Tap chi Cdng nghe Sinh hoc 10(3): 423-429, 2012
P H A T H I E N V A D J N H LlTgfNG D O T B I E N A 3 2 4 3 G T R O N G H E G E N T Y T H E Cf H Q I C I I l T N G M E L A S
T r u o n g Thj H u ? ' , Nguyen Van Li§H^ P h ? m Viin A n h \ Nguyen Thj Van A n h ' , Phan Tuan Nghia' 'Truang Dgi hoc Khoa hgc lu nhien, Dgi hgc Qudc gia Hci Ngi
^B$nh vien Bgch Mai, Bg Y te 'B4nh vien Nhi Trung irang. Bg Y te
T6M TAT
Dpt bien A3243G trong h$ gen ly the ngudi la dgt bien Ihirdng g5p nhat trong cac b^nh ty the va la nguyen nhan ehii >^eu ciia hgi ehirng ca va nao. I5ng aeid lactic va gia lai bien m^ch (dugc gpi tai la MELAS), Dpi bien A3243G t6n tai 6 dijng dj plastid hay dj DNA ly the (heteroplasmy) v4 mire dp nghiem trgng ciia bijnh lien quan vdi tj' 1? sd ban sao h? gen mang dpi bien, Trong nghien cuu nSy, ky Ihugl PCR-RFLP ket hgp vdi ky thugt real-time PCR su d\ing mdi Taqman huynh quang LNA (acid nucleic dugc khoa l^i) da dugc sir dyng de phat hiijn va djnh lugng dpi bien A3243G. Theo phuong phap PCR-RFLP, ngudi binh thudng chi c6 mgt bang DNA nhan ban 198 bp trong khi bC'nh nh3n co bang 198 bp (ciia ban sao khong bj dpt bien) va bang 111 bp Cling bang 87 bp (cua ban sao mang dgt bien). 82 bC'nh nhan vdi eae iri^u ehirng bC'nh co va nSo dugc sang Ipc sy CO mat dpi bien A3243G va 4 benh nhan (3 nam. 1 nir. tuoi 11-15) da dugc phat hi^n mang dpt bien A3243G. Dgt bien nay cflng dugc phat hiC'n thay d mi; cflng nhu anh/chj em ciia cac b^nh nhan nhung khong thay cd 6 bd eua cac b^nh nhan. Ket qua nay cflng duge tai khang djnh b5ng xac dmh trinh ty nucleotide. Qua pho bang PCR-RFLP cung da phat hien thay ring me eiia benh nhan co bSng 111 bp va 87 bp it ro net hon bang 198 bp. Su dvmg real-lime PCR \ai moi Taqman huynh quang LNA, benh nhan MELAS cua gia dinh so 11 dugc phat hien mang 73,02% ban sao he gen chua dpt bien A3243G. trong khi do mi; eua b^nh nhan nay chi chira 9,24% ban sao gen co dpt bien Kel qua nay la phii hpp \'ai ket qua phat hien d muc PCR-RFLP va giai thich vi sao m? ciia bijnh nhan khdng cd bieu hien bgnh,
Tir khoa: Dgt biin A3243G. DNA ty thi. hgi chimg MELAS PCR-RFLP. real-time PCR
DAT VAN DE
Dot bien thay the nucleotide A thanh G lai vj Iri 3243 (A3243G) trong h? gen ly the ngudi dugc xem la cd tan sd xual hien cao nhat trong cac benh ty the (Masakazu et ai, 2009). Dot bien nay nSm trong viing gen ma hda cho tRNA*"'", la nguyen nhan chinh ciia hpi chimg ca va nao, t^ng acid lactic va gia lai bi^n mach (mhochondrial encephalomyopalhy, lactic acidosis, stroke like episodes, dugc ggi 151 la MELAS) (Bai, Lee, 2004; Ma et al, 2009).
D^t bien A3243G tdn lai d dang di plastid (sy cd mat dong thdi ctia nhieu loai DNA ly the Irong cimg mpt tg bao). Muc dp dj plastid la mgt yeu to quan trgng anh hudng den sir thay ddi ve kieu hinh va tinh nghiem trgng cua benh. Ty le cua ty the dgt bien phai dat den ngudng thi mdi the hien kieu hinh lam sang (Bai, Lee, 2004). Vi vay, viec phat hipn dpi bi^n A3243G va xac djnh ty le s6 ban sao mang dpi bien la co sd phSn tir cho viec chSn doan chinh xac benh do dot bien gay ra.
CJ Vi?t Nam, dgt biln A3243G gay hgi chung
MELAS cung dii dugc phat hien bang PCR-RFLP , Tuy vay, cho den nay ciing chi mdi cd 3 Irudng hgp dugc bao cao (Le Thj Bich Thao el al, 2004; Tran Thi Minh Nguyet el al, IdQl, Trinh Le Phuong et al, 2009) va chua cd cdng trinh nao di sau nghien cim mire dg di bao quan cua dpi bien gen ty the ciing nhu su lien quan giCta mite do di plastid vdi trieu chimg lam sang ciia benh. Trong nghien cim nay, chung tdi gidi thieu ket qua budc d5u phat hien va dinh luong dpi bi^n A3243G d benh nhan bang real- lime PCR cai lien.
NGUYEN LI$U VA PHUONG PHAP Nguyen li^u va hoa chat
M I U mau ngoai vi ciia 82 bpnh nhan, dugc chan doan lam sang la bj benh co va nao pho bien va dugc Benh vipn Nhi Trung uong cung cap.
Bg kit t^ch chiet DNA dugc mua lit Qiagen, cap mdi va mSu dd dSc hieu dugc mua tir Integrated DNA Technologies (IDT). Dung dich gdc (master 423
TruoTigThj Hueelal mix) cho real-time PCR va llae\\\ dugc mua lir
Fermenias. Hda ehiil cho giai trinh ti,r gen cua Beckman Coulter, Cac ho;i chiil ci>n lai di'u dai dp linh khiel dimg cho nghien ciru sinh hgc phiin lir, PCK-KFLI*
DNA long sd lir nuiti ngoai \ i dtrgc liich llico kit eiKi Qiagen, Dgt hicn Al24^(i duoc phai hipn biing PCR-RI'LP nhu dii duge ind ta Irudc d;i> ( I'rinh Li- ' Phutvng ••/ (//, 2009) ( \ i Ihi- doan gen 19S bp U'r vi Iri 31 34 ili'ii 333 I Uong hC' gen l\ the dime iili:iii len biing PCR, su dung cap mdi nilMI.L-FI v;i mlMl I - Rl clii'ra mdl diem khdng bfit liip. sail dd doan gen iihiiii ban dirge i.,il hang //(frill Dot bien dugc khang djnh boi sir \ufil hipii hai King DNA \oi kich tliirdc I 11 bp \.\ 87 bp, lioiig kill diing khdng ddi hicn chi cu mdl bring DNA l9Sbp,
Nhfiii ildn^ v:'i giai Irinh lu' DINA
S.in phfiiii PCR nghi chira dot biC'ii A3243(i dugc nhiin ddng Irire licp vao \ccliir pOLM-T vii bicn nap \ a o le biio kha bien E. loli DH5ii Plasmid nil id hop diroe tiich, ciit kiem Ira bang liae\\\. Trinh tu nucleotide duac \ac dmh Iheo phirong phap Sanger tren he ihdng m;iy dgc Irinh Ur CEO-8000 (Beckman Coullcr)
Kcal-limc PC U
Dot bicn A3243U dugc djnh lugng bang real- time PCR sir dung cap mdi mtMEL-F2 va nuMEL- R2 va mau dd Taqman LNA (locked nucleic aeid) dac hieu cho trinh tu dich (Bang 1). Cu the, doan DNA chira ddi bic-n A3243(i dai 90 bp lir vi Iri 3206- 3295 dime nhiin ban \di cap mdi miMliL-f''2 \;i miMLl,-R2 Vii nucleotide dot bicn dugc ph;it hien bangniiiudddiichieu Wt-MEL-HEX-I \a Ml-MliL- FAM-I Thanh phiin phiin irng real-lime PCR gdm qPCR master m i \ (2X), 11.4 pmol/L mdi loai mdi, O.OX pmol/L moi loiii imlu dd (Wl-MEL-HEX-I/Mi- MEL-FAM-1) va 10 ng DNA khiidn cho idng the lich 25 pi Real-lime PCR diroe thue hien vdi chC dp nhicl 95"C, 10 phiil Hep noi 40 chu ky vdi ')S'C, 1.5 giiiy de bicn linh v;i 60"C, 60 giay de bat cap vii keo dai chudi Cucmg dd tin hieu huynh quang dugc ghi nhan va phan lich bai he thong \Q5 cycler (Biorad) Sau khi hoan tat cac chu ky nhieL cac mau chuSn va mau phan lich co cac duimg bieu dien kliucch dai luong img. Tii gia trj ngudng (Cl) cua m5u nghien ciru, dua vao duong chuan dugc thiet lap bSng each 424
irgn mau DNA plasmid mang dot bien va khong mang ildl bnin vdi eae ly le khac nhau tir dd linh ra % dpi bi^n ciia mau phan lich dugc tinh ra. Cu IhO plasmid mang DNA cd dot biln va khdng dpi bit'n A3243G dugc tach va linh sach theo kit ciia (.)iiigen. So ban copy DNA chira dgt bien va khdng dgi hien dugc linh A\r.\ tren kich thudc hay trgng Itrgng phan lir v:i ndiig dp ctia DNA plasmid Day DNA plasmid id 5,10 -5 lO^ban sao/pl'(he s6 pha loiiiig 10) chira Irinh ig dgi bii-n vti khdng dgi bien dugc ch;i> eiiiig vdi ni;iii vii Irong mdi phan img co mat ddng Ihdi pliiMiiid mang ddi bien \ii plasmid khdng miiiig ildl bicn l.tnuig dgi hiin Irong mdi mau dtrae ilu 3 kin (n 3) vii dtrgc \ir !>' Iheo phtraiit;
phiip ihdng ke.
Kl I (;UA \ \ I I I A O I DAN
Phiil liiin doi hien \3243<: b^ng PC K-KFLP Doiin gen chira diit bii-n A3243(i lir 3134 3331 (19X hp) Id hi' gen I\ the cua ciie benh nh:in diroc nhiin biin bang k\ lluuil PCR vdi cap mdi dac hieu nhu dii md l;i Irong phuong phiip Irude day Kel qua dien di san pluim PCR Iron gel agarose 2"u cho ihay chimg idi dfi nhiin hiin dugc doan DNA dac hieu co kich thudc 19« bp nhir tinh loan 1\ thuyll (Hinh 1).
San phiJm PCR mang dpi bien A3243ti cd diem cat ciia llae\\\. diroe cat Ihanh 2 doan 111 bp \a 87 bp, con mail khdng mang dot bicn van giu ngu\en kich Ihirdc biin diiu 198 bp. Kel qua dien di san pham PCR Ciit biing //</<-lll (PCR-RFLP) eho thSy cd 4 irong sd S2 benh nhan eho phd bang PCR-RFLP cOa dot biL-n A^243(; ihugc hgi chimg MELAS (Hinh 2.
duimg ehii) 4-7) luong lu nhu ddi chimg duong niiing ddi biOii .\3243(i da duoc phai hien bdi Tnnh Lc Phuinig et al (2009) ben canh eae mau khong dpi bii-n chi cho mgl bang kich thudc 198 bp (Hinh 2.
diriTng chiiy 8-10) Su cd mat cua bang cd kich ihudc nhu bill! diiu {198 bp) d eiic m^u niiy la do benh nhan cd ca nhimg ban sao gen khdng mang dot bien A3243(.i (dyng di pliistid) Mire dd dam nhal ciia cac biing Ciit o ciic m^u la khdng gidng nhau (Hinh 2), mail hpnh nhiin d dutmg chay sd 4 cd bang 111 bp vii biing 87 bp il rd net h m bang 198 bp, trong khi do eae bang Uroiip img nay d cac mau 5-7 khdng sai khiie nhau nhtfiu v^ dp sang Sy sai khac nay ggi y v^ ly le sd ban sao mang dot bi^n va khdng dot bien a mau so 4 Ihap ban eae mau con lai.
Bdn benh nhan bj benh ca va nao dugc phat hien la mang dpi bi^n A3243G Ihugc hpi chung MELAS
Tgp chi Cong nghf Sinh hoc 10(3), 423-429, 2012 bao gdm 3 nam va 1 na, dg ludi tir 11-15 tudi, trong dd cd I benh nhan chua the \ky inSu ciia cac thanh vien trong gia dinh nen tu nay v^ sau chung tdi chi lap trung phan tich lugng dot bian ciia 3 benh nhan cd the lay mau cua cac thanh vien Irong gia dinh (ky hieu la gia dinh I. II va 111)
De khiing dinh tinh ehil di IruyOn tir me sang con ciia dot bien A3243G, chimg Idi dii kiem Ira phd bang PCR-RFLP eiia cac thanh \ien trong 3 gia dinh mang dot bien. Bd eiia eae benh nhiin chi cd I bfing Bang 1 . Trinh t u cua moi va mSu do dung cho real lime PCR.
DNA 198 bp (Minh 3, dudng chay 7, 10, 17), trong khi do benh nhiiii (Hinh 3, dudng chay 4, 8, 14), anh/em cua b?nh nhan (duimg chay 5, 15) va me cua benh nhiin (Hinh 3, dudng chay 6, 9, 16) deu cii phd bang PCR-RFLP tuong Ig nhau vdi 3 bang 198 bp. 111 bp Vii 87 bp. Kel qua nay khang dinh dgi bien da dugc Iruyen lii inc cho con, Dc dimg nhiin thiiy rang bang 11 I bp va 87 bp ciia me benh nhiin kem rd net him biing 198 bp riil nhicu, ehirng to ty le han sao gen mang dot bien/khdng ddt bien ciia nic bi;nh nhan Ihiip him nliicu so vdi benh nhan .
MoiMau do mtMEL-F2 mtMEL-R2 Wt-MEL-HEX-1 Ml-MEL-FAM-1
Trinh t y
5'-CCA CAC CCA CCC AAG AAC AG-3' 5'-AGG AAT TGA ACC TCT GAC TGT AAA GTT TT-3' HEX-COG GGC+CCT GCC AT-BHQ
6FAM-CCG GGC +T+CT GCC AT-BHQ
Vj tri tren DNA ty the 3206-3225 3267-3295 3237-3250 3237-3250 Ghi chu. dau + chi nucleotide LNA
b tsi:itsaa
Hinh 1. San pham PCR nhan doan gen 3134-3331 Irong he gen ty the cua cac benh nhan (3-9), thang chuSn DNA (1), d6i Chung am (2)
Hinh 2. San pham PCR-RFLP (cat bang HaeWl) cua mot s6 benh nhan nghi bi dot bi&n A3243G (4-10), thang chu^n DNA (1), san pham PCR khong cat biing Haelll (2). doi Chung d u o n g (mhu chua dot bien A3243G da duoc xac dinh b^ng giai tnnh l u gen trong nghien cuu Ituoc day (Tnnh Le Phuong et al, 2009) (3)
Hinh 3. San p h i m PCR-RFLP (cat bang Haelll) doan gen mang dot bi4n A3243G ciJa cac thanh vien trong gia dinh benh nhan I (4-7), M (8-10), III (14-17), b#nh nhSn (4, 8,14), em/anh b?nh nhan (5, 15), me benh nhan (6, 9, 16) va b6 benh nhSn (7, 10,17), thang chulln DNA (1)
425
Truong Thi Hu^ etal
T r i n h l^r nucleotide cua do^n t;cn nhSn h^n nghi chira d$( bien A3243G
De kh^ng djnh ch^c chin sg Ion t^i ciia dpi bicn A3243G, chimg tdi lien hiinh giai trinh tg dogn DNA nhan ban 198 bp ciia 4 bpnh nhan da dugc xac djnh bang PCR-RFLP va so siinh vdi Irinh Ig doijn gen luong img Iren GenBank (J0I4I5 2) (Anderson el al, 1981), Hinh 4 va 5 hi minh hga kel qua giai Irinh Ig nucleotide ciia 4 b?nh nhan dugc nghien cim (vi kel qua hoan loan gidng nhau) Do^n gen nghien cuu lir 4 mau bv'iih nhan ed phd biing PCR-RFI.P dang mang dgi bien dcu cd 3 vj tri sai kluie so vdi Irinh Ig chtiiin da cdng bd, trong dd su sai khiic o 2 vj Iri 3149 Vii 3118 la do chimg idi chu dgng lh;iy Ih6 nucleotide Irong qu;i Irinh Ihiel kc mdi. cdn vj Iri 3243 cd sg Ihay ddi A Ihanh ( i . phii hgp vdi kk quii phiin tich PCR-RFLP d tren.
Djnh lirgng dot hicn A3243G b^ng rcal-limc PCR SIT dung mau dd TaqMan LNA
Kel qua phan lieh real - lime PCR (Hinh 6) cho thay dutmg chuan mau xanh la ciia DNA cd mang dpi bien va mau do la dang khdng mang dgi bien) tao ra bang vice su dung cac ly le ndng do khac nhau giOa plasmid chira doan gen ly the mang dot bidn A3243G va khdng mang dpi bien cd do luyen tinh cao, gia Irj ciia he sd tuong quan R" giua sd ban sao DNA ty the va sd chu ky nguong vdi ca mSu dd Wl- MEL-HEX-1 va Ml-MEL-FAM-1 nAm trong khoang 0,98-0,99, chimg Id kel qua thu duge la tin cay (Genasettifr«/., 2007)
Ap dung che dp real-time PCR da Ihic-I lap d^
phan tich ly le dot bien trong gia dinh sd II (3 l^n l|ip lai), chiing tdi da linh dirge phan IrSm sd ban .sao gen mang dgi bi^n A3243G ciia benh nhan va me benh nhan tuong ung lii 73,02 ± 0,58 vii 9,24% ±1.1 Kdl qua djnh lugng ly le dot bien nay la phii hgp vdi k8l qua phan lich PCR-RFLP trudc dd, dong Ihdi cung cho phep giai thich vi sao me ciia b^nh nhan khdng cd bieu hi?n ciia hgi chimg MELAS (iheo k^l qua kham ciia benh vi^n) miie dau cd mang dgt biin nay.
Singh va ddng tac gia (2006) cung da sii dyng real-lime PCR dg phat hi^n va djnh lugng dgt bifin A3243G. Tuy nhien cac tac gia diing cac m l u dd gSn huynh tjuang (VIC va FAM) d d^u 5' cdn diiu 3' dugc gSn vdi protein lien kit khe nhd ciia DNA (minor groove binding protein-MGB). Vifc gSn MGB vao dau 3' giup thu nhan tin hipu huynh quang ciia VIC/ FAM khi chimg d dang m l u dd, dong thoi 426
Idm ISng nhi^l dg l^ch chudi (J,„) ciia mau dd. Trong nghien ciru nay, dga Iren d$c linh ciia cac oligonucleotide khi chira nucleotide cai bien vdi cAu methylene giCJa vj Iri oxy 2' vii carbon 4' ciia g6c dudng (ggi 131 la mau dd acid nucleic bj khoa LNA) Ihi van gSn d$c hi^u voi nucleotide bd sung va eo nhi^l dg Tm cao hem so vdi dgng ihwig ihuimg lir 3 - 8"C/nucleolide b| cat bien (Leiertre el al., 2003), nhd dd eiic mau dd g9n huynh quang ciia chiing toi Ihi^
ke chi can d^i 14 nucleotide nhung van co Tni cao han Tm eua moi (dai 20 - 30 nucleotide), dam bao cho vi^c gSn d^c hi^u vao dogn gen dich.
Ue giai Ihich hipn lugng vi sao ly le sd ban sao niiing dgt bicn d m^ va con khac nhau mSc dau chinh m? dii Iruyen dpi bicn cho con cai, mgl sd nghien ciru cho rang chi cd mgl lui^mg nhd phan tu DNA ly the lir ngudi m^ dugc Iruyen cho the h^ sau (thuyel
"niil cd chai ly the") va ly the lir te bao lign than cua ngudi me dugc phan phdi ngau nhien d cac Irung, vi vay cac trimg khac nhau cd the chira mdl lugng DNA ly the mang dot bien khac nhau (Poullon et al.
1998). Thuc \k. Ma et al (2009) khi nghien cuu doi bien A3243G a cac ngudi than Iheo dong me ttr cac nguon mau khac nhau da phai hi^n thiy rang Irong 3 trong sd 5 gia dinh cd ngudi me mang dot bi&i A3243G deu cd cac ngudi con (nam va niir) khong mang ddi bien nay (ben cgnh cac ngudi con khac mang dot bien).
Nghien ciru eua Bai va Lee (2004) cho th^y su lac dgng lain sang ciia benh ty the ta khong giong nhau. phu Ihugc vao ngudng bieu hi^n d timg md va su phan bd ciia mlDNA dgi bien d cac md khac nhau.
Ty Ip cua ly the dot bien phai dat den nguong thi moi Ihe hi?n kieu hinh lam sang. Ty le mlDNA co lien quan den ludi lac va muc dp nghiem Irgng ciia benh (Tajima et al. 2007). Nhu vgy, vi^c phat hi^n dot bien va mire dg di plastid ciia dpi bien la rai quan trgng trong chan doan Kel qua phat hign va dinh lugng dgi bifin A\km (A3243G) biing real-time PCR Irong nghien ciru cua chimg tdi la dang tin cay. Tuy nhien. can cd sg nghien cuu tren d i ^ rpng vdi so lugng b^nh nhan nhieu hem dk co khang djnh chinh xac ve su lien quan giixa miJc dg di plastid va linh trang bpnh ly do dgt bien gay ra.
Sir dyng PCR-RFLP chimg toi da phat hi?n 4 frudng htjp dpt bifin A3243G trraig h? gen ty the trong sd 82 bpnh nhan bi hgi chimg co va nSo, chiem
Tap chi Cong nghf Smh hgc 10(3)' 423-429, 2012
4,9%. Dot bien A3243G dugc ttuyen tit me sang con vdi mau dd Taqman LNA da cho phep phiil hi^n va va ton tgi d dgng dj plastid. Sir dyng real-time PCR djnh lugng dugc muc dp di plastid ciia dpi biin
[Se.flBS* / 198 bpl IHT/OPT.score ! < 774/ 774 >
1' CAAGMiA AATAAGG<7iy ACTTCACAAA GCGCCTTCCC CCGTAAATGA 3121- CTSTACGJUJL GfiRCAAG&Gft A&TAiGGCCJ ACTTCACAAA GCt^CTXCCC CCGTAAATGA 46" TAICATCTCA ACTTAGTATT ATACCCACAC CCACCCAAQA ACAGGGTTTG TTAAGATGGC 3131" TATCATCTCA ACTTAGTATT ATACCCACAC CCACCCAAGA ACAGGGTTTG TTAAGATGGC 108" AtjSsCCCGGFT AATCGCATAA AACTTAAAAC TTTACAGTCA GAGGTTCAAT TCCTCTTCTT 3241" ACjAfecCCGGT AATCGCATAA AACTTAAAAC TTTACAGTCA GAGGTTCAAT TCCTCTTCTT
166' AACAACATAC C C A T G G C I ^ CCTCCTACTC C
3301- AACAACATAC C C A T G G C C J A CCTCCTACTC CTCATTGTAC CCATTCIJIAT CGCAATGGCA
H i n h 4 . So sdnh trinh t i / nucleotide doan gen 3134-3331 cua ty t h ^ b$nh nhSn gia dinh so 11 nghi mang d^l bi^n A3243G voi Irinh t i / gen c h u i n (GenBank J01415 2)
a T T T T T - - - ' C - • C C C T - - r r r - r A C ^ * - - -
,1 A 3 2 4 3 G I
ii i I
Hinh 5. Mpt phan tnnh t u nucleotide ciia doan DNA ty the chua d6t b i e n A 3 2 4 3 G d benh nhan gia dinh 11
H i n h 6, Bi^u d6 k h u ^ h dai b i n g reall-lime PCR doan gen ly th4 mang dot bi^n A3243G sir dung DNA tSch t u c^c thanh vi6n gia 3inh benh nhart II vi plasmid mang doan gen d c^c n6ng d ^ khac nhau A: Cac d u d n g cong b i l u thj mi^c dp nhan ban ciia cac dogn gen dich, B' T u o n g quan giiJa nong dO bin sao gen mang dOt bien A3243G (xanh) v^ khdng mang d^t b i l n (do) vdi g i ^ In chu ky ngu&ng,
427
I ruang Thi Huf el ai
Loi cam ffn: Cac tac gia chan thanh cam ctn (Jiiv Phat tnen Khoa hgc vd Cdng ngh^ Quoc gut (NAFOSTED) da Idi ti<f kinh phi (Di tdi ma so 106.06.123.09) de llnrc hii-n nghien cihi ndv
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D E T E C T I O N A N D Q U A N T I T A T I O N O F A 3 2 4 3 G M U T A T I O N IN T H E HUMAN M I T O C H O N D R I A L G E N O M E W I T H M E L A S S Y N D R O M E U S I N G P C R - R F L P AND R E A L - T I M E P C R W I T H L N A P R O B E S
Truong Thi Hue', Nguyen Van Lieu^ Pham Vnn .\nh', Nguyen Thi \ ' a n Anh', Phan Tuan Nghia University of Science. Vietnam National Unnersitv Hanoi
' Department of Neurology. Bach Mai Hospital Department of Neurology, National Hospital of Pediatrics
A3243G mutation in the mitochondrial genome is the most frequent muiaUon of mitochondrial diseases in humans and the major cause of mitochondrial encephaloniyopathy lactic acidosis, stroke like episodes (MELAS). The A3243G mutation exists in the heteroplasmic form (heteroplasmy) and the seventy of the
Author for correspondence: TtV. +84-4-35575497, E-mail: phantn(a).fpt vn 428
Tgp chi Cong nghe Sinh hoc 10(3): 423-429, 2012
diseases is correlated with the percentage of ihe mitochondrial genome copies which carry the mulaiion. In ihis study, the RFLP-PCR was used in combination with real-lime PCR using Taqman LNA (locked nucleic acid) fluorescent probes to detect and quanlitate the percentage of the A3243G mutation. According to the PCR- RFLP analysis, the normal subjects have only 198 bp amplified band while the patients have the 198 bp band (of the umnutated copies) and 111 bp and plus 87 bp bands (of the mutated copies) Eighty two patients with the encephalopathy symptoms were screened for the presence of the A3243G mutation and 4 patients (3 male and I female, 11-15 years old) were found to carry the A.'24.iG mutation The A3243G mutation was also detected in their mother and siblings but not in their father of the palienls. The obtained results were reconfirmed by DNA sequencing. It was also found in the PCR-RFLP banding patterns thai the mother of the patients had rather faint and tiny bands of 111 bp and 87 bp compared to the 198 bp band. Using real-time PCR with Taqman LNA fluorescent probes, the MELAS patient of family II was shown to carry 73.02% of the A3243G-carrying genome copies compared lo 9.24 % in his mother This finding was in good agreement with the PCR-RFLP bandingpattcm and explained why his mother did not sho\\ any symptom of ihe disease.
Keywords: A3243G ttiulation. imtochondt lal DNA MEl IS syndrome. PCR-RFLP. real-time PCR
