Nghiên cứu xác định người lành mang gen và ứng dụng chẩn đoán trước sinh bệnh Hemophilia A

Header Page of 161 B GIO DC V O TO B Y T CễNG TRèNH C HON THNH TI TRNG I HC Y H NI TRNG I HC Y H NI Ngi hng dn khoa hc: TS Trn Võn Khỏnh PGS.TS Nguyn Th H BI TH THU HNG Phn bin 1: nghiên cứu xác định người lành mang gen ứng dụng chẩn đoán trước sinh bệnh hemophilia a Chuyờn ngnh Mó s : Húa sinh : 62720112 TểM TT LUN N TIN S Y HC Phn bin 2: Phn bin 3: Lun ỏn s c bo v trc Hi ng chm lun ỏn Tin s cp Trng hp ti Trng i hc Y H Ni Vo hi gi ngy thỏng nm 2014 Cú th tỡm hiu lun ỏn ti: - Th vin Quc gia Vit Nam - Th vin Trng i hc Y H Ni H NI - 2014 Footer Page of 161 - Th vin Thụng tin Y hc Trung ng Header Page of 161 T VN Tớnh cp thit ca ti Bnh mỏu khú ụng hemophilia A l bnh di truyn alen ln liờn quan n gii tớnh, gen bnh nm trờn nhim sc th X khụng cú alen tng ng trờn nhim sc th Y, vy ngi m mang gen bnh cú th truyn bnh cho 50% trai v truyn gen bnh cho 50% gỏi Bnh cú th di truyn qua nhiu th h v cú nhiu ngi mc bnh cựng mt gia ỡnh Bnh nh hng n tõm sinh lý, th cht tr nh v l gỏnh nng cho gia ỡnh v xó hụi Ti Vit Nam, c tớnh cú khong 6000 ngi b bnh hemophilia v khong 30.000 ngi mang gen bnh hemophilia Mc dự thi gian qua, cụng tỏc chm súc bnh nhõn hemophilia A ti Vit Nam ó cú nhiu tin b, s lng bnh nhõn c chn oỏn v qun lớ ó tng lờn ỏng k, nhiờn mi ch chim cha ti 30% tng s ngi b bnh v a s ngi mang gen bnh cha c chn oỏn v qun lớ Vic phỏt hin ngi lnh mang gen bnh úng vai trũ quan trng cụng tỏc t di truyn v chn oỏn trc sinh cú th giỳp ngn nga sinh b bnh v gim t l mc bnh Mc tiờu ca ti: Phỏt hin ngi lnh mang gen bnh cỏc thnh viờn gia ỡnh bnh nhõn hemophilia A ó xỏc nh c t bin gen F8 ng dng k thut I-PCR v gii trỡnh t gen chn oỏn trc sinh cho nhng thai ph cú nguy c cao sinh b bnh hemophilia A í ngha khoa hc v thc tin ca ti: Phỏt hin ngi ph n mang gen bnh bng cỏc xột nghim thụng thng nh xỏc nh hot tớnh yu t VIII mỏu cũn gp khú khn vỡ hot tớnh yu t VIII ca h khụng gim hoc gim ớt, cú th dao ng t 50 150%, ch cú khong 10% tng s nhng ph n ny cú hot tớnh yu t VIII huyt tng A (c.1010 27G > A) cng vi a hỡnh STR intron 13 v 22 Phng phỏp phõn tớch liờn kt thc hin nhanh, tng i r tin, ỏng tin cy phỏt hin cỏc trng hp b bnh hemophilia A ph h gia ỡnh cú nhiu ngi b bnh Tuy nhiờn, phng phỏp phõn tớch liờn kt khụng th phỏt hin c ngi lnh mang gen cỏc gia ỡnh cú t bin mi phỏt sinh quỏ trỡnh hỡnh thnh giao t Chn oỏn trc sinh bnh hemophilia A Kt qu phõn tớch gen cho bit thai ph cú mang gen bnh hay khụng l yu t sng lc quan trng nht Sau quỏ trỡnh phõn tớch sng lc, nhng thai ph cú nguy c cao sinh b bnh hemophilia A c tin hnh ly mu t bo thai nhi phõn tớch v xỏc nh t bin Cỏc nghiờn cu v ngi lnh mang gen bnh Vit Nam Cỏc nghiờn cu v cn bnh ny nc ta mi ch trung ch Footer Page of 161 Header Page of 161 yu vo t l mc bnh, c im lõm sng, cn lõm sng, iu tr v S thit k nghiờn cu: nhng tỏc ng tõm lý ca bnh i vi bnh nhõn v thnh viờn gia 50 bnh nhõn hemophilia A ó xỏc nh t bin gen F8 ỡnh Nh vy cũn ng vic ng dng cỏc k thut sinh hc phõn t vic phõn tớch, phỏt hin t bin gen F8, ngi lnh mang gen Xõy dng 50 ph h bnh v chn oỏn trc sinh Chng 2: I TNG V PHNG PHP NGHIấN CU 2.1 i tng nghiờn cu Gm 50 gia ỡnh bnh nhõn hemophilia A ó c xỏc nh t bin gen F8 (ti Trung tõm nghiờn cu Gen Protein, Trng i hc Y Phỏt hin ngi lnh mang gen bnh chc chn (M, ch, em gỏi ) Khụng mang Mang thai Xột nghim gen F8 - K thut I-PCR(o on int22) - Gii trỡnh t gen (t bin im, mt on nh) H Ni) bao gm: - 50 ngi m bnh nhõn Xỏc nh ngi cú nguy c cao mang gen bnh (M, ch, em gỏi ) Khụng xột nghim gen - 116 thnh viờn n (b ngoi, b h, bỏc gỏi, dỡ, ch, em gỏi ) cú cựng huyt thng vi bnh nhõn Mang gen bnh Khụng mang gen - 12 thai ph/166 thnh viờn gia ỡnh bnh nhõn ú 6/12 thai ph l ngi lnh mang gen bnh, ang mang thai tun thai th 12-18 - 20 ngi (10 nam, 10 n) khe mnh, tin s gia ỡnh khụng cú T di truyn v phũng bnh Khụng chn oỏn trc sinh Khụng t bin ngi mc bnh di truyn dựng chun húa k thut v lm mu i Cú t bin chng cựng vi mu nghiờn cu thc hin cỏc k thut sinh hc phõn t phõn tớch gen Cỏc i tng nghiờn cu c ly mu nghiờn cu: Mỏu tnh T ỡnh ch thai Chn oỏn trc sinh T gi thai mch cú chng ụng EDTA, dch chc i (thai ph) 2.2 Phng phỏp nghiờn cu: S dng phng phỏp nghiờn cu tin cu v mụ t ct ngang 2.3 a im nghiờn cu + Trung tõm nghiờn cu Gen Protein, trng i hc Y H Ni + Thi gian t 1/2012 6/2014 Footer Page of 161 2.4 Quy trỡnh v cỏc k thut s dng nghiờn cu - Phõn tớch ph h gia ỡnh cỏc bnh nhõn - Tỏch chit DNA tng s t mu nghiờn cu - Phn ng PCR khuch i 26 exon gen F8 - K thut gii trỡnh t gen phỏt hin t bin im v mt on nh - K thut I-PCR (Inversion-PCR) xỏc nh t bin o on intron 22 - Nuụi cy t bo i 2.5 ti tuõn th cht ch o c nghiờn cu Y hc Header Page of 161 Chng 3: KT QU NGHIấN CU 3.1 c im ca cỏc i tng nghiờn cu Bng 3.1 Phõn b i tng nghiờn cu theo quan h vi bnh nhõn Quan h vi bnh nhõn n T l % M bnh nhõn 50 30,1 Cỏc thnh viờn n (b, bỏc, dỡ v ch em gỏi) 116 69,9 Tng 166 100 Nhn xột: Trong 166 thnh viờn n ca 50 gia ỡnh bnh nhõn hemophilia A cú 50 ngi m, chim t l 30,1% v 116 ngi bao gm b, bỏc, dỡ v ch em gỏi ca bnh nhõn, chim t l 69,9% 3.2 Kt qu phỏt hin ngi lnh mang gen bnh hemophilia A 3.2.1 T l phỏt hin ngi lnh mang gen bnh qua phõn tớch ph h Bng 3.2 T l ngi lnh mang gen bnh phỏt hin c da vo ph h Tỡnh trng mang gen Mang gen Cú nguy cao Tng bnh bt buc mang gen bnh (n,%) Thnh viờn gia ỡnh (n,%) (n,%) M bnh nhõn 20 (40%) 30 (60%) 50 Thnh viờn n khỏc 23 (20%) 93 (80%) 116 Tng 43 (26%) 123 (74%) 166 Nhn xột: Phõn tớch ph h cho thy 20/50 ngi m (chim t l 40%) v 23/116 thnh viờn n (chim t l 20%) mang gen bnh bt buc; 30/50 ngi m (chim t l 60%) v 93/116 thnh viờn n ngi cú nguy c cao mang gen bnh (chim t l 80%); 43/166 ngi chc chn mang gen bnh (chim t l 26%) v 123/166 ngi cú nguy cao mang gen bnh (chim t l 74%); 3.2.2 Kt qu tỏch chit DNA DNA tng s t mỏu ton phn c tỏch theo phng phỏp phenol/chloroform Nng DNA tng s tỏch c cú giỏ tr t 150 1200 ng/l v tinh sch ca cỏc mu t yờu cu vi t l mt quang o c bc súng 260/280 nm luụn nm khong 1,8-2,0 Footer Page of 161 3.2.3 Kt qu phỏt hin ngi lnh mang gen bnh bng cỏc phng phỏp phõn tớch gen 3.2.3.1 Kt qu phỏt hin ngi lnh mang gen F8 t bin o on intron22 559 bp 500 487 bp M: Marker 100bp : M b nh nhõn (II3) 1: Ng i bỡnh th ng (ch ng õm) 4: Bỏc gỏi b nh nhõn (II1) 3: B nh nhõn (III4) 5: Dỡ b nh nhõn (II5) 6: Ch gỏi h b nh nhõn (III2) Hỡnh 3.4 Hỡnh nh multiplex PCR xỏc nh t bin intron 22 ca gia ỡnh bnh nhõn HA02 Nhn xột: DNA ca ngi bỡnh thng v trớ ging s cú bng kớch thc tng ng 487 bp DNA ca bnh nhõn HA02 b t bin o on intron 22 ging s cú bng kớch thc tng ng 559 bp DNA ca ngi m, bỏc gỏi v ch gỏi h ca bnh nhõn ging s 3, v cú vch kớch thc tng ng l 487 v 559 bp, nh vy ngi m bnh nhõn (II3), bỏc gỏi (II1) v ch gỏi h ca bnh nhõn (III2) l ngi lnh mang gen bnh trng thỏi d hp t DNA ca ngi dỡ ging s cú bng kớch thc tng ng 487 bp ging ngi bỡnh thng, nh vy dỡ ca bnh nhõn (II5) khụng mang gen bnh, ú gỏi ca dỡ (em h bnh nhõn-III5) cng khụng mang gen bnh Header Page of 161 - Ph h ca gia ỡnh bnh nhõn mó s HA02 (sau phõn tớch gen) I II II Chỳ thớch: - Kt qu phỏt hin ngi lnh mang gen bnh 3 N bỡnh th ng N mang gen B nh nhõn tham gia nghiờn c u Nam bỡnh th ng Nam b b nh Nam b b nh ó t vong Hỡnh 3.5 S ph h gia ỡnh bnh nhõn mó s HA02 (sau phõn tớch gen) Nhn xột: Cú 2/5 thnh viờn n (II1, III2) gia ỡnh bnh nhõn HA02 cú nguy c cao mang gen bnh, c xỏc nh cú mang gen F8 t bin o on intron 22 trng thỏi d hp t 3.2.3.2 Kt qu phỏt hin ngi lnh mang gen F8 t bin im * Kt qu phõn tớch gen gia ỡnh bnh nhõn mó s HA16 Bnh nhõn HA16 cú t bin thờm nucleotid A trờn exon 14 ti v trớ c 4997insA gõy lch khung dch mó (Thr1904Asn fs*2) Hỡnh 3.7 Hỡnh nh gii trỡnh t gen ca gia ỡnh mó s HA16 - Ph h ca gia ỡnh bnh nhõn mó s HA16 (trc phõn tớch gen) Nhn xột: Hỡnh nh gii trỡnh t exon 14 gen F8 ca ngi m, bỏc Ph h gia ỡnh bnh nhõn mó s HA16 cú mt trai b bnh v ch h ca bnh nhõn HA16 xut hin cỏc nh chng lờn sau hemophilia A (bnh nhõn mó s HA16 IV6), cú tin s gia ỡnh nhng im t bin c.4997insA, chng t m, bỏc v ch h bnh nhõn khụng rừ rng, vỡ vy b ngoi (II6), b h (II1), m (III9), bỏc gỏi (III3, mang gen F8 t bin trng thỏi d hp t III5, III7 ), dỡ (III11, III13) v cỏc ch h bnh nhõn (IV1, IV2, IV4, IV5) cú th l ngi lnh mang gen bnh Footer Page of 161 Header Page of 161 - Ph h ca gia ỡnh bnh nhõn mó s HA16 (sau phõn tớch gen) bin ó phỏt hin c bnh nhõn (c 468 480del15bp), chng t ngi m bnh nhõn mang gen F8 t bin trng thỏi d hp t - Ph h ca gia ỡnh bnh nhõn mó s HA66 sau phõn tớch gen Hỡnh 3.8 Ph h ca gia ỡnh bnh nhõn mó s HA16 3.2.3.4 Kt qu phỏt hin ngi lnh mang gen F8 t bin xúa on nh - Kt qu phõn tớch gen gia ỡnh bnh nhõn mó s HA66 Hỡnh 3.17 Ph h gia ỡnh bnh nhõn mó s HA66 (sau phõn tớch gen) Nhn xột: Trong thnh viờn n (I1, II1) ca gia ỡnh bnh nhõn HA66 cú nguy c cao mang gen bnh ó xỏc nh c: thnh viờn n cú mang gen F8 t bin trng thỏi d hp t (II1), thnh viờn n (I1 b ngoi bnh nhõn) khụng mang gen F8 t bin trng thỏi d hp t * Kt qu phõn tớch gen gia ỡnh bnh nhõn mó s HA92 Bnh nhõn HA92 cú t bin thờm nucleotid A trờn exon 14 (c 3864-70 insA) gõy thay th acid amin Glycin thnh Arginin v trớ codon Hỡnh 3.16 Hỡnh nh gii trỡnh t gen ca gia ỡnh mó s HA66 Nhn xột: Hỡnh nh gii trỡnh t exon gen F8 ca ngi m (II1) bnh nhõn HA66 xut hin cỏc nh chng lờn bt u t v trớ t Footer Page of 161 1271 v lch khung dch mó ton b cỏc acid amin cũn li (P Gly1271Argfs*7) Header Page of 161 - Ph h ca gia ỡnh bnh nhõn mó s HA92 bin trng thỏi d hp t T kt qu ny khụng cn phõn tớch gen ca cỏc ch h bnh nhõn (III2, III3) vỡ t bin bnh nhõn khụng phi l t bin di truyn 3.2.3 T l phỏt hin ngi lnh mang gen bnh Bng 3.3 T l phỏt hin ngi lnh mang gen bnh Mang gen bnh Thnh viờn gia ỡnh Phõn tớch Phõn tớch Tng ph h gen (n,%) M bnh nhõn 20 14 34 (68) Thnh viờn n khỏc 23 32 55 (47,4) Tng 43 46 89 (54) Hỡnh 3.18 Ph h gia ỡnh bnh nhõn mó s HA92 Nhn xột: Ph h gia ỡnh bnh nhõn mó s HA92 cú mt trai b bnh hemophilia A (bnh nhõn mó s HA92), tin s gia ỡnh trc õy khụng cú b bnh ging nh bnh nhõn, vỡ vy b ngoi (I1), m (II1), dỡ (II3) v cỏc ch h bnh nhõn (III2, III3) cú th l ngi lnh mang gen bnh - Kt qu phỏt hin ngi lnh mang gen bnh Khụng mang gen bnh (n,%) Tng 16 (32) 61 (52,6) 77 (46) 50 116 166 Nhn xột: 34/50 (68%) ngi m mang gen F8 t bin trng thỏi d hp t v 16/50 ngi m (32%) khụng mang gen F8 t bin 55/116 (47,4%) thnh viờn n khỏc (bao gm: b, bỏc gỏi, dỡ, ch em gỏi ca bnh nhõn) mang gen F8 t bin trng thỏi d hp t; 61/116 (52,6%) thnh viờn n khỏc khụng mang gen F8 t bin 89/166 thnh viờn n l ngi lnh mang gen bnh, chim t l 54%; 77/166 thnh viờn n khụng mang gen bnh, chim t l 46% 3.3 Kt qu chn oỏn trc sinh bnh Hemophilia A 3.3.1 c im chung ca cỏc thai ph tham gia nghiờn cu Hỡnh 3.19 Hỡnh nh gii trỡnh t gen ca gia ỡnh mó s HA92 Nhn xột: Hỡnh nh gii trỡnh t exon 14 gen F8 ca ngi m (II1 ) v dỡ (II3) ca bnh nhõn HA92 ging nh trỡnh t gen ca ngi bỡnh thng, chng t m v dỡ bnh nhõn khụng mang gen F8 t Footer Page of 161 Bng 3.7 Mt s c im chung ca cỏc thai ph tham gia nghiờn cu c im n Tng s thai ph tham gia nghiờn cu 12 15 tun S thai ph khụng mang gen F8 t bin S thai ph mang gen F8 t bin Header Page 10 of 161 Nhn xột: Cú 12 thai ph tham gia vo nghiờn cu, ú cú thai ph c xỏc nh l ngi lnh mang gen bnh ang mang thai tun 12 18 Chn oỏn qua siờu õm cho thy thai nhi gii tớnh nam v thai nhi gii tớnh n Nhng thai ph ny sau c t cú nguyn vng lm chn oỏn trc sinh - Kt qu xỏc nh t bin ca thai nhi (III2) Da vo v trớ t bin ch im trờn mu DNA bnh nhõn HA04, mu DNA tỏch chit t t bo i ca thai ph II1 (thai nhi III2 ) c phõn tớch xỏc nh t bin v a tr sau sinh ó c kim tra kt qu chn oỏn trc sinh bng phõn tớch gen 3.3.2 Xỏc nh tinh sch DNA tỏch chit t t bo i 6/12 thai ph thc hin chn oỏn trc sinh c tin hnh chc i tun th 15 ca thai k ti Bnh vin ph sn Trung ng di s hng dn ca siờu õm DNA c tỏch chit t t bo i v c tin hnh kim tra tinh sch trờn mỏy Nano drop Mu DNA thu c cú tinh sch khỏ cao, dao ng 1,8 1,9 v nng khong 200 540 ng/l 3.3.3 Kt qu chn oỏn trc sinh * Kt qu chn oỏn trc sinh ca gia ỡnh bnh nhõn HA04 Bnh nhõn HA04 cú t bin thờm nucleotid A trờn exon 14 (c.4550insA) lm lch khung dch mó ton b cỏc acid amin t v trớ codon 755 trờn protein yu t VIII - Ph h gia ỡnh bnh nhõn HA04: Gia ỡnh bnh nhõn HA04 cú mt trai b bnh hemophilia A (bnh nhõn mó s HA04 III1), ph h gia ỡnh ó cú mt ngi cu (II3) b bnh, vỡ vy thai ph - ngi m bnh nhõn (II1) l ngi lnh mang gen bnh bt buc, thai ph cng ó c phõn tớch gen cho kt qu mang gen bnh trng thỏi d hp t Vic chn oỏn trc sinh cho thai ph ny l cn thit nu thai nhi cú gii tớnh l nam - Kt qu xỏc nh gii tớnh ca thai nhi (III2) K thut PCR c tin hnh vi cp mi cho phộp khuych i vựng gen xỏc nh gii tớnh SRY (cú kớch thc 254 bp) c hiu trờn NST Y Kt qu cho thy thai nhi (III2) cú gii tớnh nam Footer Page 10 of 161 Hỡnh 3.25 Hỡnh nh gii trỡnh t exon 14 gen F8 ca DNA thai nhi (III2) trc v sau sinh Nhn xột: Hỡnh nh gii trỡnh t exon 14 gen F8 ca DNA thai nhi III2 trc sinh khụng cú t bin ging nh t bin ó phỏt hin c trờn bnh nhõn v kt qu kim tra sau sinh cho phộp khng nh ngi em trai bnh nhõn HA04 (III2) khụng b t bin exon14 gen F8 Bng 3.9+10 Kt qu phỏt hin t bin trờn mu i trc sinh v chn oỏn sau sinh Kt qu n Gii tớnh Thai nhi b bnh Nam Chn oỏn trc sinh Thai nhi khụng b bnh Nam (n=6) Thai nhi khụng mang gen bnh N Cú t bin gen F8 Chn oỏn sau sinh Khụng t bin gen F8 Nam (n=4) Cha thc hin chn oỏn sau sinh nam, n Nhn xột: Cú 2/6 mu DNA tỏch chit t t bo i mang gen F8 t Header Page 13 of 161 khe núi chung v sc khe sinh sn núi riờng nhm nõng cao cht lng cuc sng Nghiờn cu cng ó khng nh 77/166 ngi chc chn khụng mang gen bnh 4.3 V kt qu chn oỏn trc sinh Trc õy, nhng thai ph mang gen bnh thng c t ch nờn sinh gỏi, nu thai nhi l trai thỡ c khuyn cỏo nờn ỡnh ch thai nghộn trỏnh sinh cú th b bnh Hemophilia A Ngy nay, nhng tin b v sinh hc phõn t ó cho phộp phỏt hin chớnh xỏc nhng ngi lnh mang gen bnh trng thỏi d hp t thc hin t hụn nhõn v di truyn gia ỡnh cú nguyn vng Cỏc thai ph cú nguy c cao sinh b bnh hemophilia A (ngi lnh mang gen bnh) c khuyn khớch thc hin chn oỏn trc sinh v cú c hi sinh trai khe mnh Kt qu chn oỏn trc sinh thc hin nghiờn cu ny: Cú 2/6 mu DNA tỏch chit t t bo i mang gen F8 t bin; 4/6 mu khụng mang gen F8 t bin; 2/4 mu khụng t bin thc hin chn oỏn sau sinh u cho kt qu phự hp vi chn oỏn trc sinh (khụng cú t bin gen F8) Trong trng hp thai ph l ngi lnh mang gen bnh mang thai gii tớnh nam nhng quyt tõm sinh cho dự cú b bnh hay khụng thỡ chn oỏn trc sinh cú t hay khụng? Chỳng tụi cho rng nờn lm chn oỏn trc sinh bi nu bit trc thai nhi b bnh thỡ thai ph ny sinh khụng c can thip th thut ly thai vỡ nh vy d gõy chy mỏu cho thai nhi Bit trc thai nhi b bnh mỏu khú ụng giỳp bỏc s sn khoa cú quyt nh ỳng n khi thai ph chuyn d Vic phỏt hin ngi lnh mang gen bnh qun lý, iu tr cng nh ỏp dng cỏc phng phỏp sng lc, chn oỏn trc sinh l iu vụ cựng cn thit, nhm ci thin cht lng cuc sng cho nhng ngi ph n mang gen bnh v quan trng nht l lm gim s tr sinh b bnh hemophilia A di truyn Footer Page 13 of 161 KT LUN T kt qu nghiờn cu cú th a mt s kt lun sau: Phỏt hin ngi lnh mang gen F8 b t bin Trong 50 gia ỡnh cú b bnh hemophilia A ó xỏc nh c t bin: - 34/50 ngi m mang gen F8 t bin dng d hp t, chim t l 68% v 16/50 ngi m (chim t l 32%) khụng mang gen F8 t bin - 55/116 thnh viờn n (gm b ngoi, bỏc, dỡ, ch, em gỏi ) mang gen bnh trng thỏi d hp t, chim t l 47,4 %; 61/116 thnh viờn n khụng mang gen bnh, chim t l 52,6% Chn oỏn trc sinh bnh hemophilia A 6/12 thai ph l ngi lnh mang gen bnh c thc hin chn oỏn trc sinh: 4/6 mu i (thai nhi) khụng b t bin gen F8 nờn thai ph ny c t di truyn gi thai v 2/4 trng hp chn oỏn sau sinh cho kt qu khụng t bin gen F8 phự hp vi chn oỏn trc sinh; 2/6 mu i (thai nhi) mang gen yu t VIII t bin nờn thai ph ny c t ỡnh ch thai nghộn KHUYN NGH Cn sm phỏt hin ngi lnh mang gen bnh cỏc gia ỡnh bnh nhõn hemophilia A cú k hoch qun lý, theo dừi v t di truyn Cn thc hin chn oỏn trc sinh i vi tt c trng hp m l ngi lnh mang gen bnh mang thai gii tớnh nam xỏc nh thai nhi cú b bnh hay khụng t ú cú quyt nh ỡnh ch thai nghộn cng sm cng tt hoc nu khụng cn cú k hoch d phũng chy mỏu cho thai ph v thai nhi lỳc sinh Header Page 14 of 161 DANH MC CC CễNG TRèNH KHOA HC CễNG B LIấN QUAN N LUN N MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY Bựi Th Thu Hng, Trn Võn Khỏnh, Nguyn Vit Tin, Nguyn Th H, T Thnh Vn (2013) Nghiờn cu phỏt hin ngi lnh mang gen bnh Hemophilia A, Tp Nghiờn cu Y hc, Tp 83, s 3, 1-8 BUI THI THU HUONG Bựi Th Thu Hng, Trn Huy Thnh, Nguyn Th H, Nguyn c Hinh, T Thnh Vn, Trn Võn Khỏnh (2014) Phỏt hin ngi lnh mang gen bnh v chn oỏn trc sinh bnh Hemophilia A, Tp Nghiờn cu Y hc, Tp 88, s 3, 1-9 CARRIER DETECTION AND PRENATAL DIAGNOSIS HEMOPHILIA A Bựi Th Thu Hng, Trn Huy Thnh, Nguyn Th H, Nguyn c Hinh, T Thnh Vn, Trn Th Oanh, Trn Võn Khỏnh (2014) Phỏt hin t bin gen F8 v xỏc nh Major : Biochemistry ngi lnh mang gen bnh trờn ph h gia ỡnh mt bnh Code : 62720112 nhõn Hemophilia A, Tp Nghiờn cu Y hc , Tp 89, s 4, 1-9 MEDICAL DOCTOR DISSERTATION SUMMARY Ha Noi - 2014 Footer Page 14 of 161 Header Page 15 of 161 THE DISSERTATION IS COMPLETED AT BACKGROUND HANOI MEDICAL UNIVERSITY Urgency of topics Hemophilia hemophilia A is a recessive genetic disease related to gender, disease gene located on the X chromosome has no corresponding allele on the Y chromosome, so the mother can pass the disease gene to 50% of patients son and gene transfer treatment for 50% of girls The disease can be inherited through many generations and many people get sick of the same family The disease affects the physiological, physical, and young children are a burden to family and society In Vietnam, an estimated 6,000 people with hemophilia and about 30,000 carriers Although in recent years, the care of patients with hemophilia A in Vietnam has made progress, the number of patients diagnosed and managed has increased significantly, but only accounted for less than 30% of the total some people are sick and most of the gene carriers have not been diagnosed and managed The detection of the genetic carriers of the disease plays an important role in genetic counseling and prenatal diagnosis can help prevent disease and reduce the birth incidence Objectives of the research: 1/ To detect the F8 gene mutation carrier status in family members of patients hemophilia A 2/ Initial prenatal diagnosis for women with high-risk babies with hemophilia A The meaning of scientific and practical subjects: The woman discovered gene carriers by conventional tests such as determining the activity of factor VIII in the blood also had difficulty because of the factor VIII activity they not reduce or reduce less, can range from 50 - 150%, only about 10% of the women with factor VIII activity plasma A) plus polymorphism STR in intron 13 and 22 linkage analysis method implemented fast, relatively inexpensive, reliable way to detect cases of hemophilia A in the family tree has many sick people However, linkage analysis methods can not detect carrier status in families with genetic mutations arise in the process of forming gametes Prenatal Diagnosis of hemophilia A Results genetic analysis that pregnant women with the disease gene screening factor is most important After the screening process Footer Page 17 of 161 Header Page 18 of 161 analysis, women with high-risk babies with hemophilia A will be conducted sampling fetal cells to analyze and identify mutations Studies of healthy people carry the disease gene in Vietnam The study of this disease in our country has been concentrated mainly on the incidence, clinical features, pre-clinical, treatment, and the psychological impact of the disease on patients and family members So are open for application in molecular biology techniques to analyze and detect the F8 gene mutation, genetic disease carrier status and prenatal diagnosis Diagram of the study design: Chapter 2: SUBJECTS AND METHODS 2.1 Research Subjects Of 50 patients with hemophilia A families were identified F8 gene mutation (Gen Research Center - Protein Hanoi Medical University) includes: - Mother of 50 patients - 116 female members (grandmother, their grandmother, her aunt, aunt, sister, sister ) have the same blood with the patient - 12 pregnant women/166 family members of patients in which 6/12 healthy women who carry genetic diseases, are pregnant at 1218 weeks of gestation - 20 people (10 male, 10 female) healthy, with no family history of the disease genetic engineering used to standardize and control samples and samples from carrying out research in molecular biology techniques for genetic analysis 2.2 Research Methodology: Using a prospective study and cross-sectional descriptive 2.3 Study sites + Center for Gene Research - Protein, Hanoi Medical University + Time from 1/2012 - 6/2014 Footer Page 18 of 161 50 patients with hemophilia A mutation identified F8 50 Construction pedigree Detecting obligate carriers (Mother, sister, sister ) No Detecting possible carriers (Mother, sister, sister ) pregnant No genetic testing F8 gene tests: - I-PCR technique (inversion int22) - Award gene sequences (point mutations, small deletions) Carrier Non carrier Genetic counseling and prevention Non prenatal Diagnosis Normal Mutations Advice abortion Prenatal Diagnosis 2.4 The process and techniques used in the study - PCR amplification F8 gene exon 26 - Technical sequenced advice keep Header Page 19 of 161 - I-PCR technique (Inversion-PCR) to identify the intron 22 inversion mutation - Analysis of family genealogy patients - Prenatal Diagnosis of Hemophilia A patients 2.5 Thread adhere research ethics in medicine Comment: Based on the results of phylogenetic analysis detected 20/50 maternal gene carriers (accounting for 40%); 30/50 mothers who have high-risk gene carriers (accounting for 60%); 23/116 female members of gene carriers (accounting for 20%); 93/116 female members who are at high risk gene carriers (accounting for 80%); 43/166 patients who definitely gene (accounting for 26%); 123/166 people with highrisk gene carriers (accounting for 74%) 3.3 Findings the F8 gene mutation carriers 3.3.1 DNA extraction results Total DNA from whole blood is separated by the method of phenol/chloroform The concentration of total DNA extracted value from 150 - 1200 ng/ml and purity of the samples with satisfactory optical density ratio measured at 260/280 nm wavelengths are in the range of 1.8 -2.0 3.3.2 Findings healthy people carry the disease gene by gene analysis methods 3.3.2.1 Findings the F8 gene mutation carriers intron22 inversions - The results of the detection of carriers of the disease gene in the patient's family HA02 Chapter 3: RESULTS 3.1 Characteristics of the study subjects Table 3.2 Distribution of study subjects according to sex with patient Relationship to patient n Ratio The mothers 50 30,1 The female members (grandmother, 116 69,9 uncle, aunt and sister) Total 166 100 Comment: In the women's 166 members of 50 families with 50 patients with hemophilia A mother, accounting for 30.1% and 116 people, including her uncle, aunt and sisters of patients, accounting for 69.9% rate 3.2 Detection rate for genetic disease carrier status through pedigree analysis Table 3.2 The proportion of carriers of the disease gene discovery is based on pedigree Status gene Obligate cariers Possible carriers Total (n,%) (n,%) Family members (n,%) Mother of patient 20 (40%) 30 (60%) 50 Other female members 23 (20%) 93 (80%) 116 Total 43 (26%) 123 (74%) 166 Footer Page 19 of 161 559 bp 500 bp 487 bp M: Marker 100bp : Patient's mother (II3) 1: The normal (negative control) 4: Uncle younger patients (II1)3: Patients (III4) 5: Aunt patients (II5) Figure 3.4 Picture multiplex PCR identified mutations intron 22 of the patient's family HA02 Header Page 20 of 161 10 11 Comment: normal human DNA in wells No position with band size 487 bp, respectively HA02 patient's DNA mutated intron 22 inversions in wells band size 559 bp, respectively DNA of the mother, her aunt and sister of the patient in their wells 3, and with bar size respectively 487 and 559 bp, so the patient mother (II3), her aunt (II1) and their sister of the patient (III2) are carriers of the disease gene in the heterozygous state DNA aunt in wells No with band corresponding to 487 bp in size like a normal person, so the patient's aunt (II5) does not carry diseases, so the aunt's daughter (cousin patient-III5) also did not carry the disease - Genealogy of the patient's family code HA02 (after the genetic analysis) (Results are conducted at the Center for Gene-Protein Research, Hanoi Medical University) - Genealogy of the patient's family code HA16 (before genetic analysis) The family code HA16 patients had a sick son hemophilia A (patient numbers HA16 - IV6), have a family history but did not clear, so grandma (II6), their grandmother (II1) , mom (III9), her aunt (III3, III5, III7), aunt (III11, III13) and the patient's cousin (IV1, IV2, IV4, IV5) can be the carriers of the disease gene - The results of the detection of carriers of the disease gene I II II Note: 1 2 3 Normal female 4 5 Normal male Carrier Patient Study patient Men were fatal disease Figure 3.5 Family tree diagram code HA02 patients (after genetic analysis) Remarks: 2/5 female members (II1, III2) family HA02 patients at high risk for the disease gene, identified F8 gene intron 22 inversion mutation in the heterozygous state 3.3.2.2 Findings healthy carrier F8 gene point mutations * Results genetic analysis of the patient's family code HA16 Patients HA16 add nucleotide mutation in exon 14 A at position c 4997insA cause translational frame difference (Thr1904Asn fs * 2) Footer Page 20 of 161 Figure 3.7 Sequenced pictures of the family code HA16 Comment: Picture sequenced exon 14 of the F8 gene mother, uncle and cousin HA16 patients appeared after the peak overlap c.4997insA point mutations, indicating mother, uncle and cousin patients with F8 gene mutation in the heterozygous state 3.3.2.3 Findings healthy people carry the gene deletion mutant factor VIII gene F8 small segment - The results of the detection of carriers of the disease gene family HA66 patients Header Page 21 of 161 12 13 Comment: In female members (I1, II1) family HA66 patients at high risk of carrying disease genes have been identified: one female member F8 gene mutation in the heterozygous state (II1, II3, III2 ), female (I1 - grandmother patients) did not carry mutations in the F8 heterozygous state 3.3.2.4 The genetic analysis of case mothers family hemophilia A patients not carry disease genes* Kt qu phõn tớch gen gia ỡnh bnh nhõn mó s HA92 Patients HA92 add nucleotide mutation in exon 14 A (c 3864-70 INSA) causing amino acid replacement Arginin Glysin the codon position bias in 1271 and translated the entire frame of the remaining amino acids (P Gly1271Argfs * 7) - Genealogy of the patient's family code HA92 Normal Normal c.465-480del15bp p.101-105delYDTVV Alen 1: bỡnh thng Alen 2: 468-480del 15bp Patient HA66 (III1) Mother of patient Figure 3:16 Sequenced pictures of the family code HA66 Comment: Picture sequenced exon of the F8 gene mother (II1) of patients HA66 peaks appear superimposed starting position mutations were detected in patients (c 468 - 480del15bp), demonstrated the parent patients F8 gene mutations in heterozygous state - Genealogy of the family code HA66 patients after gene analysis I II I III II Note: Normal female Carrier III Patient Study patient Note: Normal male Normal female Carrier Study patient Normal male Patient Men were fatal disease Figure 3:17 Patient's family pedigree code HA66 (after genetic analysis) Footer Page 21 of 161 Figure 3:18 Patient's family pedigree code HA92 Comment: The family code HA92 patients had a sick son hemophilia A (code HA92 patients), family history before nobody like ill patients, so her grandmother (I1), mother (II1), aunt (II3) and cousin patient (III2, III3) who may be carriers of the disease gene Header Page 22 of 161 14 15 - The results of the detection of carriers of the disease gene Comment: Of 50 mothers with 34 F8 gene mutation in the heterozygous state, accounting for 68% and 16 people (32% rate) is not bringing F8 gene mutation 55/116 female members (including her, her aunt, aunt, sister of the patient) F8 gene mutation in the heterozygous state, accounting for 47.4%; 61/116 members who did not carry the F8 gene mutation, accounting for 52.6% rate 89/166 female members of the family are carriers of genetic diseases, accounting for 54%; 77/166 female members did not carry the disease, accounting for 46% 3.5 Prenatal diagnosis disease Hemophilia A Figure 3:19 Sequenced pictures of the family code HA92 Comment: Picture sequenced exon 14 of the F8 gene mother (II1) and aunt (II3) of patients HA92-like sequence of the normal gene, suggesting that the mother and aunt patients F8 gene mutation in the heterozygous state From this result without genetic analysis of the patient's cousin (III2, III3) because mutations in patients is not caused by a genetic mutation 3.4 Detection rate for genetic disease carrier status Table 3.4 The rate of gene discovery healthy people sick manng Family members Carrier Non carrier (n,%) Total Phylogenetic Total Gene tets analysis (n,%) Mother of patient 20 14 34 (68) 16 (32) 50 Other female 23 32 55 (47,4) 61 (52,6) 116 members Total 43 46 89 (54) 77 (46) 166 Note: other female members including her uncle, aunt and sisters patients Footer Page 22 of 161 3.5.1 General characteristics of the women participating in the study Table 3.6 Some common characteristics of the women participating in the study Characteristic n Total number of women in the study Gestational age 12 15 weeks Non carier Carrier Comment: There are 12 women participating in the study, including women who were identified as carriers of genetic diseases are pregnant weeks 12-18, the fetus is diagnosed through ultrasound male cases and case the female sex These women after consultation with aspirations to prenatal diagnosis 3.5.2 Determination of purified DNA extracted from amniotic cells Header Page 23 of 161 16 17 6/12 women presenting diagnosis was conducted before amniocentesis in the 15th week of pregnancy at the Central Obstetrics Hospital under the guidance of ultrasound DNA extracted from amniotic cells and examined on Nano purity drop DNA samples obtained with high purity, ranging from 1.8 to 1.9 and the concentration range 200-540 ng / ml 3.5.3 Results for prenatal diagnosis * Results for prenatal diagnosis of the patient's family HA04 Patients HA04 add nucleotide mutation in exon 14 A (c.4550insA) deflects the entire frame translation from the amino acid codon position 755 on the protein factor VIII (Results are conducted at the Center for Gene-Protein Research, Hanoi Medical University - The patient's family pedigree HA04 Comment: HA04 patient's family has a sick son hemophilia A (patient numbers HA04 - III1), in the family tree had a boy (II3) is sick, so pregnant women - mothers of patients (II1 ) is a genetic disease carrier status compulsory prenatal diagnosis during pregnancy is necessary if the fetus is male gender - The results of sex determination of the fetus (III2) PCR was conducted with primers to amplify genomic regions allows sexing SRY (size 254 bp) on chromosome Y-specific results showed that the fetus (III2) were male - The results of determination of fetal mutations (III2) Based on mutations in DNA marker HA04 patients, DNA samples extracted from amniotic cells of II1 pregnant women (fetal III2) were analyzed to determine the mutation and then 1-year test results by gene sequencing techniques c.449A I c.4550 ins A I Normal c.4549A III Patient HA04 (III1) c.4549A N bỡnh th ng N mang gen Thai Nam bỡnh Nam b b nh B nh nhõn tham gia nghiờn Figure 3:24 Patient's family pedigree HA04 Footer Page 23 of 161 Figure 3:25 Picture sequenced exon 14 of the F8 gene Fetal DNA (III2) before and after childbirth Header Page 24 of 161 18 19 Comment: Picture sequenced exon 14 of the F8 gene DNA III2 fetus before birth like no mutation was detected mutations in patients and test results after birth to allow his brother confirmed patients HA04 (III2) is not mutated gene exon14 F8 Table 3.7 Results revealed a mutation in amniotic fluid samples for prenatal and postnatal diagnosis Result n Sex Fetal disease 2/6 Nam The fetus does not get sick 3/6 Nam The fetus did not carry disease 1/6 N There postnatal diagnosis F8 gene mutation 0/2 Non F8 gene mutation F8 2/2 Nam Unrealized diagnosed after birth 2/4 1nam,1n Comment: There are two sixths DNA extracted from amniotic cells F8 gene mutation; 4/6 sample F8 gene mutations: samples did not perform mutation are diagnosed after birth results in accordance with prenatal diagnosis (no F8 gene mutation); samples unrealized diagnosed after birth because the unborn and help the scientific basis for prenatal diagnosis and genetic counseling, childbirth avoid being hemophilia A 4.2 V E ratio to detect the gene carriers based on pedigree analysis For those cases where the patient has a family history clear, based on the results of phylogenetic analysis can ascertain gene status of women in family members of patients Based on the results of phylogenetic analysis of our study was to detect 20/50 maternal gene carriers (accounting for 40%); 30/50 mothers who have high-risk gene carriers (accounting for 60%); 23/116 female members of gene carriers (accounting for 20%); 93/116 female members who are at high risk gene carriers (accounting for 80%) The study results also showed that 43/166 people carries the gene certainly, accounting for 26% % Thus, the detection rate of the gene carriers of this technique is low and does not apply to individual cases in the family 4.3 Findings about the F8 gene mutation carriers 4.3.1 On the technical process to extract DNA from peripheral blood and amniotic cells: DNA extraction is a critical first step in the process of applying the techniques of molecular biology If possible DNA extraction, ensuring the purity, the DNA molecule, unbroken, no contamination, the next response will get results with high accuracy With the purified DNA, PCR reactions will be inhibited due to contamination or generate nonspecific products The DNA samples in the study have a high concentration and purity in the range from 1.8 to 2.0 allowed (Table 3.1) It is a prerequisite to ensure the technical results of the procedure followed in the study 4.3.2 Findings on healthy people carry the disease gene by gene analysis method CHAPTER 4: DISCUSSION 4.1 Characteristics of the study subjects Of 166 members of 50 female patients with hemophilia A family study participants had 50 mothers, accounting for 30.1% and 116 people, including her uncle, aunt and sisters of patients, accounting for 69.9%, many of which are those of childbearing age and children under 18 years of age The early identification of gene status of the child will help mothers care about their children early from the first menstrual cycle lasts not see, not have menorrhagia, abdominal pain and help the children as adults have the knowledge and plan for their future, actively prevent the risk factors for bleeding Footer Page 24 of 161 Header Page 25 of 161 20 21 4.3.2.1 Detecting the F8 gene mutation carriers intron22 inversions Intron 22 inversion mutation is caused by recombination between copies of the int 22h (repeat region consisting of 9.5 kb) of intron 22 and one of two identical copies of the telomere is located at position 400 kb at the 'end of the F8 gene Inversion phenomenon leading to fracture F8 gene and the disease can cause severe consequences for the patient This mutation accounts for 45-50% of patients with severe Hemophilia A For patients who have been identified with the intron 22 inversion mutation, based on just this point mutation analysis we discovered gene carrier status by I-PCR technique Figure 3.4 shows the results for the mother, her aunt and cousin healthy patients who carry the disease gene in the heterozygous state Aunt of patients did not carry the disease Although the mother has been identified as gene carriers is mandatory, but we are still analyzing DNA samples from the mother, as it is proof positive samples for gene status in the heterozygous state for female members other family members This proves that there is no difference in terms of implementation of process steps to detect mutations and inversions, such analysis results are reliable The research results provide information to clinicians advising mother, uncle, sister, patients need to prenatal diagnosis for future pregnancies 4.3.2.2 Detecting the F8 gene mutation carriers point F8 gene point mutations in patients with hemophilia A mutation markers to detect gene status of women in family members of patients We use gene sequencing techniques to analyze all 26 exons F8 gene in order to determine the exact status of gene carriers in the state zygote This is one of the modern techniques and the most accurate 4.3.2.3 Detection of gene carrier status F8 gene deletion mutants For the case of patients with mutations add or remove small sections below 50bp, in this study we use techniques F8 gene sequencing to detect the disease gene carriers in the heterozygous state in order to save costs charge Picture sequenced exon of the F8 gene mother (II1) of patients HA66 peaks appear superimposed starting position mutations were detected in patients, suggesting that the mother's DNA sample with allele normal allele and one mutated nucleotide at position 15 c 468 - 480del15bp and so this mother F8 gene mutation in the heterozygous state 4.3.2.4 In case of hemophilia A patient whose mother did not carry disease Picture sequenced exon 14 of the F8 gene mother (II1) and aunt (II3) of patients HA92-like sequence of the normal gene, suggesting that the mother and aunt patients carrying mutations in the F8 gene status heterozygotes From this result without genetic analysis of the patient's cousin (III2, III3) because mutations in patients is not caused by a genetic mutation Thus maternal gene completely normal F8, F8 gene mutation in exon 14 patients HA92 is a new mutation arises 4.4 The percentage of healthy people carry the disease gene The study was conducted with 50 patients with Hemophilia A family has identified point mutations showed only 34/50 mothers F8 gene mutations in heterozygous form accounting for 68% and 16/50 people (accounting for 32%) did not carry F8 mutations, this result is based on a small sample size can not conclude, however tentatively suggest conformity with the law of hereditary diseases (2/3 of cases due to genetic mutation, new mutation third is incurred) The results identify gene status of the mother is very important: if the mother carries the gene diseases such as hereditary nature of the disease, Footer Page 25 of 161 Header Page 26 of 161 22 23 genetic diseases son received from the mother, the maternal genetic disease can get from one generation before The study results demonstrate the new mutation rate arising in patients with hemophilia A Vietnam is 32% This can be explained by external factors impact on the development process in the body gametes father or mother as habitat conditions, due to eating habits, because war has left many toxic substances in the environment, Genetic testing results showed 55/116 female members in the heterozygous form (accounting for 47.4%); 61/116 members who did not carry the mutated gene (accounting for 52.6%) S Shetty's research (2001) on 102 patients with hemophilia A families (India) show a healthy proportion of gene carriers is higher than our study (64.5%), possibly due to rate family medical history of two different studies, but the female members of the family there is a clear medical history which may carry a higher genetic diseases We detected 89/166 female members (including my grandmother, mother, uncle, aunt, sisters patients ) are carriers of genetic disease hemophilia A The result of this discovery is important because with 89 this woman is no longer a suspect, but was certainly gene, so they need to more quantitative assay of factor VIII activity plasma to determine the risk of bleeding This result suggests that this woman needed a contingency plan to ensure overall health and reproductive health in particular to improve the quality of life The study also confirmed that the 77/166 certainly does not carry the disease 4.5 On the results of prenatal diagnosis In the past, women often carry genetic disease should be counseled daughter, if the fetus is the son shall be advised to avoid pregnancy termination may be ill baby Hemophilia A Today, advances of molecular biology has allowed the accurate detection of the disease gene carriers in the heterozygous state to perform marriage counseling and genetic family wishes Pregnant women are at high risk babies with Hemophilia A patients (healthy people carry the gene) are encouraged to perform prenatal diagnosis and the chance for a son healthy Results for prenatal diagnosis performed in this study: There are two sixths DNA extracted from amniotic cells F8 gene mutation; 4/6 sample F8 gene mutation; 2/4 samples did not perform mutation are diagnosed after birth results in accordance with prenatal diagnosis (no F8 gene mutation) In the case of pregnant women are carriers of genetic diseases of male pregnancy, but determined to bear a child whether or not the patient had been diagnosed before birth problems pose or not? We believe that there should be diagnosed before birth by the anticipation of the fetus is sick, pregnant women giving birth are not intervene because the tricks to get pregnant so easily cause bleeding to the fetus Know before baby has hemophilia help obstetricians have right decisions when the pregnant woman in labor The detection of the genetic carriers of the disease to manage, as well as treatment methods applied screening, prenatal diagnosis is essential, in order to improve the quality of life for women who carry genetic diseases and most importantly, reduce the number of children born with hemophilia A hereditary disease Footer Page 26 of 161 CONCLUSION From the research results can offer some conclusions: To detect carrier status F8 gene mutation In 50 families with a child with hemophilia A mutations have been identified: Header Page 27 of 161 24 25 - 34/50 mothers F8 gene mutation in the heterozygous form, accounting for 68% and 16/50 mothers (32% occupancy rate) is not bringing F8 gene mutation - 55/116 female members (including my grandmother, uncle, aunt, sister, sister ) gene carriers in the heterozygous state, accounting for 47.4%; 61/116 female members did not carry the disease, accounting for 52.6% rate prenatal diagnosis of hemophilia A 6/12 pregnant women who are carriers of the disease gene was performed prenatal diagnosis: 4/6 samples of amniotic (fetal) F8 gene mutations were not so pregnant women are four genetic counseling pregnant and keep 2/4 cases diagnosed after birth outcomes F8 gene mutation is not suitable for prenatal diagnosis; 2/6 samples of amniotic (fetal) factor VIII gene mutations should be advised pregnant women to abortion LIST OF PUBLIC SCIENTIFIC WORKS RELATED TO RECOMMENDATIONS For early detection of the disease gene carriers in patients with hemophilia A families to have management plans, monitoring and genetic counseling For prenatal diagnosis performed in all cases the mother is healthy gene carriers male pregnancy to determine fetal or illness from which there is no abortion decision as soon possible or if not needed a backup plan bleeding during pregnancy and the fetus at birth Footer Page 27 of 161 THE DISSERTATION Bui Thi Thu Huong, Tran Van Khanh, Nguyen Viet Tien, Nguyen Thi Ha, Ta Thanh Van Researchers found the gene carriers of Hemophilia A patients, Journal of Medical Research, Volume 83, No 3, 1-8 (2013) Bui Thi Thu Huong, Tran Huy Thinh, Nguyen Thi Ha, Nguyen Duc Hinh, Ta Thanh Van, Tran Van Khanh Detecting genetic disease carrier status and prenatal diagnosis of diseases Hemophilia A, Journal of Medical Research, Volume 88, No 3, 1-9 (2014) Bui Thi Thu Huong, Tran Huy Thinh, Nguyen Thi Ha, Nguyen Duc Hinh, Ta Thanh Van, Tran Thi Oanh, Tran Van Khanh F8 gene mutation detected and identified the disease gene carriers on a family genealogy Hemophilia A patients, Journal of Medical Research, Volume 89, No 4, 1-9 (2014) ... lệ phát người lành mang gen bệnh qua phân tích phả hệ Bảng 3.2 Tỷ lệ người lành mang gen bệnh phát d a vào phả hệ Tình trạng mang gen Mang gen Có nguy cao Tổng bệnh bắt buộc mang gen bệnh (n,%)... x a đoạn gen chiếm khoảng 10 – 15% Tùy thuộc vào kiểu vị trí đột biến gen F8 mà gây thể bệnh nặng nhẹ khác Người lành mang gen bệnh hemophilia A Người lành mang gen bệnh hemophilia A người mang. .. Thai nhi bị bệnh Nam Chẩn đoán trước sinh Thai nhi không bị bệnh Nam (n=6) Thai nhi không mang gen bệnh Nữ Có đột biến gen F8 Chẩn đoán sau sinh Không đột biến gen F8 Nam (n=4) Ch a thực chẩn đoán