Tri~u Ti~n Sang*, Tr5n van Khoa*, Dinh Doim Long**, Nguy~n Duy Bic"" CHUYEN DE: HiNH THAI H9C lrNG Dl}NG PHAN rtcn ADN PHDI THAI Tty DO DE SANG LQC DI TRUYEN TRlf6'C SINH TOM TAT Ma dau:
Trang 1Tri~u Ti~n Sang*, Tr5n van Khoa*, Dinh Doim Long**, Nguy~n Duy Bic""
CHUYEN DE: HiNH THAI H9C
lrNG Dl}NG PHAN rtcn ADN PHDI THAI Tty DO
DE SANG LQC DI TRUYEN TRlf6'C SINH
TOM TAT
Ma dau: Vi~cphat hi~nra sutontal cua ADN
phoi thai tlf do va te beo phoi thai tlf do trong
huyet tu'dng cua me da rna ra mQt hu'ong chan
doan tru'oc sinh moi b~lng bi~n phap khong can
thi~p Ban chat cua vi~c chan doan khong xam
Ianu~StIdl,!ngADN phoi thai luu hanh trong rnau
ngoGlivi cua me de chan doen va sang IQcmQt so
b~nh di truyen Boi tu'Qng va phu'dng phep
nghien ciru: 34 thai phu dang mang thai tU'tuan
thU' 8 den tuan thU' 9 cua thai kyoTrong do, 11
m~u cua gia dlnh mang gen b~nh tang san
thu'Qng th~n bam sinh, 10 m~u cua gia dlnh
mang gen b~nh Teo cd Duchenne du'QClay tU'
BGlihQCY Ha NQiva 13 m~u cua ngu'ai m~ mang
nhom mau Rh(-) lay t9i trung tam Nghien cU'u
Sinh Y du'QchQc- HQcvi~n Quan y Be kiem tra
nhom mau Rh cua thai nhi, va sang IQc b~nh
tang san thu'Qngth~n va teo cdDuchenne chung
toi thu lay ADN du'QCtach chiet tU' huyet tu'dng
cua m~ Ket qua: Boi voi sang IQCb~nh tang san
thu'Qngth~n bam sinh co 5/11 tru'ong hQp khong
phai dieu trl thuoc nua, 6 tru'ong hQp tiep tl,lC
dung thuoc den khi chQc oi de chan doan chinh
xac thai nhi co mang gen CAH hay khong Sang
IQc b~nh Teo cd Duchenne co 4/10 tru'ong hQp
am tinh v(1i gen SRY (khong mang b~nh), con
6/10 tru'ong hQp du'Qcchan doan (1tuan thU' 16
chQcoi thay 3 tru'ong hQp bl b~nh va 3 tru'ong
hQp du'dng tinh vdi gen SRY nhu'ng khong mang
gen DMD Chan doim du'Qcchinh xac 13tru'ong
*HQc vi~n Qulin Y
** Khoa Y DuO'c- Dai Hoc Q1I6cGia Hit NQi. .'
Chju tnich nhi~m chinh: TrAn Van Khoa
Email: tvkhoabio@gmail.com Dt: 01684552737
Ngay nh~n bai: 5110/2014
Ngay dlly~t bai: 16/10/2014
130
hqp nhorn mau Rh thai nhi, trong do 3 tntong hqp nhom rnau thai nhi Rr( -), con 19i 10 tru'on\; hop nhorn rnau Rh (+).
SUMMARY APPLICATION OF CELL FREE FET AL DNA ANALYSIS ON PRENATAL GENETIC SCREENING Introduction: The noninvasive nature of the detection of fetal DNA in the maternal peripheral blood represents the greatest advantage over the conventional methods of prenatal diagnosis The applications of this metrodology involve the detection of the fetal sex, diagnosis, and evaluation of the prognosis of many diseases Objects and methods: We selected the 34 pregnant women in the 8th week to the 9th wee;;'
of pregnancy including 11 samples of gene carrier parents of congenital adrenal hyperplasic (CAH), 10 samples gene carrier parents c; Duchenne muscular dystrophy (DMD) obtained at the Hanoi Medical University and 13 samples ofc mother with Rh (-) obtained at the Military Medical Academy To examine the performance
of the Fetal RHD,CAH, DMD, we use tree circulating cell-free fetal DNA (ceff-DNA:; extracted from maternal plasma Results: Fe!.'" patients screened for' congenital adrer~~ hyperplasia with 5/11 cases of non-medicatic·-' treatment, 6 cases continued dosing urr::, amniocentesis for fetal diagnostic accuracy w;:: CAH gene ,or not Screening for Duchen;-~ muscular dystrophy with 4/10 cases negative f~ SRY gene (non-medical), and 6/10 cases we:; diagnosed in the 16th week of amniocentes~,··· found3casesof the disease and 3 cases of Dt·D gene with positive SRYgene, but not DMD ger ;
Trang 2y HQC VI~T NAM THANG 11 • 56 ()~C BI~T/2014
.Accuretely diagnosed 13 cases of Rh between
mother and baby, including 3 cases of fetal Rh
jlood group (-) and the remaining 10 cases Rh
,+).
Keywords: fetal DNA; RhD; CAH, DMD;
non-interference
I £>J;\TVAN DE
Nam 1969, Walknowska va cong sir da
phat hien ra str co m~t cua t~ bao phoi thai W
do trong mau ngoai vi cua ngiroi me Error!
Reference source not found Day la phat
hien vo cung quan trong mo ra mot huong
m6i d~y tri~n vQng trong chdn do{m tnr&e
sinh b~ng bien phap khong can thi~p
Di tat bd~ sinh da va dang la m(>t vAn d~
:6n kh6~g chi d6i v&i nganh sim khoa rna con
L~U hut S\l' quan tam eua to an xa h(>i Hi~n
~v viee di~u tri cae di tat bdm sinh v~n con
i~~~ue'kho kh~ va phfre·tl;lp Tang san tuy~n
:huqng th~n bdm sinh (gQi t~t la
CAH-Congenital Adrenal Hyperplasia) lit m(>t 10l;li
t~nh di truy~n lien quan d~n r6i IOl;ln
:Drmone cua tuyen thugng th~n (cortisol
~dosterone) B~nh nay khong ph6 bi~n vai
sm1t m~c b~nh lrl 1112.000-15.000 tre em
sinh.
B~nh lo~mduong caDuchenne (Duchenne
ular dystrophy - DMD) lit m(>t trong
benh thAn kinh ca - di truy~n ph6 bi~n
~, "ai' ty l~ merim~c Ja 113.500 tre trai de
Benh DMD co d~c tnmg la thmH hoa va
-suy 'y~u ca m(>t each tuftn ti~n d~n d~n ta,n
-,:1tu vong do suy tim va b(>i nhi~m ph6i
dAu hieu Him sang ctm b~nh duqc nh~n
a giai dOl;lntre tu 2, d~n 3 tu6i C~c b~n~:
duae coi nhu mac the n~ng neu phm
xe Ian mAt kha nang tv di ll;litnr&c tu6i
dph\1 nu Rh-, lay ch6ng Rh+, cac con co
la Rh+ ho~c Rh- N~u thai nhi Rh+ trong
hong cau cua mau thai nhi eo th~ lot qua nhau thai vao co th~ me, trong mau cua me se xwlt hien khang the Rh- chong Rh+. al~n eo thai d~u tien lUQ11g khang the khang Rh+ trong rnau ella me con it,nhirng a nhirng l~n
eo thai sau IUQ11g khang th~ khang Rh+ trong mau ella ngiroi me tang len nhieu, neu thai nhi la Rh+, khang th~ se khuyech tan qua nhau thai sang gay ngung k~t h6ng cdu cua thai nhi Vi v~y, rftt d~ gay xay thai, de non, thai chet hru,
Voi cac yell cau cftp thi~t tren chung toi ti~n hanh nghien ciru va chdn hoa quy trinh sang IQc va chftn doan cae b~nh di truy~n nay qua mau m~ Thai di~m chung toi l\ra chQn la
a8 tudn Cllathai kY·
II DOl TU'Q'NG, HOA CHAT VA PHU'ONG PHAp NGHIEN C(rU
2.1. DBi tU'C~)'Ognghien cU'u
d nghien Clru Clla chung t6i, chlmg toi Iva ch9n thai di~m cac tu6i thai til' tll~n thu 8 cua thai ky d cac gia dinh co mang gen bi benh CAH, DMD va gen RhD (-) Cac m§u
mau mau ngo?i vi eLla m~ acae gia dinh c6
mang gen b~nh Tang san thlrQ'flgth?n b§m sinh Cae mfiu teo cO' Duchenne chung t6i thu
th~p auqc 10 m~Ll mall ngo{li vi ella ngtrai
m~ mang thai aeac gia alnll ell mang gen teo
t6i thu dtrqc 13 m~u mall ngol;li vi Ctta cac ph\1 nCr mang nhom mali Rh(:J man~Athai, a
tuan thlr 8 va thlr ,9 Cac mau nghlen CUll sang 19C b~nh DMD va CAH duqc chl;ly nhfm gen tren gen SRY va gen GAPDH lam n6i chlrng Cac m~u ch~n do an nhom mau Rh c(1a thai nhi se du<;ycnh~n gen RhD va gen GAPDH la gen n(>i chlrng Tftt ca cac P~\l nfr nay dugc ti~n hanh Itty 5ml mau tofm phan va
Trang 3B' 1 B' 1 ki h h
Huy~t nrong sau khi 1y Him ti~n hanh l~y 200pl huy~l nrong tach chi~t bang bQ kit cua Quiagen. ADN sau khi i.ach chi~t dircc hoc tan trong 50~t1H20 deion
Thirc hien phan irng nhan gen voi gen SRY gen RhD va gcn GAPDH noi chimg.
Cac mfill ADN sau khi tach xong a trer
se duoc nhan gen bang ky thuat Nested PCR (peR lbng) cho gen SR Y va nhan gen PCR thucmg, cho g,en GAPDH Voi trinh tv m6; cua 2 gen nay nhu sau:
ly tam 5000 vong/phllt trong 5 phut rbi lAy
huy~ttirong.
2.2 H6a chAt nghien ctru
_ H6a chAt tach cbi~t ADN: Ethanol 70%.
Ethanol 100%, Proteinase K Niroc cat, B(>
kit Quiagen
_ Hoa chAt cho peR: PCR Reaction Mix
DNA Polymerase, Reverse primer, Forward
primer, Probe, Nuac cftt klnr ion va v6 trung
2J PIIU'Ol1g phapnghien cuu
aug : ang tnn1W va IC t iroc san p am cua gen va
SRY ngoai
eTA NNNCGe AGA GGC GeeAT 239 5511C TAG NNN CCA CGC eTG eTC CGG
SRY trong
Nested CAT NNN GAG CGT CCC TGG CTT 198
57°C CTT NNN ACA GCC ACA TTT GTC
6011C
va elm trinh nhiet nhan gen GAPDH
94"C 1 phllt -Chu trinh nhi~t ch~y nhan 5511('
1 phllt 22 ehu ky
94"C 1 phllt Chu t1'i11hnhi~t eh?y nhfm 57llC
1 phut 30 elm ky
941>C 1 phllt 45 chu ky Chu t1'1nhnhi~t Ch~lYnhf111 60°(' I phllt
1 chu ky
A
' • A A
v6"i thai gian 30 phttf trong TEE IX.
132
Trang 4Y HQC VI$T NAM THANG 11· so£)~C BI$T/2014
~ 'J ,
1II.KET QUA VA BAN LU;N
3.1 K~t qua sang loc b~nh tang san
thU'Qngth~n bim sinh
Sau khi dachuan hoa diroc ky thuat tach
.::hi~tADN phoi thai W do va k5' thuat PCR
nhfm gen SR Y chung toi tien hanh thir
nghi~m sang loc benh tang san thuong th~n
bam sinh tnroc sinh atuftn tlnr 8 cua thai ky.
D6i voi cac truemg h9'P thai duong tinh vui
gen SRY sau khi nhan PCR thi kheng phai
dimg thu6c Dexamethason, cae b~nh
nhan am tinh voi gen SRY se duoc cac bac si tiep we eho dung thuoc Dexamethason d~n tuan tlur 16 cua thai ky d~ tranh tnrong hop nam hoa khi thai nhi mang gen b~nh CAH D~n twln nay cac bac si sieu am lai va choc 6i voi cac thai am tinh voi gen SRY dechAn doan co bi benh C AH hay khong de tiep we phac d6 di~u tri benh bang Dexamethason ntra hay dung thuoc khi khong m~c benh Chtmg t6i thu duQ'ck~t qua di~n di san phdm peR nlm sau:
E
I; C,ic mJu l-llllrang t'mg vai mftu CAHI, CAH2, CAH3 CAH.J, CAH5 CA/-/6 CAH7 CAH8,
t-" CAH9, CAH10,CAHl1 hI cae mftu h?nh nhan nghien etcu
~:.
t. va 6/11 trlrong hgp am tinh viri gen SRY T5t cil II truimg hgp nay dbu cho ktt qua duang
~.
[ tinhv6i gen nQichung GAPDH.
t, I
Trang 5CHUYEN DE: HiNH THAI HQC
Biz
Mii
BN
AHI
CAH2
CAH3
Til qua tren chung toi nh~n ding
trong 11 benh nhan sang lee tang san thuong
th~n bam sinh nay co 5 tnrong hQ'Pduong
tinh voi gen SR Y va khong phai dung thuoc
nira, Con 6 tnrong hQ'Pam tinh voi gen SRY
ti~p we phai dung thuoc d~n tu~n thir 16cua
thai kyoTheo tac gia Du, Zou Pan va cong Sl!
nam 2009 da sang loc va chAndoan thanh
cong cac tnrong hQ'Pmang thai b] benh tang
san thlTgng th~ bAm sinh thong qua ADN
phoi thai tl! do trong mau ngo~i vi ella mtr·
3.2 K~t qua sang IQc b~nh Teo co'
Duchenne
Cae gia dinh, dong hQ co ti~n su sinh con
bi teo co Duchenne se dugc sang IQc b~nh
nay tru6c khi sinh b~ng ADN phoi thai t\f do
trong mau ngo~i vi Clta m~ atuAl~thu 8 clm
MaBN CAH7 CAH8 CAH9
thai ky Cac gia dinh nay co ong ngoai ~ benh va truyen gen benh eho me thai nhi \i·
thai nhi nay co kha nang mang gen b~r-~ diroc di truyen nr ongngoai, D6i voi cac thzi nhi rna am tinh vci gen SRY se khong ~ benh Con nhtmg thai nhi dirong tinh voi gea
SR Y se co th~ mang gen benh va bi b~nr~ Vi~c chan doan chinh xac thai nhi co man; gen benh hay khong se duoc ti~n hanh choc 6i va la xet nghi~m atu~n thtr 16eua thai k::_ Ch(mg toi l\ra chQn duqc 10gia dinh co ng~:·
cosinh con bi Teo coDuchenne dugc gui rj Trung tam Cong ngh~ cao - D~i hQc y H3 NQi Ch(mg toi nhfm gen SRY va ge:: GAPDH nlm quy trinh tren v6i lOmb nghien ClTll nay va thu duge k~t qua nhu sau:
b?nh nht1n nghien cteu
134
Trang 6y H9 c VI~T NAMTHANG11 - 56 £)~cBI~T12014
DMDJ, DMD2, DMD3, DMD4, DMD5, DMD6, DMD7, DMDR, DMD9, DMDIO, III cue mJu
ChQc
01
thai 16 chAn
Tir hinh di~n di san pham PCR nhan gen
SRY d~ sang lee benh Teo co Duchenne
mroc sinh chung t6i nh~n th~y r~ng cac mau
len bang dep, TO net, chirng am kh6ng bi
nhi~m, clurng dirong len bang t6t Co 4/10
mau am tinh voi gen SRY, nhimg gen nQi
B· 4 B' k"
Qua bang s6 li~u tren chung t6i nh~n thay
rang trong 10 tnrong hop nay co 6 tnrong
hQ'pdtrong tinh v6i gen SRY va 4 truang hQ'p
am tinh v6i gen SRY 86n trtremg hgp am
tinh v6i gen SRY nay se kh6ng c~n ch9C 6i
d~ ch:ln doan b~nh Teo co Duchenne nay
nua Con 6 truemg hgp duong tinh v6i gen
SRY se duQ'cch9C 6i d~ ti~n himh xet nghi~m
b~nh Teo co Duchenne nay t~i f)~i h9C Y Ha
NQi va cho k~t qua 3/6 tnrang hgp mang
b~nh Teo co Duchenne 3 tnrang hQ'pkh6ng
chirng van duong chirng to chung t6i van tach duoc ADN trong huy~tnrong Con 6/10 mau duong tinh voi gen SRY va duong tinh voi ca gen nQi chirng GAPDH do v~y kh6ng
co m&unao duong tinh gift v6i gen SRY
h
Tac gia Sekizawa, A • T Kimura M Sasaki
va cong sv (1996) da chan dofm thanh c6ng B~nh teo co Duchenne tren cac m~u hfmg
duco nhan Cllaph6i thai lUllhimh trong mall ngo~i vi Cllamy
3.3 K~t qua chan doan Slf bit dAng nhom mau Rh gill'a m~ va thai nhi
Chlmg toi chAn do{mbitt dfmg nh6m mall gifra my va can thong qua ADN ph6i thai W
do trong mall ngo~i vi Clla my d6i v6i 13 tnrang hqp chAn doan va duQ'c chAn doan
Trang 7CHUYEN DE: HiNH THAI H9C
kiem tra chinh xac lai sau sinh Viec chan
doan Rh con sau sinh diroc thuc hien tai
phong 107 Trung tam nghien ciru Sinh Y
Duoc - HQcVi~n Quan Y.
RhO-FS-AAAGGGGTGGGT AGGGAA TA-3' RhD-R:5'_AGGTAGGGGCTGGACAGAA-3' San phflm phan irng cua gen RhO nay 10 366bp
Chu trinh nhie: cua phan irnu: ~
Buoc 1 94"C - 4 phLlt
I chu ky 94"C - I phllt
30 chu ky 7211C- 1phut
1chu ky
1 chu ky
-vo cung
M am dVD'ns rhl 1hZ rhl rh4 IhS rn6 rit7 ,hi 1M ,hlO rhll rh l~ Itln
}G(.hp
IO!U
Bang . ang et qua e an oan n om mau 1CLtat1aln 1
-,j
.
Rh sau
Rh Thai
,
nhan
J
(+) (+) ;
j
(-j i
(+) (+) '1
1l
1 h'
136
Trang 8Y HQC VI$T NAM THANG 11 • 56 D~C 81$T/2014
I.
Tir k~t qua tren cho chung ta thay: trong
J3 benh nhan nghien ciru nay c6 3 benh nhan
mang thai mang nhom mau Rh( -) , cho thay
khong co sir b~t d6nb nh6m mau gitra me va
'con (m~u Rh4, Rh 11va Rh 12). C6 10trong
tong s6 13m~u nghien ciru c6 thai nhi mang
nh6m mau Rh(+), 10m~u nay c6 sir b~t d6ng
nh6m mau gitra me va con Cac k~t qua nay
da duoc xac djnh llili bang test nhanh nh6m
mall Rh cua con sau sinh Theo cac tac gia
Aykut A,Onay H, Sagol S, Gunduz
C,Ozkinay F, Cogulu 0 et al (2013) nghien
cuu chftn dean nh6m mall Rh cua thai nhi
cho 29 tnrong hop me mang nh6m mau Rh
(-) thAy xUc1thien 21 tnrong hqp con nh6m
mall Rh(+)va 8 truang hgp mang nh6m mau
Rh (-) Theo HlC gia Hahn S van der Schoot
CE, Chitty LS (2008) dB thanh eong trong
chAn doan nhom mall Rh Clla thai nhi a cae
I ph\! nir mang thai co nhom mall Rh( -}
[ Akolekar Finning, Kl1ppl1samy va e¢ng s\r
t
f (20II) ch~ndOlin nhom mau Rh thai nhi cho
cac ba m~ mang nhom ImilJ Rh( -) co thai tu
I tll~n thu 11 d~n tu6i thai 13 tu~n eho 502
! truang hqp trong do 496 trubng hgp ch§n
doan chinh xae nhom mau eua thai nhi vai d¢
nh~y la 98.8% trong 338 tnrang tra lai
duong t1nh thi co 6 truang hqp la duang tinh
gia Bombard 1 Akolekar, Farkas va c¢ng s\r
(2011) da nghien elm eh§n doan nhom mall
Rh Clla thai nhi qlla mall m~ tren 207 tnrcmg
hqp k~t qua 140truemg hqp duang t1nh mang
nhom mall Rh(+) nhlmg trong do co 2 tnrcmg
hqp duong Hnh gia va 67 truang hgp am tinh
trong d6 co 4 tnrbng hqp am tinh gia
'" A
IV KETLU~N
Trong nghien elm nay chtmg toi dll sang
19C dugc 11 truang hqp gia dinh mang gen
b~nh tang san thuqng th~n b§m sinh, 10 gia
: dinh mang gen Teo co Duchenne va chfm
doan duoc nh6m mau ella thai nhi a 13 phu nCrmang nh6m mall Rh (-) dang mang thai a
tuoi thai tuan thir 8va thir 9
TAl L1~UTHAM KHAo
1 Akolekar, Kirstin Filming Ramesh Kuppusamy et al (2011) Fetal RHD Genotyping in Maternal Plasma at 11-13 Weeks of Gestation. Fetal Diagn Ther
;29:301-306.
2 Aykut A,Onay H, Sagol S, Guncluz C,Ozldnay F, Cogulu 0 et al (2013) Determination of fetal rhesus d status by maternal plasma DNA analysis. Balkan f A4ed(iellel.16(2):33-8
3 Bombard, Akolel,ar, Fnrkus et HI (2011) relal RHD gl:notypc detection Ii'om circulating ccii-free fetal DNA in maternal plasma in non-sensitized RhO negative
women frenal Diagl1.31: 802-808
4 Du ••J, ZOli X, Pan Y ct al (2009) Noninvasive prenatal diagnosis of congenital adrenal hyperplasia in cell -free fetal DNA with cold-peR. de ES Ramos, P·395
5 Hahn S, van der Schoot CE, Chitty LS (2008) Non-invasive prenatal diagnosis and determination of fetal Rh statlls Semin Fetal
Neollala/ Med. 13:63-8.
6 Seldzawa A., T Kimura, M Sasaki, S Nakamura, R Kob~lyashi, and T Sato (1996) Prenatal diagnosis of Duchenne muscular dystrophy using a single fetal nucleated erythrocyte in maternal blood.
Neurology 46: 1350-1353.
7 Walknowslm, J., F A Conte, and M M Grumbach (1969) Practical and theoretical impl ications of fetal-maternal lymphocyte
transfer Lancel, I: I II 'i-I 122.