l A P C H I S l N H H O C . 2 0 1 2 . 3 4 ( 2 ) ^ 4 0 ^
PHAT HIEN MAT DOAN 9 BP TRONG HE GEN TY THE O MOT SO BENH NHAN NGHI H Q I CHlTNG CO NAO
TmoTig Thi H u e ' , P h a m M i n h H u e ' , Lc N g o c Y e n ' , P h a m Thi Van A n h ^ . N g d Di^m Ngoc", P h a n T u 5 n NghIa''
" ' T r u o n g dai hoc Khoa hgc tu nhicn, D H Q G Ha Ndi, '"'[email protected] '"'Benh vien Nhi Trung U(,Tng
TOM T A T Cac dot bien trong he gen ty the la nguycn nhan ciia nhieu benh khac nhau, dac biet la cac benh co nao d ngudi Mile dii vay, viec chan doan cac bC-nh nay thudng kho chinh xac neu chi thong qua cac xet nghiem lam sang Phan tich DNA la phuong phap chinh xac nhai dtj phat hien cac dot bien trong he gen ty the. Trong nghicn ciru nay, 72 benh nhan tuoi dirdi 20 nghi mJic hoi chirng benh ca nao da duoc kiem Ira su c6 mSl ciia dot bien A8344G ciia hoi chimg MERRF. dot bien A3243G oua hoi chirnj!
MELAS va su mat doan 9 bp giQ-a gen COll va gen ma hoa cho IRNA'"^'bang ky thuat PCR-RFLP \ a \ac dinh Irinh tir nucleotide DNA cua mau ngoai vi duoc su dung lam khuon cho phan irng PCR nhan doan gen 212 bp Tir vi tri 8155 den vi tri 8366 irong he gen ty the. Bang di?n di gel poiyacrylamide, 23 man dugc phat hien la co bang DNA nhan ban chay nhanh hon cac mau cdn lai, Cac san pham PCR da dugc cat bang enzyme BanU va kh^ng dinh sy sai khac giua cac mau San pham PCR cua 23 m3u benh nhiin nghi ngd chira dot bien ciing vdi bo me ciia benh nhan da du'oc nhan dong vao vector pGEM. plasmid tai to hgp da dugc tach va xac djnh trinh tu Ket qua giai irinh tu cho thay, 23 m5u benh nhan nghi ngo co chiia dot bien mat doan 9 bp CCCCCTCTA so vdi Irinh tu chuan da cong bo (J01415 2), dot bien mat doan dugc di truyen tii me cho con va tat ca deu a dang dong nhai, Ket qua nghicn ciru cung chi ra lang, khong CO benh nhan nao tiong so 72 benh nhan mang dot bien A8344G ciia hoi chirng MERRF. chi c6 mgt benh nhan nam \ u a mang dot bien A3243G cua hoi chirng MELAS vita mang dot bien mat doan ') bp, chirng to la khong co tuong quan tin cay nao giCra hien tugng mat doan 9 bp CCCCCTCTA v;i dot bien A8344G cua hgi chirng MERRF hay dot bign A3243G ciia hoi chiing MELAS.
Tir khoa: benh co nao ty the, DNA ly the, mat doan 9 bp.
MO DAV
Ty the la e g quan tir cd mat trong tat ca cac le bao CO nhan vdi c h u c nang chinh la san xuat nang lugng d u d i d a n g A T P cho li; bao. He gen ly the la D N A d a n g vdng, cd kich t h u d c 16,569 bp, gdm 37 gen ma lida cho 13 ehuoi polypeptide, 22 IRNA va 2 r R N A khac nhau lien quan den hoat d d n g chirc nang cua ty the [11 So \ d i D N A ciia nhan te bao thi D N A ciia ly the de hi tdn t h u o n g d o mdi t r u d n g giau chat oxy phan u n g t r o n g ly the va d o thieu c o d i e sira chira hieu qua. H o n 300 dot bien kliac nhau irong he gen ty the da d u g e xac dinh ke lir klii dot bien dau ticn d u g c phat hien vao n a m 1988 [8]. Nghicn cim cae ddt bien Irong hg gen ty the cho p h e p phat hien ra nguycn nhan cua nhieu benh khac nhau.
Hicn t u g n g mat d o a n 9 b p ( C C C C C T C T A ) d vung gen giiia c y t o c h r o m e o x i d a s e II va I R N A ' " trong he gen ty the d u g c x e m la mgt dang dot bien ciia h e gen ty the \ a cd ty le c a o d
9 b p trong h e gen ty the d u a c di truyen theo d o n g me, la c d n g cu hiru ich de kiem tra moi quan he di truyen giCra cac ca the [5, 6], Tuy nhicn, c h o den nay, c h u a cd thdng tin nao \ e su lien quan ciia mat d o a n 9 b p vdi cac tinh trang benh ly dac t r u n g .
Ivanova et al. ( 1 9 9 9 ) [2] b u d c dau da nghien ciru D N A ty thi; eua n g u d i Viet N a m va phtil hien mgt sd t r u d n g h g p mat d o a n 9 bp neu tri:n, tuy vay, cac tac gia cting c h u a cd nhan .\cl gi ve hien t u g n g m a t d o a n vdi cac tricu chirng benh ty the. T r o n g nghien c u u nay, chiing tdi ket hgp viec sang lgc dot bien A3243CJ thugc hgi c h u n g M E L A S va dot bien A 8 3 4 4 G ihugc hdi c h u n g M E R R F d cac b e n h nhan nghi bi benh c a n a o vdi hien l u g n g mat doan 9 bp vdi hy vgng tim ra d u g c s u lien quan nao dd giiia cac loai dgt bien nay trong cac hdi chirng benh ty the.
PHUONG PHAP NCHIF.N CVV N g u } c n lieu hda c h a t va thiet bi
Truong Thi Hue el al.
Mau mau ngoai vi cua 72 benh nhan, tudi dudi 20 duge chan doan lam sang ia bj mgt trong cac benh ca nao phd bifin va dugc Benh vien Nhi Trung uang cung cSp, Bd kit tach chiet DNA dugc mua tir hang Qiagen, cap mdi dac hieu dugc dat mua tir hang Integrated DNA Technologies (IDT). Enzyme gidi ban dugc mua lir New England Biolabs va Fermentas. Hda chat cho giai trinh tu gen cua hang Beckman Coulter.
Cac hda chat cdn lai dhu dat dd tinh khiet dimg cho nghien cim sinh hgc phan tu.
Phuong phap
DNA tdng sd dugc tach theo kit cua Qiagen.
De phat hien dot bien A8344G thugc hdi chiing MERRF va dot bien mat doan 9 bp (CCCCCTCTA) d viing gen giil'a cytochrome oxidase II va tRNA^*''' trong he gen ty the, doan DNA ty thg (chiia trinh tu CCCCCTCTA "d viing gen giiia cytochrome oxidase II va IRNA'-*') dai 212 bp tir vi tri 8155-8366, dugc nhan ban bang phan ung chudi polymerase (PCR) vdi cap radi DELFw-5'GGT ATA CTA CGG TCA ATG CTC 3' (8I55-8I75). DELRv- 5' TTT CAC TGT AAA GAG GTGTGG G 3' (8366-8345) theo trinh tu ciia Anderson et al.
(1981) [1], nucleotide khdng ghep cap dugc gaeh chan. Mdi ngugc chiia 1 nucleotide khdng bat cap (T dugc thay bang G) tai vi tri 8347, tao diem cat cho enzyme BanW khi tRNA*"-^' bi ddt bien de hoc Id su sai khac khi cd va khdng cd dot biSn A8344G Cac thanh ph§n PCR (25 pi) gdm 30 ng DNA khudn, 50 ng radi xudi va
50 ng mdi ngugc, • dan vi Taq DNA polymerase va dNTP d ndng do cudi cung la 0,2 mM. PCR dugc thuc hien vdi che dd nhiet:
94"C, 2 phiit, tifip ndi 35 chu ky vdi 9 4 ^ , 10 giay de biSn tinb, 58°C, 1 phut de gan mdi va 72''C, 1 phiit de keo dai cbudi. San pham PCR dugc phan cat bang enzyme BanU, DNA khdng dot bien chua 2 vi tri nhan biet cua BanU. dugc cat thanh 99 bp, 41 bp va 72 bp. N^u DNA dot bien thi bang 72 bp dugc cat thanh 2 bang cd kich thudc 52 bp va 20 bp. San pham cat dugc dien dl tren gel poiyacrylamide 10% trong dcm Tris- Boric- EDTA, pH 8,0.
Dot bign MELAS A3243G dugc phat hien bang PCR-RFLP nhu da dugc md ta trong nghien ciiu trudc day [4],
San phara PCR nghi chiia dot bien raat doan dugc nhan ddng vao vector pGEM va bien nap vao te bao E. coll kha bien DH5a. Plasmid tai td hgp dugc tach, cat kiem tra bang cac enzyme gidi ban va xac dinh trinh tu bang he thdng may tirdgng CEQ 8000 (Beckman Coulter).
KET QUA VA THAO LUAN
Phat hien dot bien mat doan bang phirong phap PCR-RJFLP
Dau tien, chung tdi da tien hanh dieu ira su- ed mat eua dot bien A8344G d 72 benh nhan nghi bi hdi chiing eg nao (bang I). Chung tdi da sir dung ky ihuat PCR vdi cap radi dugc thiet ke d tren de nhan ban doan gen lir 8155-8366 (212 bp) tir he gen ty the cua cac benh nhan.
Hinh 1. K8t qua dien di san pham PCR nhan doan gen chiia dot bien Giang I: Thang chu^n DNA; GiSng 2: Ddi chiing am, Gieng 3-9: San phSm PCR tir cac benh nhan.
I DNA cua
Hinh I la kSt qua dien di san pham PCR cua mot sd benh nhan duoc nghien ciiu, cu the.
chiing tdi da nhan ban thanh cdng doan DNA dac hieu vdi kich thudc 212 bp nhu tinh loan ly
247
TAP CHi SINH HQC, 2012. 34(2) 2-^_t;:lJl ihuyet (cac mau d gieng 4 va 5), ddng thdi
chung tdi cung da phat hien thay 23 trudng hgp (gdm 15 nam va 8 nu-) ed cac bang nhan ban chay nhanh han (dugc minh hga d cac mSu cua gieng 3, 6-9, hinh 1), cluing td cd kicb thudc ngan hon va ggi y ve kha nang mat doan 9 bp trong cac mau nay.
De phat hien dot bien A8344G, san phara PCR dugc cat true tiep bang enzyme BanU. Theo each thiet ke eua thi nghiera, san phara PCR cua ngudi klidng raang dot bien A8344G se ed 3 bang 99 bp, 72 bp va 41 bp, cdn neu cd dot bien A8344G thi san pham PCR se cd 4 bang la 99 bp, 52 bp, 20 bp va 41 bp (bang 72 bp dugc cat thanh bang 52 bp va 20 bp). Chung loi da khdng
phat hien dugc su sai khac nay trong tdng sd 72 mSu nghien eiiu, diSu nay chimg td cac ddi tugng nay khong mang dot biSn A8344G. Tuy vay, ket qua dien di san pb4m PCR ck bang BanU (hinh 2) cho th^y, cac mlu cd bang nhan ban chay cham thu dugc d hinh I (giSng 4 va 5) cd 3 bang la 99 bp, 72 bp va 41 bp theo nhu dnh loan ly thuygt, cdn cac mhi cho bang nhan ban chay nhanh hon (giSng 3, 6-9) cd 3 bang la 99 bp, 72 bp va I bang ngSn hon bang 41 bp. Ket qua nay mgt lan niia ggi y ve kha nang mat doan 9 bp, Ngoai ra, ket qua d binh 2 cung cho thay, cac mau nghi mat doan deu khdng cd bang 41 bp, chiing td tinh ddng nhat ve loai cau tnic nay d tat ca eac ban copy ciia DNA ty the.
Hinh 2 Ket qua dien di san phara PCR dugc cat bang BanU ciia ragt sd benh nhan I. Thang chuan DNA, 2. San pham PCR khong cat bang Ban\l; 3-9. San pham PCR til mau DNA ciia cac benh nhan dugc cat bang BanU.
Hinh 3. Kk qua dien di san phSm PCR dugc cat bang enzym BanU
I Thang chu^n DNA; 2. San ph^m PCR khong ck bSng BanU, 3A-8A: San pham PCR duoc c5t bSng BanU tuang ling cua bcnh nhan. me benh nhan va bo benh nhan ciia gia dinh I va gia dinh II, 3B-5B: San pham PCR dugc c^t bing BanU luong irng ciia bcnh nhan, me benh nhan va bd benh nhan cua gia dinh III.
Dg khang dinh tinh chSt di truyfin theo ddng cac gia dinh cd benh nhan mang dau hieu mdt nic cua cac gen tren he gen ty the. Chimg tdi da doan phat hien dugc quaPCR tren day. Kk qua
Truong Thi Hue elal mau DNA khuon cua b6 benh nhan cr 3 gia dinh va benh nhan.
sau khi cat bang Banll co 3 bang DNA 99 bp, x a c dinh t n n h tir nucleotide ciia doan gen 72 bp ya 41 bp giong nhu mau binh thucmg ^^an ban nghi mil doan
(hinh 3A, gieng 5 va gieng 8; hinh 3B, gieng 5). „ ; , . i . . . , ; , ^ .> . • A;
Trong khi do, san phim PCR v*i DNA khuon . B<= .Hiang d,nh chac chan sir ton tai cua dpt ciia me benh nhan a 3 gia dinh duac c4t bang ^ien mat doan va kich thuoc cua <to?n mat, Bonn dJu cho ph6 bang DNA co kich thuoc fhu"g <o. tien hanh giai trinh tl. doan DNA co giang nhu mlu benh nhan chua dot biSn mit '"?h taoc 212 bp tu vector pGEM ta, to hop.
doan (hinh 3A, gi6ng 4 va gilng 7; hinh 3B, ^ e t qua giii tnnh tu dugc so sanh voi tnnh tu gieng 4). K^t qua nay chung to su tuong dSng f"?'" Sen t u ^ g tog d J d u y c cong bo tren ngan vk kich thuoc oua doan gen nhan ban giua me ha-ig gen the gioi vol ma so J01415.2 bang phan
mem Genetyx.
A [95.283%/212 bp] INT/OPT.Score <504/762
1 ' GGTATA 8101" AGATGCAATTCCCGGACGTCTAAACCAAACCACTTTCACCGCTACACGACCGGGGGTATA
7' CTACGGTCAATGCTCTGAAATCTGTGGAGCAAACCACAGTTTCATGCCCATCGTCCTAGA B161" CTACGGTCAATGCTCTGAAATCTGTGGAGCAAACCACAGTTTCATGCCCATCGTCCTAGA
67 ' ATTAATTCCCCTAAAAATCTTTGAAATAGGGCCCGTATTTACCCTATAGC^^^^^^^^^^^^^
8221" ATTAATTCCCCTAAAAATCTTTGAAATAGGGCCCGTATTTACCCTATAGCACCCCCTCTA U S ' CCCCCTCTAGAGCCCACTGTAAAGCTAACTTAGCATTAACCTTTTAAGTTAAAGATTAAG 8281" CCCCCTCTAGAGCCCACTGTAAAGCTAACTTAGCATTAACCTTTTAAGTTAAAGATTAAG
178- AGAACCdACACCTCTTTACAGTGAAA
8341" AGAACCHACACCTCTTTACAGTGAAATGCCCCAACTAAATACTACCGTATGGCCCACCAT B [ 9 9 . 5 2 8 % / 2 1 2 b p ] I N T / O P T . S c o r e 8 4 2 / 8 4 2
1 ' GGTATA 8101" AGATGCAATTCCCGGACGTCTAAACCAAACCACTTTCACCGCTACACGACCGGGGGTATA
7' CTACGGTCAATGCTCTGAAATCTGTGGAGCAAACCACAGTTTCATGCCCATCGTCCTAGfl 8161" CTACGGTCAATGCTCTGAAATCTGTGGAGCAAACCACAGTTTCATGCCCATCGTCCTAGA 67' ATTAATTCCCCTAAAAATCTTTGAAATAGGGCCCGTATTTACCCTATAGCACCCCCTCTA 8221" ATTAATTCCCCTAAAAATCTTTGAAATAGGGCCCGTATTTACCCTATAGCACCCCCTCTA 127' CCCCCTCTAGAGCCCACTGTAAAGCTAACTTAGCATTAACCTTTTAAGTTAAAGATTAAG 6281" CCCCCTCTAGAGCCCACTGTAAAGCTAACTTAGCATTAACCTTTTAAGTTAAAGATTAAG
187' AGAACdOACACCTCTTTACAGTGAAA
8341" AGAACCAACACCTCTTTACAGTGAAATGCCCCAACTAAATACTACCGTATGGCCCACCAT
Hinh 4. Kk qua so sanh trinh tu nucleotide doan gen 8155-8366 ciia ty the benh nhan so vdi trinh tu gen chuan da dugfc cdng bd
A. Trinh tu nucleotide ciia trudng hgp co bang PCR nhan ban chay nhanh;
B. Trinh ty nucleotide ciia trudng hpp co bSng PCR nhan ban chay cham.
Ket qua so sanh do?n gen quan tam (hinh 4) nhan cd sy: sai khac so vdi trinh tu chuan da cho th5y, doan gen nghien ciiu tir eac mlu b?nh cong bo, vj tri 8347 do chung toi chu dpng thay
249
TAP CHi SINH HQC, 2012, 3 4 ( 2 ) - ^ ' : ^ the nucleotide trong qua trinh thiet ke mdi nen
cd mdt su sai khac so vdi trinh tu gen chuan (C dugc thanh bang A), mau ed bang DNA nhan ban chay nhanh bi mat 9 bp vdi trinh tu CCCCCTCTA (8272-8280) so vdi mau chuin ciing nhu so vdi mau cd bang DNA nhan ban chay cham duoc phat hien d hinh I. Chiing tdi ciing giai trinh tu doan gen tren tir bd va me benh nhan, ket qua thu dugc (khdng neu d day)
cho thdy, hien tugng mSt do?n cbi cd d DNA ty the ciia m? va khdng cd d bd benh nhan, phii hgp vdi kel qua PCR cung nhu PCR-RFLP ban diu. Diku nay da k h k g djnh tinh chit di truyen theo ddng me cua cac gen trcn be gen ty the.
Kk qua giai trinh tu do^n DNA (8155-8366) tren he gen ty the da chimg td rang viec phat hien cae b?nh nhan cd dot bien mat doan theo phuang phap PCR-RFLP la tin cay.
Hinh 5.Mdtphan trinh tu nucleotide cua doan DNA ty the (8155-8366) A. Doan DNA ty the chiia dot bien mat doan; B. Doan DNA ty thi khdng chiia dot biin m4t doan.
Bdng 1. Thdng tin benh nhan va ket qua xac djnh cae dot bien gen ty th^
So lugng
72
Gidi tinh Nam
42 Nir
30
Ket qua xac djnh mat doan 9 bp
23(31,9%) (15 nam va 8 nir)
Ghi chu
1 nara cd chira mat doan 9 bp va dong thdi raang dot bien MELAS A3243G Ket qua xac dinh dot bien mat doan 9 bp
cua 72 benh nhan duge nghien ciin (bang 1) cho thay rang, trong sd 72 benh nhan nghi bi benh CO nao cd tdi 23 hay 31,9% benh nhan cd hien tuong mat doan 9 bp trong he gen ty the. Trong so nay cd tdi 15 benh nhan nam va chi cd 8 benh nhan nir. Ngoai ra, khi dieu tra su cd mat cua dot bien A3243G thugc hgi chung MELAS, chiing toi ehi phat hien thay mot benh nhan nara vua mang dot bien A3243G vua ed hien tuong mat doan 9 bp.
Zhuo et al. (2010) [8] khi xac djnh su lien quan giiia hien tugng mh doan 9 bp CCCCCTCTA d cae benh nhan da nang budng trung thi thay ty le ed mk doan la 23,5%, so vdi ngudi khde manh thi ty le nay chi ed 7,l%i va cac tac gia nhan dinh cd su lien quan giira rait doan va benh da nang budng trimg Theo nghien cuu trudc day cua Ivanova et al. (1999) [2] cho tb§y, ty le mat doan 9 bp d vung gen giua COII va tRNA''*' d ngudi Viet Nam khde manh la 20%), eon nghien cuu cua Liu ct al. (2005) [3] d
Truong Thi Hue et al cac benh rdian ngudi Dai Loan thudc hgi chung
MELAS va MERRF cho thiy ty le mat do^n 9 bp la 47%, trong khi do, ty le nay d ngudi khde manh la 21%. Mdt sd tac gia cho rang cd sir bat on dinh cua viing gen COII/tRNA''^' d cac benh nhan bi benh ty the va la diem ndng cho su mat doan [8]. Trong nghien ciiu nay, chiing tdi chua phat hien thay su lien quan tin cay nao giira h4n tugng mat dojin 9 bp vdi mire dg bi dgt biin A3243G trong hgi chirng MELAS hay dot bien A8344G thugc hdi chimg MERRF.
Hien tugng mat doan 9 bp trong h? gen ty the cd le dugc di truyen cimg vdi mgt so dot bi6n DNA ty the gay benh trong qua trinh di truyen va tien hda lau dai cua he gen ty the. Tuy nhien, can cd su nghien cuu dich te hgc phan tii benh ty the de khang dinh chac chan ve mdi Hen quan giira su mat doan 9 bp trong he gen ty the va su xuat hien benh ty the d ngudi Viet Nam.
KET LUAN
Hien tugng mSt doan 9 bp trong he gen ty the cd the phat hien nhanli va chinh xac bang phuong phap PCR-RFLP, Su dung phuang phap thiet lap dugc, chung tdi da phat hien 23 trudng hgp mSt do?n 9 bp (8272-8280) trong he gen ty the trong tdng sd 72 benh nhan nghi hgi chirng ca nao ty the, chiem 31,9%). Cae trudng hop mat doan 9 bp trong he gen ty the phat hien duge d 23 benh nhan deu the hien tinh chat di Iruyen theo ddng me va d dang ddng nhat (homoplasmy).
Lot cam on: Cae tac gia chan thanh cam an Quy Phat triln Khoa hgc va Cdng nghe Qudc gia (NAFOSTED) da tai trg kinh phi (De tai ma so 106,06.123.09) de thuc hien nghicn cun nay, cam on B^nh vien Nhi Trung uong da cung cap cac mau benh pham.
TAI Ll£U THAM KHAO
I. Anderson S., Bankier A. T., Barrell B. G., deBruijin M. H., Coulson A. R., Drouin J., Eperon I. C , Nierlich D. P., Rose B. A., Sanger F., Schreier P. H,, Smith A. J. H., Staden R. and Young I. G„ 1981. Sequence
and organization of the human mitochondrial genome. Nature,, 290: 457- 465
2. Ivanova R., Astrinldis A., Lepage V., Djoulah S., Wijnen E., Vu-Tricu A., Mors J., Charron D., 1999. Mitochondrial DNA polymorphism in the Vietnamese population Eur. J. Immunogenet,, 26: 417-422.
3. Liu C. S., Cheng W. L., Chen Y. Y., Ma Y.
S,, Pang C. Y., Wei Y. H., 2005. High prevalence of the COII/tRNALys intergenic 9-bp deletion in mitochondrial DNA of Taiwanese patients with MELAS or MERRF syndrome, Ann. N.Y. Acad. Sci., 1042: 82-87.
4. Trinh Le Phuang, Chu Van Man va Phan Tuan NghTa, 2009. Phan tich dot bien gen A3243G cda hgi chimg MELAS bang phuong phap PCR-RFLP cai ti6n. Tap chi Dl tmyen hgc va Ung dung, 4: 6-9.
5. Vassihna A. S., Vadim B. V., Thomas D., Catherine H., Dominique R , Catherine G , 2002. A Russian family of Slavic origin carrying mitochondrial DNA with a 9-bp deletion in region V and a long C-stretch in D-loop. Mitochondrion, 1: 479-483.
6. Watkins W. S., Bamshad M., Dixon M. E„
Rao B. B., Naidu J. M., Reddy P. G., Prasad B. V. R., Das P. K., Reddy P. C , Gai P. B., Bhanu A., Kusuma Y. S., Lura J. K, Fischer P., Jorde L. B., 1999. Multiple Origins of the mlDNA 9-bp Deletion in Populations of South India. Ara. J. Phys, Anthropol., 109: 147-158,
7. Yao Y. G., Watkins W. S„ Zhang Y. P., 2000. Evolutionary history of the mtDNA 9- bp deletion in Chinese populations and its relevance to the peopling of cast and southeast Asia. Hum. Genet., 107: 504-512, 8. Zhuo G., Feng G., Leng J., Yu L., Jiang Y.,
2010. A 9-bp deletion homoplasmy in women with polycystic ovary syndrome revealed by mitochondria! genome-mutation screen. Biochem. Genet,, 48: 157-163,
251
TAP CHI SINH HOC, 2012, 3 4 { 2 ) 2 ; l 6 : ^ D E T E C T I O N O F 9 - B P D E L E T I O N I N M I T O C H O N D R I A L G E N O M E I N V I E T N A M E S E P A T I E N T S W I T H S U S P E C T E D M I T O C H O N D R I A L
E N C E P H A L O M Y O P A T H Y T r u o n g Thi Hue*, P h a m M i n h H u e \ L e N g o c Y e n ' , P h a m Thi Van A n h ^ N g o D i e m N g o c ^ P h a n T u a n N g h i a '
" ' H a n o i University of Science, V N U '^'National Hospital of Pediatrics SUMMARY
Mutations in the mitochondrial genome are the cause of many diffenrent diseases, especially enccphalomyophathies in humans However, it is difficult to diagnose Ihe diseases with cimal examinations, DNA analysis is the most reliable method for detection of the mutations. In this study, 72 patients under 20 years old with enccphalomyopathies were checked for the presence of the A8344G mutation of MERRF syndrome, the A3243G mutation of MELAS syndrome and a 9-bp deletion between the COII and tRNA''^'genes in their mitochondrial genome by using PCR-RFLP and DNA sequencing methods, DNA from peripheral blood samples was used as templates for PCR amplification of mitochondrial DNA (212 bp) fragment starling from 8155 to 8366 position of the human mitochondria! genome. By the poiyacrylamide gel electrophoresis, 23 samples were shown to have a bit faster running amplified DNA band than the others The PCR products were then digested with BanU restriction enzyme, the Sanll-digested PCR products again confirmed the difference between the samples. The PCR products of the 23 suspected samples and those from their parents were cloned into pGEM vector, recombinant plasmids were isolated and used for nucleotide sequencing analysis The obtained nucleotides of the 23 suspected samples were shown to have a 9 bp deletion (CCCCCTCTA) compared with the published reference sequence (J01415,2) and this deletion was inherited from the mother to her child(rcn), and appeared in homoplasmy in all the cases It was also found from this study that none of the 72 subjects carried the A8344G mutation of MERRF syndrome, but only one male patient carried both the 9 bp deletion and A3243G mutation of MELAS syndrome, indicating thai there is no reliable relation between the 9 bp (CCCCCTCTA) deletion and AS344G or A3243G mutations
Keywords: Mitochondrial DNA, 9-bp deletion, mitochondrial encephalomyopathy.
Ngdynhdnbdi: 15-7-2011
