DOI 10 31276/VJST 64( 12),01 04 Khoa hoc Y DUtic I Y hoc cd sd Ung dung ky thuat Multiplex ligation dependent probe amplification f xac djnh nguoi lanh mang dot bien gen o thalassemia Nguyen Minh Ngoc[.]
DOI: 10.31276/VJST.64( 12),01-04 Khoa hoc Y - DUtic I Y hoc cd sd Ung dung ky thuat Multiplex ligation-dependent probe amplification xac djnh nguoi lanh mang dot bien gen o-thalassemia f Nguyen Minh Ngoc, Le Thi Phirong, Tran Huy Thjnh, Tran Van Khanh* Truong D a i hoc Y H a N o i N g a y n h a n b a i /1 /2 ; n g a y c h u y e n p h an b ie n /1 /2 ; n g a y n h a n p h a n b ie n /1 /2 2 ; n g a y ch a p n h an d a n g /1 /2 2 Tom tat: Benh a-thalassemia phan Ion la dot bien xoa doan gen HBA1 va HBA2 gay thieu hut chuoi a-globin cau nen phan tu Hemoglobin (HGB) Tuy theo so luong chuoi a bj thieu hut ma mire bieu hien lam sang cua benh a cac cap khac Benh di truyen lan tren nhiem sac the thuong nen viec xac djnh nguoi lanh mang dot biln gen a-thalassemia co y nghia quan trong tu van di truyen trirdc sinh va truoc hon nhan nham giam ty le mac benh cong dong Bang ky thuat Multiplex ligation-dependent probe amplification (MLPA) tren mau mau cua 15 nguoi nghi ngo' mang dot bien gen a-thalassemia, ket qua xet nghiem cac chi so hong cau (RBC) va huyet sac to da xac djnh dirge 12/15 ngudi mang dot bien ~SEA, 1/15 nguoi mang dot bien -a3 ,1/15 nguoi mang dot bien -a42 va 1/15 ngudi mang dot bien diem Hb constant spring (-aHbCs) Tir khoa: gen a-thalassemia, Hb constant spring, MLPA, ngutri mang dot bien, —f ', -af ', -a42 Chi so nhan loai: 3.1 Bitvinfle % V i v a y v ie c x a c d jn h d o t b ie n tren n g u d i lan h m a n g d o t b ie n a -th a la s s e m ia la m o t b en h tan m a u b a m sin h d o g ia m sa n x u a t c h u o i a -g lo b in c u a h u y e t sa c to B e n h d u g c d m th a y tren to a n th e gicri tro n g d o p h a n Ion d c a c k h u v u c : D ja T rung H (1 % ), ch a u Phi (4 % ) v a D o n g N a m A (4 ,6 % ) [ ], g e n b en h h u d n g td i m u c tie u t u v a n d i tru y en , tir d o n g a n ngira v a g ia m ty le m a c b e n h tr o n g c o n g d o n g la rat c a n th iet K y th u at M L P A c o d o c h in h x a c c a o v a k e t q u a n h an h c h o n g , H ie n n ay, k h o a n g 5% la p h u o n g p h ap h u u ic h tro n g v ie c x a c d jn h c a c d o t b ie n x o a d o a n d an s o th e g id i m a n g d o t b ie n g e n a -th a la ss e m ia , b a o g o m d a n g d o n g h g p tu c u n g n h u dj h g p tu D o n g th d i c u n g x a c d jn h c a c d o t c a c v itn g v a b ie n lap d o a n v a m o t v a i d o t b ie n d ie m th u d n g g a p tro n g c u m g e n a -th a la ss e m ia v a a °-th a la ss e m ia , p h a n b o ro n g rai D o n g N a m A ) T a i V ie t N a m , ty le a -g lo b in X u a t p h at tu th u c tie n n e u tren , n g h ie n c u u d u g c tien n g u d i m a n g d o t b ie n g e n b e n h d u g c p h at h ie n v b i tan s o c a o nhat h a n h v d i m u c tieu : “X a c d jn h c a c d o t b ie n g e n d n g u d i lan h m a n g tro n g k h u v u c [2 , ] d o t b ie n g e n b en h a -t h a la s s e m ia b a n g k y th u at M L P A ” q u o c g ia k h a c n h a u (c h u y e u B e n h a -t h a la s s e m ia la b e n h d i tru y en la n tren n h ie m sa c th e th u d n g g a y d o d o t b ie n g e n a - g lo b in (H BA1 va H B A 2) m en n h ie m sa c th e s o 16 d an d e n g ia m h o a c th ie u h u t to n g h g p c h u o i a -g lo b in M o i g e n g o m a le n q u y d jn h to n g h g p c h u o i a -g lo b in , n h u v a y m o i p h a n tu H G B g o m c h u o i a -g lo b in , tu y th u o c v a o s o lu g n g a le n b i d o t b ie n v a d a n g d o t b ie n m a c a c b ie u h ie n la m sa n g c u a b e n h a -th a la s s e m ia x a y d c a c m u c d o k h a c nh au % c a c tr u d n g h g p la d o d o t b ie n x o a d o a n g e n , c o th e xay tren hoac ca gen HBA1 g e n a -g lo b in , b a o g o m c a g e n l va H B A 2, g lo b in ; Doi tieong N hom nghien cuu: cong th u c 15 n g u d i d u g c c h o n d u a tren x e t n g h ie m m a u b a t th u d n g : R B C > ,5 T /L , M C H < ,0 10 % c o n la i la d o t b ie n g e n a tren c u n g n h ie m s4 c th e d an d i n th u d n g C o n g th u c m a u c u a n g u d i lan h m a n g d o t b ie n g e n b en h d u g c to n g h g p d b a n g Bang Cong thirc mau va chi so dien di huyet sac to cua nhom nghien cdu 11=15 Trung binh (±SD) R BC (T/L) ,73± 0.54 H G B (g/L ) 127.61= 17.62 N g u d i la n h m a n g d o t b ie n g e n b e n h la n h u n g n g u d i c h i m a n g M C V (fL) 6721=3.47 d o t b ie n a 0, h o a c 1-2 d o t b ie n a + N h u n g n g u d i n a y tu y k h o n g M CH (pg) 21 5 = H b A l (%) 97j07=:1.43 H bA (%) = 1 k b o n g to n g h g p d u g c c h u o i a - g lo b in ) v a a +-th a la ss e m ia ( m i t h o a c g e n a tren n h ie m sa c th e d an d e n g ia m to n g h g p c h u o i a - g lo b in ) [ ], b it hoat c d b ie u h ie n la m sa n g , c h i x u a t h ie n th ie u m a u n h e k h i la m x e t n g h ie m c o n g th u c m a u , n h u n g k h i n g u d i m a n g d o t b ie n g e n la y n h au h g c d n g u y c o sin h n h u n g d u a c o n b i b e n h v d i x a c su a t la T jc gia lien he: Email: tranvankhanh@hmu.edu BSSJSSe\ A L 64,,2),2'2°22 pg, M C V < fL , H G B < g/1 v a k e t q u a d ie n d i h u y e t sa c to b in h h o a c to a n b o cu m d ie m [4 ], N h u n g d o t b ie n n a y s e ta o c a c a le n d o t b ie n d d a n g a -t h a la ss e m ia (m a t c a Doi tiffing va phiffing phap nghien ciffi n am Khoa hoc Y - Dude I Y hoc cd sd K y thuat M LPA: N g h ie n Applying Multiplex ligation-dependent probe amplification technique to identify a-thalassemia carriers Minh Ngoc Nguyen, Thi Phuong Le, Huy Thinh Tran, Van Khanh Tran* H a n o i M edical U niversity c u u sir d u n g b o k it S A L S A M L P A cu a H a n g M R C H o lla n d (H a L a n ) v d i b o d i u d o P I d an h c h o ch a n d o a n a -th a la ss e m ia T iin hanh: u l D N A c o n o n g d o to i u u tron g k h o a n g - n g d a khir R N A d u p e s u d u n g la m k h u o n c h o p h an u n g M L P A g o m b u d e c h in h la b ie n tin h , la i h o a d a u d o , P C R v a d ie n di m a o quan C a c b u o c th i n g h ie m v a c h u trinh n h ie t cu a p h an u n g d u p e th u c h ie n tu an th e o q u y trinh c h u n g c u a n h a sa n xuat K St qua M L PA d u p e phan tich tren phan m em C O F F A L Y SE R N et R e c e iv e d 21 D e c e m b e r 2 ; a c c e p te d Jan u ary 2 d ! tin h g ia tri D o s a g e Q u o tie n t (D Q - th u o n g s o c u a tin h ie u d in h Abstract: m au n g h ie n c u u s o v d i m au d o i c h u n g ) N h u n g d in h c o g ia tri D Q a-thalassemia disease is mostly caused by mutations in the tro n g k h o a n g ,8 - ,2 la b in h th u d n g ; D Q = tu em g d u em g v d i d o t HBA /a n d HBA2 genes that lead to the deficiency in the a-globin b ie n m a t d o a n d o n g h o p tir; D Q tro n g k h o a n g ,4 - ,6 d u p e x a c chain, which builds up the haemoglobin molecule Depending on the number of missing a chains, the clinical manifestations of the disease are at different levels This disease is inherited in an autosomal recessive manner, hence identifying healthy individuals carrying mutations in the a-thalassemia gene is essential for prenatal and premarital genetic counselling to reduce the incidence in the community Multiplex ligationdependent probe amplification (MLPA) technique was used to determine 15 people who were suspected of carrying the a-thalassemia gene based on their complete blood count This study identified that there were 12/15 people carrying —SEAmutation, 1/15 carrier having -a37mutation, 1/15 carrier having -a?-2mutation and 1/15 person carrying point mutation Hb constant spring -aHhC\ djnh la x o a d o a n d i h o p tu ; D Q ,3 -1 ,6 la c o d o t b ie n lap d o a n D ao d u e nghien cm t: N h u n g n g u d i m a n g d o t b ie n g e n b en h a -th a la s s e m ia th am g ia v a o n g h ie n c u u n a y m o t e a c h tu n g u y e n H o d u p e th o n g b a o k e t q u a x e t n g h ie m g e n v a b a o m a t th o n g tin c a n h an N g u d i th a m g ia c o th e n it k h o i n g h ie n c u u b a t c u lu c n a o Ket qua Dot bien phat hien dm c tren ngu&i lanh mang dot bien gen benh D o t bien m d t doan lan: B a n g k y th u at M L P A , n g h ie n c u u d a x a c d in h d u p e /1 n g u d i la n h m a n g d o t b ie n m a t d o a n ld n v u n g g e n a -g lo b in T ron g d o , /1 (8 % ) n g u d i m a n g d o t b ie n - SEA, 1/15 (6 ,6 % ) n g u d i m a n g d o t b ie n - a J v a /1 (6 ,6 % ) n g u d i Keywords: Hb constant spring, MLPA, mutation carriers, a-thalassemia, —sc \ -a37, -a42 m ang d ot b iln -a4 K e t q u a p h an tic h M L P A c u a tu n g n h o m d o t b ie n d u p e th e h ie n d h in h Classification number: 3.1 Tieu ch u rn loai trie: N hdm chung: N h u n g n g u d i th ie u m a u d o th ie u sat M a u m a u cu a n g u d i tn r b n g th an h c o c o n g th u c m a u b in h th u d n g : s o lu p n g R B C : ,0 - ,5 T /L ; M C H : ,0 - ,0 p g; M C V : -9 fL ; H G B : - g/1 K et q u a d ie n d i h u y e t sa c to b in h thu-OTig: H b A l ,5 -9 ,5 % v a H b A la ,5 -3 ,5 % C a c m a u tro n g n g h ie n curu d u p e th u th ap v a tien h an h c a c k y th u at sin h h o c p h a n tur tai T rung tarn N g h ie n c u u G e n - P ro tein , T r u o n g D a i h o c Y H a N o i Phieang phap nghien cm K y thuat tach chiet D N A : D N A d u p e ta ch tir m a u m a u to a n p h a n b a n g b o k it W iz a r d ® G e n o m ic D N A P u rifica tio n K it c u a H a n g P r o m e g a , H o a K y Q u y trinh ta ch c h ie t tu an th e o h u d n g dan c u a n h a san xu at N o n g d o v a d o tin h sa ch cu a D N A sau tach c h ie t d u p e k ie m tra b a n g p h u o n g p h ap d o q u an g tren m a y N a n o D ro p : n o n g d o D N A -2 0 n g /p l, danh g ia d o tin h sa ch b a n g ty le A /A = l ,8 -2 ,0 KHOA o n g HOC \ / 64( 12) 12.2022 Hinh Ket qua MLPA cua nhdm chirng va nhom nghien cu>u co dot bien mat doan lorn Tryc tung b & i Ihi gia tri DQ cua cac mlu, true hoanh bieu thj cac dinh cua d^u bo kit MLPA P140 (A) Nhom chirng; (B) Nhdm mang d$t bi^n - SEA; (C) Nhom mang dot bidn -a3 7: (D) Nhdm mang d$t bi^n -a ** Khoa hoc Y - DUpc I Y hoc c0 sd K e t q u a M L P A c u a n h o m c h u n g (h in h 1A ) c h o th ay, c a c d in h Ban luan c u a c u m g e n q u y d jn h to n g h o p c h u o i a - g lo b in (tro n g k h u n g m au H ie n n ay, c o n h ie u k y th u at s in h h o c p h an tu d a d u p e p h a t trien d o ) c o c h ie u ca o d e u n h au v a g ia tri D Q d e u n a m tro n g g ia tri b in h c h o p h e p x a c d in h c h in h x a c d o t b ie n g e n a -g lo b in p h o b ie n h o a c th u b n g (k h o a n g ,7 - ,3 th e o n h u k h u y e n c a o c u a n h a sa n xu at) h ie m g a p , g iu p g ia i th ic h c a c k ie u h in h k h a c n h au c u a b en h M o t H in h I B c a c d in h tir H B M den H B Q (tro n g k h u n g m a u d o ) c o g ia s o n g h ie n c u u s u d u n g c a c p h u o n g p h ap k h a c n h au n h u G a p -P C R tri D Q ,4 - ,6 , th ap h o n k h o a n g 1/2 s o v b i n g u b i b in h th u b n g , v a g ia i trinh tu D N A [ , ] M b i k y th uat d e u c o u u d ie m v a n h u p c - SEA T u o n g tu n h u v a y , h in h 1C th e h ie n d o t b ie n g a y m a t d o a n g e n H B A c u a d o t biCn - a 17 v a hinh 1D c o tin h ie u c a c d in h tai v im g g e n H B A th ap h o n v o i n h o m c h u n g , tu o n g u n g v b i d o a n g e n m at g a y d o t b ie n -a4 d ie m c u a n o T u y th e o d ie u k ie n , m u c d ic h v a d o i tu p n g n g h ie n tu o n g u n g v b i k h o a n g m a t d o a n D o t bien diem -aHbCs: C o 1/15 (6 ,6 % ) m a n g d o t b ie n d ie m -aHKs tro n g n g h ie n c u u c u u k h a c n h a u m a m b i c o n g trin h lu a c h o n k y th u at p h u h o p , h o a c k e t h o p n h ie u k y th u at g iu p c h o k e t q u a c h in h x a c n h at tro n g th b i g ia n n g a n n hat, v b i c h i p h i h o p ly n hat H o n 12 lo a i d o t b ie n g e n a - g lo b in g a y b e n h a -t h a la s s e m ia d a d u p e tim thay Sir tu o n g tac g iu a c a c d o t b ie n n a y c o k h a n a n g n g a y c a n g ta o c a c k ie u h in h n g u b i d u p e x a c d in h n a y (h in h ) k h a c n h au M u c d o n g h ie m tro n g c u a b e n h tu o n g q u an ch a t c h e v b i v ie c g ia m h o a t d o n g c u a g e n a -g lo b in [7 ], E E o S - ,_ «_-eeI I e_ _ - I I 1*_ liS S iiiiiS I I S ; ! ! § ? ? ? !? !!: I h i ! I: liiiiiiiiiii = iiilh 'ilH , - a d « n n SI T T T T T T T t T T 'T t t 'T'TT Hinh Ket qua MLPA cua ngu’d’i ianh mang dot bien diem -a HbCs N g u b i m a n g d o t b ie n -aHbCs c o k e t q u a M L P A g a n g io n g v b i N g h ie n c u u n a y sir d u n g k y th u at M L P A , g io n g v b i n g h ie n c u u k et q u a c u a n h o m c h u n g , g ia tri D Q c u a c a c d a u d o d e u n&m tron g c u a J.Z L iu v a c s ( 0 ) [ 8], M L P A d u p e c h o n la m k y th u at ch in h k h o a n g ,7 - ,3 n h u n g x u a t h ie n th e m d in h H B A 2-C S d e x a c d in h d o t b ie n g e n a - g lo b in tro n g n g h ie n c u u v i n o c o m o t T o n g h o p k e t q u a M L P A c u a 15 n g u b i lan h m a n g d o t b ie n g e n b en h d u p e th e h ie n b b a n g s o u u d ie m s o v b i c a c k y th u at th o n g th u b n g k h a c d u p e sir d u n g d e p h a t h ie n b e n h a -th a la ss e m ia K y th u at G a p -P C R c h i c o th e x a c d in h c a c d o t b ie n m a t d o a n d a b ie t v a k h o n g th e x a c d in h d u p e c a c Bang Cac dang dot bien va kieu gen phat hien tren gen a-globin d o t b ie n d ie m M L P A la m o t k y th u at c o d o n h a y v a d o d a c h ie u c a o , k h o n g c h i d u n g la i b v ie c x a c d in h d u p e c a c d o t b ie n m a t d o a n p h o b ie n d a b ie t tr u b c , n o c o n c o th e x a c d jn h d u p e c a c d o t b ie n D f i g d $ t b ie n T e n d § t b ie n K i i u g en S l u u n g (n = ) D i h o p tu -a 17 ~a37/a a (6 ,6 % ) a -th a la ss e m ia N g o a i ra, M L P A c o n c o th e p h a t h ie n d u p e d o t b ie n D i h o p tur -o f2 -a 42/a a (6 ,6 % ) la p d o a n v a d ie m C h u a x a c d in h ducrc -aHbC‘ a a /a a (6 ,6 % ) D i h o p tu SEA - SEi/a a 12 (8 % ) m a t d o a n h ie m h o a c m b i p h a t s in h c u a c a c g e n a - g lo b in g a y b en h -aHbCs [ 8, ], T ron g n g h ie n c u u n ay, c a c ta c g ia q u an sa t th a y d o t b ie n c h ie m 80% to n g s o m a u p h a n tich , tro n g k h i -a3 7, -a 42 - 5Lt v a - a Hbc’ c o c u n g ty le ,6 % C H o c k h a m v a c s ( ) [5 ] d a phat h ie n T at c a 15 n g u b i th a m g ia n g h ie n c u u d e u c o d o t b ie n g a y b en h ran g, - SEA la d o t b ie n -th a la s s e m ia p h o b ie n n h at b k h ap k hu \ tjc A, tu o n g tu n h u n h u n g g i d a dupe tim a -th a la s s e m ia , tro n g d o c o /1 (9 ,3 % ) n g u b i m a n g d o t b ie n D ong N am m at d o a n lb n v a 1/1 (6 ,6 % ) n g u b i m a n g d o t b ie n d ie m g a y b en h cutu n a y N g o a i ra, m u c d o x u a t h ie n c u a -th a la ss e m ia la - -a >,K' tro n g d u g c g a y b d i sir th a y H B A ( c T > C ) V ie c n g h ie n c u u -aHKs th e T A A - > C A A d v i tri c o d o n k e t th u c c u a g e n g a y sir k h e o d c h u o i p ro tein a -g lo b in tro n g c o n g d o n g la rat q u a n tro n g T e b a o R B C c o lie n k e t c h u o i g lo b in -a"bCs d a d u g c c h u n g m in h s e g a y h ie n tu o n g tan h u y et -a HiCs k h o n g c o trieu c h u n g , n h u n g - SEA c o th e g a y b e n h H b H [1 ] V i T h o n g th u d n g , d i h o p tu c u a ket h op v d i su x o a d oan v a y , v ie c x a c d in h c a c d o t b ie n n a y d o n g v a i tro rat q u an tro n g tro n g v ie c tu v a n d i tru y en tr a d e sin h v a sa n g lo c tr a d e sin h K y th uat M L P A c o d o n h a y v a d a c h ie u c a o n e n h o a n toan c o th e u n g d u n g tro n g c h a n d o a n x a c d in h c a c d o t b ie n x o a d o a n v a d o t b ie n a HbCs tren g e n a -g lo b in g a y b e n h a -th a la ss e m ia T u y n h ie n , d o k in h p h i h a n h e p , n g h ie n c u u m b i c h i tie n h an h c d m a u [7] D.R Higgs, W.G Wood, (2008), “Long-range regulation of alpha globin gene expression during erythropoiesis”, C urr O p in H e m a to l., 15(3), pp.176-183 [8] J.Z Liu, et al (2008), “Detection of a-thalassemia in China by using multiplex ligation-dependent probe amplification”, H e m o g lo b in , 32(6), pp.561-571 [9] C.L Harteveld, et al (2005), “Nine unknown rearrangements in 16pl3.3 and llp l5 causing alpha- and beta-thalassaemia characterised by high resolution Multiplex ligation-dependent probe amplification”, J M ed G e n e t., 42(12), pp.922-931 [10] R Ahmad, et al (2013), “Distribution of alpha thalassaemia gene variants in diverse ethnic populations in Malaysia: Data from the institute for medical research”, I n te r n a tio n a l J o u r n a l o f M o le c u la r S c ie n c e s , 14, pp.18599-18614 n h o , c h u a d a n h g ia d u g c tan su a t n g u d i lan h m a n g d o t b ie n g e n b en h a -th a la s s e m ia tro n g dan so N g o a i ra, c o n k h o a n g 10% d o t b ie n d ie m tren c u m g e n a -g lo b in k h o n g th e p h at h ie n b a n g p h u o n g p h ap M L P A V i v a y , ca n k e t h g p th e m p h u em g p h ap k h a c n h u [11] Q.W Qiu, et al (2013), “Evidence of recent natural selection on the Southeast Asian deletion ( - s“ ) causing a-thalassemia in South China”, B M C E v o lu tio n a r y B io lo g y , 13(1), DOI: 10.1186/1471 -2148-13-63 [ 12] S Farashi, C.L Harteveld (2018), “Molecular basis of a-thalassemia”, 70, pp.43-53 g ia i trinh tu g e n S a n g e r h o a c N e x t-g e n e r a tio n se q u e n c in g tren c d m a u lb n d e x a c d in h d u g c to a n b o c a c d o t b ie n g e n g a y b en h a -th a la s s e m ia v a d u a b a n d o d o t b ie n g e n a -g lo b in c u n g n h u tan su at n g u d i la n h m a n g d o t b ie n g e n b e n h a -t h a la s s e m ia tren q u an B l o o d C e lls, M o le c u le s a n d D is e a s e s , [13] S Fucharoen, P Winichagoon (2011), "Haemoglobinopathies in Southeast Asia”, I n d ia n J M e d R e s , 134(4), pp.498-506 th e n g u d i V ie t N a m Ifth a a B a n g k v th u at M L P A n g h ie n c u u d a x a c d in h d u g c lo a i d o t b ie n tro n g 15 m a u n g u d i la n h m a n g d o t b ie n g e n b e n h a -th a la s s e m ia B a o g o m d o t b ie n x o a d o a n ( —s/: l, d o t b ie n d ie m - a H'1 ' D a n g x o a —SEA d u g c -a17, -a4 2) v a tim th a y n h ie u n h a t tro n g 64( 12) 12.2022 [14] N.T Nga, et al (2013), “Hemoglobin constant spring is markedly high in women of an ethnic minority group in Vietnam: A community-based survey and hematologic features”, B lo o d C e lls M ol D is , (4 ), pp 161 -165 [15] W Jomoui, et al (2015), "Hemoglobin constant spring among Southeast Asian populations: Haplotypic heterogeneities and phylogenetic analysis”, P L O S O N E , 10(12) DOI: 10.13” ! joumal.pone.0145230 ... mutations in the a -thalassemia gene is essential for prenatal and premarital genetic counselling to reduce the incidence in the community Multiplex ligationdependent probe amplification (MLPA)... Y - Dude I Y hoc cd sd K y thuat M LPA: N g h ie n Applying Multiplex ligation- dependent probe amplification technique to identify a -thalassemia carriers Minh Ngoc Nguyen, Thi Phuong Le, Huy Thinh... globin gene expression during erythropoiesis”, C urr O p in H e m a to l., 15(3), pp.176-183 [8] J.Z Liu, et al (2008), “Detection of a -thalassemia in China by using multiplex ligation- dependent probe