] Qe tai d y a c t i l n hanh vai muc tieu Ung dung ky thuat MLPA phat hien ngu'ai lanh mang gen benh loan daang ca Duchenne II. D 6 I TU'O'NG VA PHU'ONG PHAP
1. Do! tu'O'ng
- Nhdm d l i chang 10 ngaoi nO hoan toan binh thaang, daac lly ngau nhien tu cong dong
- Nhom nghien CLPU 68 thanh vien nu trong gia dinh benh nhan loan du-ang co Duchenne, t i t ca cac benh nhan nay da daac c h i n doan CO dot bien m l t doan gen dystrophin
2. Phu'O'ng phap 2.1. Ky thuat tach chiet DNA DNA du'ae tach chiet tu bach cau mau ngoai vi b l n g phenol/chloroform Tat ca cac mau DNA d u a c t i l n hanh do nong do va do
TAP CHi N G H l i N CO'U Y HQC •
tinh sach, chl co m l u DNA dat gia trj ve mat dp quang OD280/OD260 > 1,8 mai dat yeu c l u v l tinh sach va daac SLP dung de phan tich
2.2. Ky thuat MLPA xac djnh fiieu gen dj ho'p tLF
+ Nguyen ly': Sa dung 79 doan do gen dystrophin M 5 I probe gdm hai chudi oligonucleotid, DNA (probe) lai dac hieu vai 79 exon cua mdt chudi n g l n va mpt chudi dai M6i chudi CO 2 vi tri quan trong: (1) vi tri iien k i t d i e hieu va l i l n ke nhau tren trinh t a exon dich, tao dieu kien cho enzym ligase g i n 2 chudi thanh probe hoan chmh. Neu ngad'l me mang gen a trang thai di hap t a (1 allele bj m l t doan va 1 allele binh thaong) thi probe taang ung vd'i cac exon cua allele bi xoa khdng daac hinh thanh do 2 sai oligonucleotid khong lai g i n dup'c vd'i nhau va san p h i m se khong daac khuylch dgi, trong khi do allele con lai v i n daac hinh thanh va san pham v l n daac khuylch dai, k i t qua san pham khuylch dai cua ngadi me mang gen se b l n g 1/2 so vdl m l u doi chang niJ' binh thudng, (2) vj tri g i n m i l khulch dai probe, cd trinh t a gidng het nhau, nen chi c l n mot cap mdi duy n h l t de khulch dai t i t ca probe
Rieng d chudi dai cd them mot doan dem gdm sd nucleotid khac nhau, nhd dd cac probe cd su khac biet ve kich thadc, giup phan tach khi dien di mao quan sau khuech dai probe K i t qua dien di se cho 79 dfnh co kich thadc taang ang vdi 79 probe dac hieu vdl 79 exon, exon b\ xoa doan khi dinh cua exon do khdng phat hien d a a c
+ TiSn hanh cho 5 pi DNA c l n phan tich vao dng PCR, b i l n tinh a 98°C/5 phut, Nhiet dd daac c h u y i n ve 2°C trade khi cho 3 pi hdn hap chaa cac doan oligonucleotid cua cac probe Hon hap d a a c u d 60°C/16 gid, luc
nay 2 doan lai cua 2 phan t a oligonucleotid se g i n vdi exon dich dac hreu a vi tri sat nhau.
Them 32 pi hon hp'p ligase buffer, u a 54''C/15 phut, enzym ligase noi 2 doan oligonucleotid cua probe vdi nhau Phan i>ng noi c h i m dij-t khi tang nhiet dp len 98°C/5 phut, hdn hap d a a c giO d 4°C K h u l c h dai san pham lai Them 10 pi san p h i m lai vao 30 pi hdn hap PCR buffer, gia a 60°C t r a d e khi them 10 ^ll hdn hop PCR master, thac hiSn chu trinh nhiet: [95°C/30 giay, 60°C/30 giay, 72°C/1 phut] X 35 chu ky, 72°C/20 phut; san pham d a a c bao quan d 4°C San pham khulch dai probe d a a c dien di mao quan hu^nh quang tr^n may giai trinh t a d l phan tich k i t qua.
Neu cd dot b i l n xda doan, probe khdng g i n d a a c vao gen dich, do do k i t qua dien di se khdng x u l t hien dmh t a a n g ang tai exon ddt b i l n [5],
C h i l u cao cua cac exon phan ^nh ndng dd cua chung trong san p h i m PCR Moi gia dinh d a a c lap lai thi nghiem 2 l l n ,
D l tai tuan thu chSt che dao dac nghien cau trong Y hoc,
III. K^T QUA
1. K i t qua phan t i c h cua cac thanh vien nij' t r o n g gia d i n h benh n h a n
68 thanh vien nii' trong gia dinh b&nh nhSn loan d a d n g ca Duchenne da d a a c xac dinh dot bien m l t doan gen dystrophin se dup'c lu'a chon de t i l n h i n h xac dmh ngadi ISnh mang gen benh (trang thai di hp'p ta) Ky thu§t MLPA d a p c ang dung de xAc dinh sa dung m l u d6i chang na va m i u ngadi con trai bi benh d l lam ddi chu'ng va so Scinh K i t qua cho t h l y 36/68 thanh vi^n la ngadi l i n h mang gen benh
TCNCYH 87 (2)-2014
• TAP CHI NGHJEN CO'U Y HOC
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Htnh 1. K i t qua phat hien ngu'd'i lanh mang gen 6> me benh nhan ma s 6 1A B$nh nhan nam da d a a c c h i n doan loan dadng co Duchenne b l n g ky thuat smh hoc phan ta va x i c dmh cd dot b i l n m l t doan gen dystrophin Sa dung MLPA-SALSA probe mix P034 da phit hien t h l y benh nhan cd ddt b i l n m l t doan ta exon 21 - 30 cua gen dystrophin K i t qua dien dl cho thly c h i l u cao dinh {taang ung vdi ndng dd khuylch dai cua san pham PCR) cua cac exon tu 21 - 20 cua ngaai me chi b l n g 1/2 so vdi m l u ddi chang nip K i t qua nay cho t h l y me cua bdnh nhan cd mang gen ddt b i l n
K i t qua x i c dinh tinh trang mang gen cua 35 t h i n h vien khac cung cho hinh anh tuang tu nhu k i t qua cua ngadi me d ma sd IA, tdc la c h i l u cao dinh cac exon d vung dot b i l n gen chi blng 1/2 so vdi m l u ddi chdng na
2. Ty le phat hien ngu-o-i lanh mang gen benh
Bang 1. Ty le phat hien ngu'd'i me mang gen benh
Ket qua
C6 mang gen dot bren Ktidng mang gen dpt bien Tdng s4
Me benti nhan
n % 36 6 5 8 32 44,2 68 100
TAP CHI NGHIEN C U U Y HOC •
Trong 68 thanh vien na gia dinh benh nhan loan dadng ca Duchenne tham gia nghien cau, 36/68 ngadi d dang di hp'p t a , chiem ty le 55,8%
IV. BAN LUAN
Ky thuat MLPA cd do tin cay r l t cao nhd sa dung probe ndi c h u i n k i l m tra chat lup'ng m l u , qua tnnh b i l n tinh DNA m l u va c h i t luang g i n 2 chudi oligonucleotid thanh probe hoan chmh Cac dinh taang dng cua probe kiem tra ndi chuan nam d cac vi tri dau tien v i xen ke giaa cac dinh taang ang vdi c i c exon cua gen dystrophin, chung se x u i t hien khi tdi au hda daac quy trinh, dd l i dieu kien dam bao ket qua dang tin c i y vi sa x u l t hien cac dmh chung to chat laang m i u va quy trinh thac hien phan dng dat tieu chuan. Ngoai ra, m i u chang la DNA cua ngadi na binh thadng ludn daac t i l n hanh song song ciing m i u benh n h i n d l so sanh n h l m dam bao dd tin cay tuyetddi
Hinh 1 la k i t qua minh hoa cua mdt gia dinh benh nhan cd ddt b i l n m l t doan gen Benh nhan da d a a c x i c dinh cd ddt b i l n mat doan cac exon 21 - 30 tren gen dystrophin Phan tich k i t qua cua ngadi me benh nhan cho thly chieu cao dinh taang dng vdi ndng dd san p h i m PCR cua cac exon chi b l n g /z chilu cao dinh exon taang irng d m i u doi chu'ng nu, vi vay me benh nhan la ngadi lanh mang gen benh Vdi nhOng ngudi me cho hmh anh MLPA giong nhu m i u ddi chang nd daac xac dmh khdng phai la ngadi lanh mang gen benh
Trong 68 thanh vien na cua cac gia dmh benh nhan loan dadng ca Duchenne cd dot b i l n gen dystrophin, nghien cdu da phat hien duac 36 thanh vien nu' d dang di hop t u , chiem ty le 55,8% Nha vay, 44,2% ngudi hoan t o i n binh thudng D i l u n i y cd nghla la
ty le dot b i l n m d i phat sinh tren cac benh n h i n loan dud'ng ca Duchenne d nghien cau n i y la 44,2% Theo Ligon, ty le b6nh nhSn loan d a d n g c a Duchenne thCra hadng gen benh t d ngao'i me la 2/3, chi cd 1/3 tradng hap la do dot b i l n md'i phat sinh [2], K i t luan nay t a a n g t a nha nghien c a u cua Gatta (2005) va Hung (2006) [5, 6] Nha vay, ty le ddt b i l n mdl phat smh d benh nhan loan dadng ca Duchenne d Viet Nam theo nghien c d u nay cao han so vdi cac tac gia k h i c D i l u nay d a a c giai thich la do y l u td chung tdc Cijng cd the do cac yeu td ben n g o i i tac ddng len q u i trinh p h i t sinh giao t a d ca the ngadi me ( d i l u kien sdng: an udng, mdi tradng d n h i l m . ) Theo Fortina va n h i l u t i e gia, ddt b i l n mdi phat sinh g i y benh loan d a d n g ca Duchenne t h a d n g xay ra trong qua trinh tao giao t a do t i c ddng cua cac y l u td ben ngoai [4]
Nghien c d u phat hien ngadi lanh mang gen dystrophin ddt b i l n tren cac b i me cd con bj benh loan d a d n g c a Duchenne, ddng vai trd het sdc quan trong K i t qua nay la t i l n d l giup c h i n d o i n trade smh va ta v l n di truyin n h l m ngan ngda va giam ty le m l c benh DMD d Viet Nam
V. K^T LUAN
Ky t h u i t MLPA da giup phat hi6n dace kieu gen di hap t d cua c i c t h i n h vien m trong gia dinh benh nhan bi ddt b i l n m l t doan gen dystrophin T-/ le ngadi mang gen benh l i 55,8%, d i l u nay cd nghTa la 44,2% benh nhin loan dadng c a Duchenne d nghien cdu niy cd ddt biln mdl phat smh ma khdng phai do nhin gen benh ta ngadi me
Lo'i cam en
Nghien c d u d a a c thac hi§n tai Trung t i m nghien c d u Gen-protein, T r a d n g Dai hpc Y H i
TCNCYH 87 (2)-2014
• TAP CHI NGHIEN ClfU Y HOC Ndi, vd'i s a hd trp' kmh phi cua d l tai "Nghien
cau ang dung ky thuat smh hoc phan t a d l xac dmh ddt bien va phu nij' mang gen dystrophin" thudc kinh phi hd tra cua Quy phat tnen Khoa hpc v i Cdng nghe Qudc gia Nafosted v i d l t a i " Nghien cau x i y dang quy trinh d i l u tn gen cho benh loan dadng ca Duchenne " thuoc kinh phi ho tra cua d l tai cap nha nadc - KC 04/11 -15
TAI LIEU THAM KHAO
1. Prior T.W., Barrtolo C , Paerl D.K et al (1995). Spectrum of small mutation in the dys- trophin coding region Am J Hum Genet, 57, 2 2 - 2 4
2. Ligon A.H., Kashork C D . , Richards C.S., Shaffer L.G (2000). Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach Eur J Hum Genet, 8, 293 - 298,
3. Abbs 8., Bobrow/ M (1992). Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene J Med Genet, 29, 191 - 196
4. Fortina P., Shoffner M.A., Surrey S et al (1997). Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identifica- tion of earner status by use of entangled solu- tion capillary electrophoresis. Clinical Chemis- try, A3(5), 745-75:.
5. Gatta V., Scarciolla O., Gaspari A.R. et al (2005). Identification of deletions and dupli- cations of the DMD gene in affected males and earner females by multiple ligation probe amplification (MLPA). Hum Genet 117, 92 - 98
6. Hung CC, Chen C.P., Lin C.P., Chen S.C et al (2006). Quantitative assay of dele- tion or duplication genotype by capillary elec- trophoresis system application in Prader-Willi syndrome and Duchenne muscular dystrophy.
Cim Chem, 5 2 , 2 2 0 3 - 2 2 1 0
7. Lai K.K., Lo I.F., T o n g T.M et al (2006).
Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation- dependent Probe Amplification (MLPA) Clin Biochem, 39, 3 6 7 - 372
Summary
CARRIER DETECTION OF DUCHENNE MUSCLAR DYSTROPHY USING MULTIPLEX LIGATION - DEPENDENT PROBE
AMPLIFICATION
Duchenne Muscular Dystrophy (DMD) caused by dystrophin gene mutation, is responsible for 60 • 65% of DMD patients About one third of DMD patients are earners of the new mutation and the others inherit the abnormal gene from the heterozygous mother Multiplex Ligation - depend- ent Probe Amplification (MLPA) has been commonly used to detect mutation in inherited diseases because MLPA is a rapid and an accurate technique In this study, MLPA technique was used to detect the rate of female carriers m family with DMD The results shovi/ed that 36/68 of female members have been detected as earners, accounted for 55 8%
Key w o r d s : Duchenne m u s c u l a r d y s t r o p h y , carrier, MLPA
