Summary
MULTIPLEX PCR FOR PROMOTER METHYLATION OF EBNA1 GENE
EBNA 1 protein is expressed in all latency pmgram of infected cells using different promoters including the Wp, Cp and Qp. The EBNA 1 expression is regulated by some different mechanisms and methylation ofcytosine in CpG positions of promoter region is shown The aim of the study is to design primer pairs to detect the methylation level of EBNA 1 promoters by the multiplex PCR using cell lines Namalwa and Raji.
Results: Alt unmethylation and methylation primer pairs of Wp. Cp and Qp work well with DNA of 1000 Namalwa and Raji cells, except the Cp is negative for both unmethylated and methylated primer pairs- Conclusion. Wp and Qp primer pairs and conditions for the multiplex PCR are specific with Namalwa and Raji lines, except Cp for Raji
Keywords: multiplex PCR, methylation, Wp, Cp and Qp
DOT BIEN GEN COL1A2 y BENH NHI TAO XUONG B A T TOAN
Bill Thi Hong Chau\ Nguyen Thu Thuy, On Quang Phong^, Tran Van Khanh^
Ho Cam Tu', Nguyen Thj Ha', Ta Thanh Van' i^^Trwong Bai hQC YHa Noi, (^'Benh vi^n Xanh Ron
Tao xuvng bai toan (Osteogenesis Imperfecta: 01) la mot benh dl truyen troi tren nhiim sac th4 do dot blin gen ting hap collagen typ 1 (C0L1A1,COL1A2). Nghien cuv duvc thuc hien nhim phat hien dot bien gen C0L1A2 tren b$nh nhi duqic chin doan mic benh Ol dua vao tri$u Chung lam sang, hlnh anh X quang va lien sCr gay xuang tu nhien; sir dung ky thuat PCR va giai trinh tw gen tri/c tiep. Ket qua: Phat hi?n 3/11 benh nhi 01 c6 dot biin tren gen COL 1A2.
TCr khoa: benh 01, dot bien gen C0L1A2 NhOng nghien cu'u v§ hoa sinh va di I f l A T V A N n ^ truyen phan ti> cho thay hau het benh nhan 01 (>90%) b the l+the IV c6 dot bien liSn quan Tao xuong bat toan (Osteogenesis ^^^ ^^^ C0L1A1 va C0L1A2 tham gia ma Imperfecta: 01) la mpt b^nh di truySn trpi ^^^ ^.^^ ^^p collagen typ 1- la protein chinh tren nhilm sSc the do dot bien gen tong j^ong (hanh phin cau true xu.OTg, da va khop.
hop collagen typ 1(C0L1A1,C0L1A2). Theo B j „ h Q, 4 j ^ l v v a thi VI khong lien quan din Sillence, 01 chia lam 4 t h i khac nhau (thi ^^ ^.^^ ^^^ ^ ^ , 1 ^ ^ ^ ^ ,,p .,
kthe IV) tCiy thuoc vao mu'C do nang nhe cua
benh. Nhttng the b?nh moi cOa 01 diro'c bilt Pf-Sn ti> collagen typ 1 bao gom 3 chuoi
la thi V (Glorieux va cpng sp.., 2000) va t h i VI polypeptid (2 chu6i ol va 1 chu5i a2) cd cau
(Ward va cong sy., 2000) (3) tnjc xoSn 3 vdng. Bit thu'ang trinh tu- gen diln
TCNCYH Phy trUdng 80 (3B) - 2012
hinh nhlt lien quan den Ol l i dOt biln dllm trin gen C0L1A1 v i C0L1A2 gly anh hudng din bO ba acid amin Gly-X-Y tren protein C0L1A1 ho$c C0L1A2. Nam 2006, Pollitt R. v i cOng sy tiln hlnh phin tich gen cCia 83 b§nh nhin 01 ngudi Anh d t h i typ l+t^p IV v l da phit hiOn dup'c 62 dOl biln khie nhau trSn 2 gen C0L1A1 viCOL1A2[4].
Nghiln euu nly nhlm myc tieu phit hiC'n cic dot biln trin gen C0L1A2 d b|nh nhan Ql tgi bpnh vipn Nhi Trung uong
II. D 6 I TU'P'NG VA PHU'ONG PHAP 1. Ooi tup-ng
-11 b|nh nhi dup'c chin doinmlcblnhQI tgi Khoa NOi tiet-Chuyln hda-Di Iruyin, bOnh vien Nhi Trung uong dya vio Ineu chung llm sang v i X quang dien hinh.
- 1 ngudi khde mgnh, khdng mlc b|nh ly dl Iruyen dup'c lay miu d l lim mau doi chung.
2. Phuo-ng phap - Lay mau benh pham
B§nh nhi dupe lay 2ml miu ITnh mgch, chong ddng trong EDTA1,5 mg/ml v l dim bao vd trung tuyOt doi.
- Tach chlSt DNA
DNA t6ng s6 dui?c tich chiet tu bgch clu miu ngogi vi bing dung djch phenol' chlorofonm:
isopropanol (25:24:1). Nlng dO v i dO tmh sgch DNA dup'c xic djnh IrSn miy Nano-Drop 1000, nhO'ng mlu DNA dgt Hlu chuin OD280/OD260 2 1,8 dup'c su dyng d l phin lich gen.
- Ky thu$t PCR
Su dyng cic c|p mOi dSc hi§u 6h khulch dgi gen C0L1A2. Chu ky nhi$t 940C/5phut, [940C/1phul - 590C/1phut - 720C/2phut] x 35 chu ky, 72''C/5phut. Bio quin d IS^C.
Sin phIm PCR dup'c diln di tr§n gel agarose 1%, 90V trong 30 phut.
- Giai trinh tiJ- gen
San phIm PCR dup'c tinh sgch v i giai trinh ty tren may 3100-Avant Genetic Analyzer cua hing ABI-PRISM tgi Trung t i m nghien CU'U Gen - Protein, trudng Dgi hpc Y Ha NOi- Kit qua dup'c so sinh vdi trinh ty trfin GeneBank (National Center for Biotechnology Information. NCBI).
III. K£T QUA
Kit q u i PCR khulch dgi gen C0L1A2 ti>
7 8 9 10 11 MK
— 1kl
exon 47 den exon 49 eua 11 miu DNA tich chilt tu mlu miu cua cic bOnh nhi 01 Hinh 1. Hinh anh dl^n di san pham PCR ciia gen C0L1A2 ttpexon 47 den exon 49
C' mau doi Chung, 1+11: mlu b^nh nhin, MK: Marker 100 bp
Ket qua d hinh 1 cho thiy sin phIm PCR cua 11 mau DNA tren gel agarose rd n§t, dam bao cho viOc giai trinh ty gen tiep theo d l phit hi$n dpt biln .
14
C.3688C C.36880T p.Thr1073lle
T C C T G G r . A . C A G l t G G . A C C T C C r o O i i . ^ G T T O u - i C C
- K i t qua giai trinh t y gen C 0 L 1 A 2 cua 11 m i u DNAbOnh nhi 01 M a u n g u d i b i n h t h u d n g Mau b ? n h n h i 01 Hinh 2. Hinh anh giai t r i n h t y gen C 0 L 1 A 2 cua b^nh nhi so 1 3 , 1 4 va 20 Giai trinh tytn,j'c tiep gen C O L 1 A 2 d a p h i t h i e n 3 b e n h nhi ma so 13,14 v a 2 0 c d d g t b i e n d i e m d j hp'p t u thay the C thanh G tgi vj tri 3688 thuOc exon 48, lam cho bO ba tai vj tri 1073 ma hda Threonin diuyen t h i n h Isoleucin (p.Thr1073lle): dpt b i l n d\ hp'p ti> c.3688C>T - * p , Thr1073lle gen C0L1A2
IV. B A N L U A N
B i n g p h u a n g p h i p giai trinh t y t r y c tiep.
p h i t hien 3/11 benh nhi 01 c d dOt bien tren gen C 0 L 1 A 2 , deu l i dot bien missense thay the C t h i n h G tgi vj tri 3688 thudc exon 48, lam cho bO ba tai vj tri 1073 ma hda Threonin chuyen t h i n h Isoleucin (p. Thr1073lle). Dpt bien n i y c h u a dup'c cong b d tgi ngan h i n g d u ligu ve bOnh 01 (Osteogenesis Imperfecta Variant Database).
Gen C 0 L 1 A 2 m i hda cho sp-i mpt trong hai dgng t i l n collagen type 1, do la pro-a2(l).
Gen n i y c h u a 52 e x o n , trong dd vung hinh thanh chuoi helix bgc 3 v d i hai sp'i p r o - a l ( l ) trai d i i t u exon 6 den exon 4 8 . VCing n i y cd bp ba acid amin Gly-X-Y v d i X la Prdlin va Y la Hydroxyprolin, dong vai trd quan trpng trong vigc bao ton hinh dgng ehuoi x o l n ; s y thay doi cac acid amin se anh h u d n g t d i s y bao ton dd. (y ba bOnh nhi ed dpt bien Threonin chuyen thanh Isoleucin, eau true chudi helix bac ba cd the bj thay doi. Threonin l i mpt acid
amin p h i n e y e do cd chua mOt goc - O H va Isoleucin cd tinh chat khdng phan eye; nhung acid amin khdng p h i n e y e n i m ben trong Idi chudi helix va bao bpc ben ngoai la cac acid amin phan cue, dac biet la bo ba Gly- X-Y. N h u vay dpt bien missense p.Thr1073lle cd the la nguyen nhan gay roi loan cau true C-tgn cua pro-collagen, lam giam chat lup-ng va tinh ben vu'ng cua protein n i y [4].
N a m 2 0 0 1 , Ward v i eOng s y da p h i n tich gen cua 33 b e n h n h a n 01 typ K I V n g u d i C a n a d a va phat hien dup'c 16 dot bien tren gen C 0 L 1 A 1 v i 17 dot b i l n tren gen C 0 L 1 A 2 , hau h i t cac d p i bien l i do chuyen acid amin glycin t h i n h mOt acid amin khac [5]. Nam 2 0 0 4 , Hartikka H. va cOng s y tien h i n h p h a n t i c h gen cua 54 benh n h i n 0 1 , tat ca bOnh nhan nay deu mat kha nang n g h e ; cac t i c gia da phat hien dup'c 49 dot bien khac n h a u , trong dd 38 dOt b i l n tren gen C O L 1 A 1 v i 11 dpt bien tren gen C 0 L 1 A 2 [2].
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TCNCYH Phy trUdng 80 (38) - 2012
Tu nam 2000 din nay khoa Chuyen hda- Npi tiet-Di truyin, b$nh viOn Nhi Trung uong da lien hinh nghien cuu d^c dilm lam sing, cgn llm sang, Ihed ddi v i dilu trj cho 104 bpnh nhin 01 [6,7,8]. 0 ViOl Nam chin doin xac djnh b|nh 01 cdn gap nhilu khd khin do chua ap dyng xet nghiem di Iruyen phan tU', vi$c chin doin b^nh thudng muOn, khi bOnh nhan d i xult hipn bien chu'ng v i chu yeu dya vao cic dlu hiOu llm sing nhu: thdi gian l^p di Igp Igi cua tn$u chu'ng giy xuong, giy xudng tu phit ho^c sau va chgm nh?, hinh anh l6n Ihuong tren phim XQ v l elc tin thuong khic ngoai xuo'ng bieu hipn d tai, mil, Vdi sy phat tnen eua Y hpc hipn dgi, viec thyc hipn nhung nghien cuu nhlm chan doan nhanh va ehlnh xac cac bgnh ly di truyen d l cd the dua ra phuong phap dilu tri thich hp'p v i Idi khuyin kjp thdi, vd'i myc dich hudng den cpng dong v i nang cao chat lup'ng song cua ngudi dan la mot viec lam hit sue can thiet va mang day tinh nhan vin.
V. K t T LUAN
Ciing vdi viOc phat hien cac dpt bien tren gen COL1A1. nghien euu da dua ra nhijng benh nhi 01 dau tien d Viet Nam cd dot bien tren gen C0L1A2.
TAI LIEU THAM K H A O
1. Anna GALICKA, AndrzeJ GINDZIENSKI (2002). Direct sequencing of PCR products for mutation detection in osteogenesis imperfecta, J. Appl.Genet, 43;
365 - 369.
2. Hartikka H, Kuurila K, Korkko J, et al Kokko L (2004). Lack of correlation betvireen the type of C0L1A1 or C0L1A2 mutation and hearing loss in osteogenesis imperfecta patients. Hum Mutat., 24; 147 -154.
3. Pollitt R, McMahon R, Nunn J, etal (2006). Mutation analysis of C0L1A1 and C0L1A2 in patients diagnosed w/ith osteogenesis imperfecta type l-IV, Hum Mutat., 27,716.
4. Sillence DO, Senn A, Danks DM (1979). Genetic heterogeneity in osteogenesis imperfecta, J. Med. Genet., 16(2): 101 -116.
5. Ward LM, Lalic L, Roughley PJ, Glorieux FH (2001). Thirty-three novel C0L1A1 and C0L1A2 mutations in patients with osteogenesis imperfecta types l-IV, Hum Mutat., 17:434.
6. Can Thj Bich Nggc (2010), Nghien cuu d$c dllm l i m sing, e^n l i m sing va dieu trj bpnh tgo xuang bat loan tgi B#nh viOn Nhi Trung uong 2000 - 2009; Lu^n van Thgc sy Y hpe.
7. Tran Van Khanh (2011), BOnh tgo xuong bit toin. BOnh hpe phin tu, N h i xult ban y hpc. 208-216.
8. Vu Chi Dung, Nguyen Thj Hoan va cpng si^ (2004). BOnh tgo xuong b i t toin do dpt bien gen C0L1A1 v i COL1A2: Nhgn xet cic trudng hp'p dup'c chan doin v i dieu trj tgi BOnh viOn Nhi Trung uang. Y hpc thyc hlnh, 495.42-51.
Chu thich: Nghien cu'u dup-c Ihyc hiOn tgi Trung t i m nghien cuu Gen - Protein, Trudng Dgi hpc Y H i NOi trin ea sd eua de t i i d p BO:
"Nghien euu ung dyng c i c ky thugt sinh hpc phin tu d l xac djnh dOt bien gen (C0L1A1, C0L1A2) g i y bOnh tgo xuong bit toin (Osteogenesis Imperfecta: 01) d tre em ViOt Nam", vdi sy tai trp' kinh phi td ngan sach Sy NghiOp Khoa Hpc elp BO Y t l nim 2011.
S u m m a r y
MUTATION A N A L Y S I S OF C 0 L 1 A 2 IN OSTEOGENESIS IMPERFECTA PATIENTS Osteogenesis Imperfecta is an autosomal dominant desease. Most of mutations will change the triple form Gly-X-Y in C0L1A1 or C0L1A2 gene which could harm a Collagene I structure. Mutalion analysis of COL 1A2 was carried out from DNA of osteogenesis imperfecta patients who were dignosted base on clinical characters. X-ray result, and bone-broken history using direct sequencing method. The results indicated that 3/11 patients were found to have the point mutation in COL 1A2 gene
K e y w o r d s : O s t e o g e n e s i s Imperfecta - 01, C 0 L 1 A 2 gene m u t a t i o n
N 6 N G D O CYSTATIN C MAU VA CHU'C NANG THAN 0 BENH NHAN OAI THAO DU'O'NG TYP 2
N g u y e n T h j L y \ Tran Thj Chi Mai^
f'J B0nh vi$n 19 - 8. (^)Tru-d-ng D^ihoc YHa Npi
Nghien cuv nay nhim danh gia si/ thay ddi nong dd cystatin C huyet thanh trong phat hi^n ton thuxmg th$n a b^nh nhan dai thao du'ang typ 2. 90 benh nhan dai thao duxyng typ 2 du^c chia lam 3 nhdm: albumin nieu am tinh, abumin ni$u vi luung va albumin nieu lu-ang lan. Ket qua nghien cuv cho thay: nong dQ cystatin C tang cd y nghia tLF nhdm abbumin ni$u binh thudng (0,81 ± 0,40 mg/L) din nhdm albumin ni$u vi luvng (1,59 ± 0.66 mg/L) tiSp den la albumin ni0u luvng lan (8,62
± 5.58 mg/L) (vaip < 0,001). Ndng dp cystatin C khdng phu thuQC vao giai, tudi va Btvti. Cystatin C nh^y han, thay ddi sam han creatinin trong sang IQC va theo ddi c/rt/c nSng th$n a b§nh nhan dai thao duo'ng typ 2. Nong dQ cystatin C tang cd y nghia tu- nhdm benh than man do dai thao duung giai doan 1 den giai doan 2, giai do^n 3 (tat ca cac p < 0.001); trong khi ndng dd creatinin chi khac bidt cd y nghia giO^ nhdm albumin ni$u vi luxyng va albumin nieu luxyng lan, giQ-a b$nh th$n m^n giai doan 2 va 3. Gia tri chin doan chinh xac b$nh than m^n giai doan 1 va 2 cua cystatin C tdt han ciJa creatinin. TO" dd ta cd kit luan: cac kit qua nay gai y cystatin C huyet thanh cd the la mot chi tS de phat hidn sam suy giam mCrc Ipc cau th$n a bdnh nhan dai thao du^ng typ 2.
T u k h o a : C y s t a t i n C, c r e a t i n i n , m u c loc c a u t h a n , a l b u m i n n i e u v i lucvng, b e n h t h g n man, dai t h a o d u d n g t y p 2
I. D A T V A N 0 £ O T D . Do do, viOc chan doan p h i t hiOn s d m s y Bpnh thSn do dai thao du-c^ng (OTO) c6 ^"^ 9 ' ™ "^"'^ " « " 9 * ' ' ^ ^°' " ' ''^"^ '^'^^"
dac trtmg bpi albumin nipu vi l u v n g , t i l p theo S ™ . | a r l t can thiet. Hipn nay cac xet nghiem la albumin nieu lu^gng Ion va suy giam mire Ipc a"''?c dung sang Ipc bpnh th?n do DTD la theo cau than. D a y la mpt bien chiJng du'pc coi la doi microalbumin nieu, danh gia chiic nang Ipc nguy h i l m va ton kem nhat a cac bpnh nhan c l u than b i n g dinh lu-png n I n g dp creatinin
17
