Ứng dụng kỹ thuật Multiplex Ligation Dependent Probe Amplification (MLPA) xác định đột biến gen trong bệnh lý di truyền

TCNCYH 66 (1) - 2010

L/NG DUNG KY THUAT MULTIPLEX LIGATION -

DEPENDENT PROBE AMPLIFICATION (MLPA)

XAC DjNH DO T BIEN GEN TRONG BENH LY Dl TRUYEN

Ta Minh Hie'u', Tran Van Khanh', Nguyen Thj Thanh Hai'^, Nguyen Thj Ha', Ta Thanh Van''^

' I rung lam nghien cifu Gen - Protein,^'Bg mdn I Ida sinh, Trtfiing Dai hgc Y Hi Ngi

'Tren the gidi, ky thuat MLPA vdi Uu the thdi gian phat hien dot bien nhanh (3 - 4 ngay) va do chinh xic

cao dang dugc ip dung rdng rai trong viec chan doan benh loan dtfdng trtfc cd Duchene (DMD) vii de i)enh

ly di truyen khic. Muc tieu: nghien eifu ip dung qui tnnh ky thuit MLPA xic dinh dot bien gen lrong benh

ly di truyen (bUdc dau ifng dung trong chi'n doin benh loan difdng ca Duchenne - DMD). Doi tUgng va

phuang phap nghien cdu: 06 benh nhan dugc chiin doin mic benh DMD dtfa tren lieu chuan lam sang va

can liim sang, tach chie't DNA td miu ngoai vi, sd dung ky thuat MLPA de xic djnh dot bie'n gen. Kit qua:

phat hien Of benh nhan cd dot bien xda doan gen dystrophin, trang dd mat benh nhin bi dot bien xda doan

gen nim ngoii vung "hot - spot". Kit luan: quy tnnh ky thuit MLPA da dugc ip dung thinh cdng xie djnh

dot bie'n gen trong benh loan difdng ca Duchenne.

Tii khoa: DMD, dot bidn gen, ky thuat Multiplex Ligation - dependent Probe Amplification, MLPA

I. DA T VA N DE

Cac benh ly di truyen thifdng de lai nhflng hau

(|iia nang ne cho gia dinh benh nhan va xa hpi.

Mpt lrong nhflng nghiem vu hang dau dat ra cho

nin y hpc hien dai la viec tim ra d c phfldng

phap chin doln nhanh, chinh xac va diiu trj hieu

c^iia ddi vdi d c benh ly di truyen.

"frong so' d c benh ly di truyin, Duchene

Muscular Dystrophy (LDMD) la mpt benh ly cd do

di truyin thfldng gap nhit va tien kfpng nang;

benh nhan thifdng bi tan phe, mi t khi nang di lai

va chdt trfldc tudi trifdng lhanh. Tan suit ml c II

1/3500 tre trai 16]. Benh gay nen bdi dot bie'n gen

dystrophin. Gen dystrophin cd chieu dai khoang

2400 kb gdm 79 exon, mdt Irdng nhflng gen Idn

nhi i cua loai ngifdi difdc phai hienche den nay.

I.3)ang dpi bidn thifdng gap nhit cua gen

dystrophin la dot bie'n xda doan, chiem ty le

khoang 60 - 65% 18]. Xl c djnh dpi bien xda doan

gen dystrophin cd the tien hanh d 2 mflc dp: mflc

dp DNA va mflc dp RNA. d mifc dp DNA, de xac

dinh day du d c dpi bien xda ddan tren loan bd

79 exon, d c ti c gi l phai khuech dai 79 exon

blng 79 cap mdi dac hieu, cdng viec nay ldn

kem nhieu ve tai chinh va thdi gian (3 - 5 tuln).

Do vay, d c ti c gia thifdng chi sif dung 19-2 5

cap mdi dac hieu bao phu 2 vung trpng diem (hot

- spot) la vung trung tam d gifla gen (exon 43 ^

60) va vung tan cung dau 5' (exon 1 -f 19) 11, 2,

3, 5, 8]; tuy nhien dot bien gen dystrdphin cd the

nam rai ric tren 79 exon, vl the d c dot bien gen

thtfdng bj bd xdt. O mflc dp RNA, viec sfl dpng 1 5

cap mdi dac hieu cd the khuech dai loan bp

mRNA chfla 79 exon do d c intron da difpc cat bd

trong qua trinh holn thien mRNA; qui trinh nay

phai tien hanh nhiiu bfldc: phan flng RT - PCR,

phan flng Nested - PCR, ky thuat nhan ddng va

giai trinh tfl gen vdi thdi gian lif 2 - 3 tuan 14].

Ngay nay tren the gidi, ky thuat MLI'A difdc flu

tien chpn Ifla trong viec chi n doan dot bien xda

doan gen dystrophin ndi rieng va d c benh ly di

truyin ndi chung 17]. Vdi ky thii|t MLPA, dot bien

gen dystrophin se difpc khio sat day du trong

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