KHAo SAT va DINH LUQ'NG ADN PHOI THAI TV DO TRONG HUYET TUaNG CUA Mf: (J cac TU.AN TVDI THAI. TV' 6 TVAN DEN 22 TVAN BANG PHV'aNG PHAP REALTIME PCR

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1 CHUYEN DE: HiNH THA H9C KHAo SAT va DNH LUQ'N ADN PHO THA TV DO TRON HUYET TUaN CUA Mf: (J cac TU.AN TVD THA "...","" '", ~, TV' 6 TVAN DEN 22 TVAN BAN PHV'aN PHAP REALTME PCR,.. TOM TAT (). Ma dau: Vi~e phat hi~n ra su' ton t(,licua ADN phoi thai tl/ do va te bao phoi thai tl/ do trong huyet tu'dng ella me da rna ra mqt hu'ong chan doan tnnrc sinh moi bang bi~n phap khong can thi~p. De djnh u'c;.9 du'c;cadn phoi thai tl/ do trong huyet tu'dng cue me chung toi su dl,mg phu'dng phap Realtime -PCR dl/a tren ky thu~t TaqManPCRRealtime co SU dl:mg cac dau do huynh quang. Ky thu~t nay cho phep quan sat du'c;cec tin hi~u huynh quang cue cac gen dich va biet du'c;cso ban copy cua chung a tung chu ky nhan gen. (). Doi tu'c;ngnghien oru: chung toi su d,lngla 6 the: phu dang mang thai tti tuan th(j'6 den tuan th(j'22 clla thai kyo().ket qua: Nong dq ADN tl/ do tuan th(j'6 va tuan th(j'7 co mqt so tru'ong hc;pkhong phat hi~n ra sl/ co m~t clla gen DYS4chi phat hi~n du'c;cgen nay trong tat ca cac rl'~u du'dng tinh vao tuan th(j'8. Ket qua chung toi thu du'c;ccling chi ra rang nong dq ADN plioi thai tl/ do trong mau ng9i vi clla m~ tang Can theo tuoi thai va ph,! thuqcvao tting ca the. SUMMARY NVESTATON AND CELL FREE DNA QUANTFCATON N MATERNAL PERPHERAL PLASA AT THE ESTATON FROM 6 WEEKS TO 22 WEEKS USN REALTME per * HQc vi~n Quan Y ** Khoa Y Dugc - Dl;liHQc Qu6c ia Ha N<)i Chju tnich nhi~m chinh: TrAn Van Khoa tvkhoabio@gmail.com Dt: Ngay nh~n bili: 5//2C4 Ngily duy~t bili: 6//24 38 Tri~u Ti~n Sang*, Tr~n Van Khoa*, Dinh Doan Long** (). ntroduction :Thc discovery of the existence of fetal cells in maternal peripheral blood has opened up a new direction for the early diagnosis of congenital anomalies by means of non-interference. To quantify fetal DNA circulating in maternal blood we used realtime PCR method based on TaqMan PCR technique using fluorescent probes. This technique allows to observe the fluorescence signal of the target gene ADN for us to know the number of copiesof each target gene at each time point of the PCR process. (). Objects, chemicals ADN research methods: We shall select the 6 pregnant women in the 6th week to the 22th week of pregnancy. Along with two negative control sample of 2 ADN pregnant women not yet having sex once 5 ml of blood is taken ADN conduct split ADN separated plasma free plasma DNA as the other sample. (). Results: Free DNA concentration at week 6 and week 7, there are some cases not detected the presence of the DYS4 gene was only detected genes in all positive samples in week 8 our results obtained indicated that the concentration of free fetal DNA in peripheral mother blood and increaseswith gestational age, depending on the individual. Keywords: fetal DNA; reatime-pcr; noninterference w ".... D~T VAN DE Vi~e phat hi~n ra ADN phoi thai til do trong mall ngo<;livi eua my la mqt buac phat tri~n mai eua khoa hqc, rna ra mqt hu6ng mai trong ehfm dmin di truyb truae sinh b[tng bi~n phap khong can thi~p. N6ng dq ADN xudt hi~n trong mall ngo~i vi cua my a cae thai di~m khae nhau la khong theo mqt

2 Y HQC V$T NAM THAN - 56 )~C B$T/24 ~ :. lu~t, do vay d~ t3i iru va va chon diroc ::~'ridi~m lfiy mau ngoai vi cua me d~ tach! _!J);\ ph6i thai tv do ia co mot Y nghia rfit 6 :_-,)ngchan doan va sang loc truce sinh, tranh "':'-=ljyco chan doan nham am tinh gia khi -~ i.bng tach duoc ADi.-.phoi thai. :~ f)~ dinh hrong duoc ADN ph6i thai hru bh'h trong mau me chung t6i dtng phuang ;hip realtime PCR j\!,a tren ky thu~t PCR T:!qMan su dvng dau db hu5rnh quang. Ky ±~t nay cho phep quan sat cac tin hi~u t;l5-nh quang cua cac gen dich va eho ta bi~t :C'.!yC s5 bfm copy cua timg gen dich ( ttmg ;!Kri di~m cua qua trinh PCR. Do v~y chung :.3th\!'c hi~n nghien CUll nay vai m\c tieu:?hat hi~n va dlnh tuqng ADN phoi thai W do?ng huy~t tuang cua m~ b~ng phuang phap ::~ealtime-pcr, xac d!nh thai di~m ADN phoi :hai trong mau m~ du tan d~ tach duqc ADN. ' # " J. ) TU'Q'N, HOA CHAT VA PHU'O'N PHAp NHEN C(YU 2.. DAitU'qng nghien cu'u a nghien CUll Clla chung toi, chung toi ~),rachqn thai di~m cac tubi thai ti tuftn thu 6 c~n tuftn thu 22 cua thai ky eua 6 mau nghien cuu. a m6i tuftn thai ti tu~n thu 6 d~n ru~n thu 2 la 6 mau duqc lay Sml mrm trong tat ca cac tu~n nay, cac mau duqe xac dinh chinh xac duang tinh vai gen DYS 4 va duang dnh vai gen DYS4 ( tuftn thu 2 Juqc h,ra chqn ti~p thea d~ lay mau djnh tuan, Cac m~u nghien ciru nay vua duoc chay nhan gen tren gen DYS 4 vira duoc dinh luong s6 ban copy tren gen DYS4 b~ng c~p m6i tren nhiern s~c th~ Y (gen DYS 4) va gen APDH lam nqi chirng. TAt ca cac phu nft nay diroc ti~n hanh lfiy 5ml mau toan ph~n va ly tam 5 vong/phut trong 5 phut r6i lay huy~t tuong. Cung voi 2 mau d6i chung am lit 2 ncr giai chua mang thai va chua quan h~ tinh d\lc l~n nao duq'c lay 5 ml mau va ti~n hanh tach huy~t tuang va tach ADN w do trong huy~t ttrang nhu cac mau nghien Clru khac. Olc mau nghien Clru duq'c lay t~i Trung tam nghien clm Sinh Y duq'c hqc quan S\T - HQc vi~n Quan y Hoa chat nghien cu'u H6a ch t tach chi~t ADN: - Ethanol 7%, Ethanol %, Proteinase K, Nuac ck B<) kit Quiagen. H6a chat cho per: PCR Reaction Mix. DNA Polymerase, Reverse primer. Forward primer,?robe, NtTac cat khu ion va vo trtmg. Thi~t bi may moe: May ~c 6n nhi~t (Eppendorf-f)lfc). May i tam cao t6c (Hettich-f)lre), Buf,ng thao tac per (USA), May Realtime PCR (Rotoen - Quiagene), May PCR ABl 97 (Applied Biosystem), Tl h6t hor. chat (Han Qu6c), May do n6ng d<) ADN (Nano Drop-USA) PhU'o'ngphap nghien cu'u: Huy~t tuang sau khi ly tam ti~n hanh lay 2ml huy~t tttong tach chi~t b~ng b<)kit cua Quiagen. ADN sau khi tach chi~t duq'c hoa ;u<;mgnong Q ( cac twin thai 3 en 22 tan trong SOul H2 deiof'-. Ȧ da d CCCCACACACATCACT DYS4 TCATTAATCAAAT APDH -F TACC F TCC CCTATCCCACTTT DYS4 CCCCCTATACCCTAC - APDH -R ATT R AATTATT (HEX)_AAAACT A Probe (FAM)_ TCTCCTCCCT Probe APDH AAACACAACTT DYS4 "CTCT(T AMRA) C (BHQ) 39

3 CHUYEN DE: HiNH THA HQC Hinh. Hinh anh trinh W doan gen DYS 4 CCCCCTATACCCTACAATTATTCATTCTCAACAAAACATTAATTCATA ACTTACTACTTCCCTCTATCTCCTCCCTACTCTCTACTCTTCCTA AAAATCAATTTACTTAATCCCA Hinh 2. Hinh anh trinh tu doan gen APDH CCCCACACACATCACTTACCTTCTCCCACTCCTATTTCTAAAAACTA AAACACAACTTCAAATCAAACCCTACTA Hlnh trinh W doan gen DYS 4 minh hoa vi tri g~n m6i cua gen DYS 4 bleu th] bang mau xanh, con vi tri duoc co mall do la vi tri g~n cua ddu do probe. Tuong W nhtr v~y tren gen APDH vi tri duoc boi mau xanh ii vi tri gilnm6i cua gen APDH va vi tri boi mau do la vi tri g~n ddu do probe. Qua trinh chay Pt:R duoc thuc hien theo chu trinh nhir sau: Phan irng PCR voi tong th~ tich phan ung lit 25ul trong do per master mix l Oul: Primer DYS4 (F, R, Probe): (,25:,25:,5)ul va primer APDH (F, R, Probe) : (,25:,25:,5)ul; mroc: 8ul. Chu trinh nhi~t elm ky (95 C trong phut); 55 chu ky (95 C - phut, 6 C - phut), eho may doc huynh quang i:t nhiet dq 6 (' trong phut, XL' y s6 lieu bang phan mem chuyen dung ella may chay relatime-pcr Rotoen - Quiagen cia tich hop sfin va cac thuat toan th6ng ke tren ph fin mem phan tich 56 li~u Excel Microsoft Otice. ",, "\. KET QUA VA BAN LUA.N 3.. K~t qua sau khi eh~y du'()'ogehu n eua gen DYS4 va gcn APDH o' m~u ehuan '",.. pba loang o' cae nang dq 4; 3; 2 c-~- ----,..J l_~~~~-;."qi~~?l.-;±;!s6-..._ 8:l2!!2!!i!! :,' '-_. gr- Hinh 3: Hlnb :lub plliill dcb duo'ug cbuiu cua geu APDH Hiub 4: lfulb ;lllh phau tich Quang ChU3l cua geu SRY TiT k~t qua hinh anh tren nh?n thfty r~ng tin hi~u Clla gen APDH len d<rp, duong chu~n t6t co h~ 56 R=,99997; R2=O,99993, M s6 tmmg quan rat cm!. D~ tinh dtrq"cs6 ban copy Cl3 cac mflll nghien cuu cua gen APDH thea cong thlrc: conc= OA(-.2xCT +.386) CT = -5.2Xlog( cone)

4 f: y HQCV~T NAM THAN - 56 D~C B~T/24 ~; ~.... en DYS4 voi tin hieu huynh quang mall xanh diroi day ciing cho tin hieu t6t va duong ~-.:~".dnlen dep voi ~~ s6 urong quan chat R= va R~= ~.' Chung ta co the tinh so ban copy cua gen DYS \ 4 theo cong thuc sau: cone= "lo.27"c +.647) C = -4.6 O:<log(conc)+ 4~3.86 Vad~\ b: =Qy (--- ~. Cong thirc tinh s6 ban copy/ ml mall: _. Vpe! Ve:~. Trong do Q lit gia tri tinh duoc ~. tr.!o cong tlurc duong chuan, Vadn lit th~ tich m~u duoc dua vao phan irng PCR, Vpcr fttbng ~.d:~tich phan irng PC!t, Vext th~ tich huy~t nrong d~ tach ADN phoi thai nr do. ~~ 3.2. K~t qua chgy iljnb.u'9ngcac 6 mfiu huy~t.tuong phu nu' mang thai ' cac tufin ~tiltlni' 6 d~ntuan 2 cua thai kyo ~" Ap dung quy trlnh tren d~ ti~~ ha~hdjnh u,?"g ki6m ra nbng do ADN ella thai nhi tro~g F'. ;:~yet tuang nguai m~ tren 6 mau ba~g c~p moi va probe tren gen DYS 4, clmg v6'i 2 mall ~ :.,:'~i chung am chung toi thu duq'c cac ket qua sau: ~: Bang : Bang k~t qua s6 ban copy va n6ng et(l gen DYS 4 va n6ng d<)gen APDH a ~. de luan 6 d~n 2 cua thai ky ~::- ri. 'y TS ~ Ty ~ T)'~ Ty ~ ~ s6 (cn (cn S6 Stl stl '5' ~ S6 gc gen gc ban b;in han bun biill )...S O\'S hiln gcn ban l)rs lys DrS eupy eupy cupy copy cupy :" (C gcn gell.:" (i..\' (ia' DYS A') )YS DYS )\S J)\S )YS Drs )J > J)J > ~ 4 hlli 6unll 7 tuan H lutlll ) utili Olurtn X : ~ D3 2 OJX , lJ8 5 (l (l ''l{ 2.' J7:'i.j j:'i :'i H ll() 23.X lJ X (J.O!! ~ ~ ~ ~----~--~----~--+---~~~ ~--~.9 b 38 o o (Ull, r s :~, 53 X.6X 2.!) (i (l i ;:: '\ 34 () i O.oJ (l.o (J i.. J r --'-_--l--~---' l---l----,---l--l---l.--l----.l--l l llunn 2uAn ) ) ,43!l OJl9 RO (; O.D.3 4

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6 y HQC V~T NAM THAN 56 D~C B~T/24,;,...,. o.oo,....,, 3.7 Bang qua i~u chay tren 6 phu ncr mang thai tir tudn thrr 6 dsn tudn thu 2 cua thai ky va n6ng dq ADN phoi thai W do trong mau ngoai vi cua me a cac tu~n thai khac nhau. Co 3/6 twang hop mang thai nam. Tir bang k~t qua tren chung toi nh~n th~y nong dq ADN cua thai ky ( tirng nguaj khac nhau la r~t khac nhau va co S,l' bi~n thien tang d~n n6ng dq ham hrong cua ADN ph6i thai tv do theo S\l' tang dau ella tu6i thai. Tir bang nay chung toi ciing th~y r~ng n6ng dq trung blnh ctta ADN phoi thai W do trong mau ngo{li vi ctta mt( a cac tu~n ttr tuan thtr 6 d~n tu~n thu 2 nay co dq l~ch ehll:n r~t 6n. va ian han ca ghi tri tru'g blnh di~u nay chung to n6ng dq ADN phoi thai nay dao dqng ( mqt khoang r~t on nhu a tudn 6 ctta thai ky n6ng dq trung blnh ella ADN ph6i thai W do trong mau ngo{li vi clta mt( la 5.69 ± ban copy/mt. hay a tll~n thir 2 la ± 742,55 copy/mt. Theo tac gia Lo YMD. Tein MSC, Lau TK va cqrtg s\t (998) nghien cuu tren 27 ph\! nil' mang thai a quy d~u etla thai ky, qlly thtr 2 va qlly thu 3 Clla thai kyo cac ph\l nil' nay mang tha~ b~ng ky thll~t YF. cho thdy a quy clla thai ky s6 han copy clla gen oys 4 trong hlly~t tuang Clla m~ vao khoang 4, d~n 58, ban copy/ml di~u nay ciing phu hqp voi nghien Ctru ella chung toi a tudn thu 6 clla thai k5'. m~c du ( cac tuan ti~p theo nghien cuu ella chung toi s6 him copy co cao han so voi tac gia nay. Cung theo tac gia Lo YMD. Tein MSC. Lau va S\' ( ) a giai doan muon quy thir 3 cua thai ky s6 ban copy cua gen DYS 4 trong huyet nrong dao dqng tir 76.9 d~n 769 copy/ml. Nghien ciru cua Costa M. Benachi A, autier E va cong str narn 2 cung da dung ky thuat Realtime PCR d~ chimg minh r~ng ADN phoi thai W do t6n tai trong mall ngoai vi ella me. Tac gia Alen Chan K.C. va cs. (24). tach chi~t va nghien CUlln6ng dq va ph an b6 kich thuoe ADN ph6i thai W do trong mall mt( cho thfty n6ng dq ADN trong mall m~ chi~m 3.4-6,2% so voi ADN t6ng s6. tac gia nghien Ctru trtn 56 ph\! nti' mang thai ttr tll n thu 7 d~n tu~n thtr 6 clla thai ky Trong nghien cuu Clla chtmg t6i n6ng dq ADN phoi thai a cac tll~n thai dao dqng tir,52% a 6 tufm tu6i thai d~n 3,7% a 2 tll~n tu6i thai. Chungwen Wei. Devereux N. Saller va C9ng s\r (2) du dinh ltrqng ADN tv do ctla ph6i thai trong huy~t tucmg ella m~ etla 3 ph\! nft mang thai ttt tll~n thit 9 d~n tu~n thtr 7 Clla thai ky thdy co 9 trtt{'tng hqp dmmg Hnh vai gen DYS 4, s6 ban copy clla gen nay dao d9ng ttt 3.8 d~n 29 copy/ml, v6'i n6ng d() gen DYS4 nay trong hlly~t tuang la ttt.28 d~n49.7%. voi 2 truang hq'p n6ng d9 r~t cao la 29,2% bi hqi chtmg Down. va m9t tnrang hqp n6ng d9 cao d9t bi~n 47.69% nhung bq nhi~m s~c th~ binh thuang 46XY. 43

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8 y H9c V~T NAMTHAN' 56 f)~c B~/24 Tir bang k~t qua so sanh tren cluing toi nhan thay k~t qua ella cac tnrong hop nghien dill tir tuan thir 8 d~n tufin thir 2 cua k9 thuat Nested per, Realtime per. sieu am tufin thir 6 va sau sinh la phil hop nhau. Voi oq chinh xac la % va d9 nhay la %. Voi tuan thai 6 co 4/6 tnrong hop chay Realtime - per khong phu hop voi sieu am va sau sinh do v~y dq o~c hi~u Clla ky thu~t nay la % va OQ nh\ly la 93,33% va 7/6 truang hqp ch~y Nested per khong phu hqp voi k~t qua sieu am va sau sinh do v~y oq d~c hi~u Clla ky thu~t nay la % va oq nh~y la 88,33% K~t qua bi~n thisn nang dq ADN phoi thai tl}' do trong mau ngo~i vi ciia me D~ kiern tra xem nong dq ADN phoi thai W do trong rnau ngoai vi cua me bien 6i theo tu~n thai ella thai nhi chung t6i tien hanh dinh luong cac mftll nghien ciru nay a cac tuan tir 6 tufm o~n 22 tuan tuoi. K~t qua cho thay n6ng oq bi~n thien nong dq ADN phoi thai W do trong ml huy~t tucmg co s\r thay obi gilra cac tuftn va n6ng oq nay co xu huang tang len thea tu6i thai, m~c dll trong tlmg trtrang hqp n6ng dq nay co hk giam nlmng sau do xu huang: tang tra l~i 6 cae tuan sall. -i... 5:':.:-:' ~:':,:':' l 3:.;" :;: ~:':.:': ' o ::.::., \", l-ongdq,.,.,, '!,..!' - '.' <) z lu'imthai Hinh 6. Biel! do bien tbien liang dq AD:'i til do clia tlwng hq'll~~ 5:'; ';'. 6n~do 682J822 lu~ntllai 5 SO,,})24QB22 Tuanlilai Hinb i. Bieu do bien hiellnon~ dq AD:'i J do ctia tlirn~ hqp :96 Hinh 9. BieD bien bien noug dq AD:'i :\t do ctia luintg htrp lcngo ooc -:: oo = t,,,, ~ ~ ~ ;. :: ~~!,...! \,, ', Tuanthai 6) 3,) ) (,2Cg \8:)22(; TU3!ha: Hinb S.Biell do bien hien nilllg t9 AD:"! do ella limg bqp 9" Hillb. Bieu biellihien non!!.do.\d:-; t~do ctia mim!!.hll 3 45

9 Tir hinh anh cac bieu db v~ S\Tbien thien nong dq ADN phoi thai chung toi nhan thay nong dq ADN phoi thai t\t do trong mau ngoai vi ella my bi~n thien thea tung d6i CHUYEN DE: HiNH THA H9c nrong va co tinh eh~t ca th~. Theo tac gia Lo YMD, Tein MSC, Lau TK va cong S\T(998) dll nghien cuu S\Tbien d6i ADN tv do trong huy~t tuong ella my ella 2 tnrong hop lam thu tinh bng nghiem VF a cac tu~n thai ttr tu~n d~u d~n 3 7 tu~n. K~t qua cho thdy neng dq ADN ph6i thai bi~n d6i thee tung ca th~ va ding co xu huang tang sb ban copy khi tu6i thai cao d~n. Tren bieu db bien thien nbng dq ADN tv do trong huy~t tuong cua me a tren chung t6i nh~n thdy vai k~t qua tuang tv S\l' bi~n thien nay d~c ttung cho tung ca th~ va co xu huang tang len khi tu6i thai tang. V. KETLU~N Trong nghien clru nay chung t6i da ph~\t hi~n va dinh lugng dugc nbng dq ADN W do Clla thai nhi luu hanh trong huy~t tuang clla m~ b~ng phuang phap realtime per. Khao sat duq'c nbng dq ADN ph6i thai tv do trong mau ngo~i vi cua m~ a cac tu~n tu6i thai khac nhau. Nghien ClrU miy chi cho chung toi thay duq'c s\'bi~n thien nbng dq ADN ph6i thai tlj do a tirng ca th~ lil kh6ng gibng nhau. Xu huang nbng dq ADN phoi thai tv do t~ng dan theo tu6i thai, thai diem tu6i thai thich hqp nhdt chung t6i lja chqn la 8 tu~n tu6i. d thai diem tu6i thai nay nbng dq ADN du 6n d~ co th~ tach dugc ADN phoi thai va co th~ (rng dung d~ sang loc va chfm doan benh di truyen chinh xac. TAl L~UTHAM KHAo. Alen Chan K.c. et al (24). Size Distribution of maternal and Fetal DNA in maternal Plasma. Clinical Chemistry. 5: pp Costa JM, Benachi A, autier E, Jouannic JM, Ernault P, Dumez Y (2). Firsttrimester fetal sex determination in maternal serum using real-time PCR. Prenat Diagn. 2: Chungwen Wei,Devereux N. Saller va cqng sl}' (2). Detection and Quantification by Homogeneolls PCR of Cell-free Fetal DNA n Maternal Plasma. Clinical Chemistry. 47. No. 2: Lo YMD, Tein MSC, Lnu TK, Haines CJ, Leung TN, Poon rmk, et al (998). Quantitative analysis of fetal DNA ill maternal plasma and serum: implications for noninvasive prenatal ciagnosis. Am J Hum enet, 62: Lo, Y. M., P. Patel, J. S. Wainscoat, M. Sam pietro, M. D. illmer, and K. A. Fleming () 989). Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancel, 2: olotukhina T.V. et al (25). Analysis of Cell-free Fetal DNA in Plasma and Serum of Pregnant Women.. His/ochem C:vtochem, 53: