B GIO DC V O TO B Y T TRNG I HC Y H NI NGC HI NGHIÊN CứU XáC ĐịNH ĐộT BIếN Và LậP BảN Đồ ĐộT BIếN GEN DYSTROPHIN TRÊN BệNH NHÂN LOạN DƯỡNG CƠ DUCHENNE VIệT NAM LUN N TIN S Y HC H NI - 2015 B GIO DC V O TO B Y T TRNG I HC Y H NI NGC HI NGHIÊN CứU XáC ĐịNH ĐộT BIếN Và LậP BảN Đồ ĐộT BIếN GEN DYSTROPHIN TRÊN BệNH NHÂN LOạN DƯỡNG CƠ DUCHENNE VIệT NAM Chuyờn ngnh: Húa sinh Mó s: 62720112 LUN N TIN S Y HC Ng-ời h-ớng dẫn khoa học: TS Trn Võn Khỏnh GS.TS T Thnh Vn H NI - 2015 LI CAM OAN Tụi l Ngc Hi, nghiờn cu sinh khúa 29 Trng i hc Y H Ni, chuyờn ngnh Húa sinh, xin cam oan: õy l lun ỏn bn thõn tụi trc tip thc hin di s hng dn ca TS Trn Võn Khỏnh v GS.TS T Thnh Vn Cụng trỡnh ny khụng trựng lp vi bt k nghiờn cu no khỏc ó c cụng b ti Vit Nam Cỏc s liu v thụng tin nghiờn cu l hon ton chớnh xỏc, trung thc v khỏch quan, ó c xỏc nhn v chp thun ca c s ni nghiờn cu Tụi xin hon ton chu trỏch nhim trc phỏp lut v nhng cam kt ny H Ni, ngy thỏng nm 2015 Ngi vit cam oan NGC HI DANH MC CH VIT TT BMD Becker Muscular Dystrophy BVSKTE Bo v sc kho tr em CK Creatine Kinase Duchenne Muscular Dystrophy DMD (Bnh lon dng c Duchenne) DNA Deoxyribo Nucleic Acid KB Kilo Base MLPA Multiplex Ligation Probe Amplification NST X Nhim sc th X NST Y Nhim sc th Y PCR Polymerase Chain Reaction RT RCR Reverse transcription PCR (PCR mó ngc) RNA Ribo Nucleic Acid T VN Bnh lon dng c Duchenne (Duchenne Muscular Dystrophy-DMD) l mt bnh lý thn kinh c di truyn thng gp nht, c phỏt hin tt c cỏc chng tc khỏc trờn th gii Tn sut bnh vo khong 1/3500 tr trai Duchenne l ngi u tiờn ó mụ t chi tit bnh ny vo nm 1861 [1] õy l mt bnh lý c rt nng, mang tớnh cht tun tin, nng dn theo thi gian Hu ht tr trai mc bnh u cú du hiu lõm sng vi triu chng suy yu c, biu hin bng khú i li, khú ng lờn ngi xung v khú khn leo cu thang Trong giai on nng, bnh nhõn tr nờn tn ph, mt kh nng i li vo la tui 12 v thng t vong ngoi 20 tui tn thng c tim v ri lon hụ hp [2, 3] Mt th bnh nh ca DMD rt hay gp l lon dng c Becker (BMD) Bnh nhõn BMD xut hin triu chng lõm sng mun hn DMD, biu hin lõm sng nh hn v thi gian tin trin chm hn Vi nhng biu hin nng n, cựng vi ri lon v tinh thn, DMD/BMD thc s khụng ch l mt tai i vi bn thõn ngi bnh m cũn l gỏnh nng ca gia ỡnh cng nh ca c cng ng DMD/BMD l bnh di truyn ln liờn kt gii tớnh, gõy nờn bi t bin gen dystrophin Gen dystrophin nm v trớ Xp21 trờn nhim sc th X, cú chiu di hn 3000 kb, gm 79 exon vi promotor khỏc v l gen ngi ln nht c phỏt hin cho n Gen dystrophin mó hoỏ 14 kb mRNA v tng hp sn phm tng ng l protein dystrophin Protein ny cú mt mng ca t bo c v cú chc nng chớnh vic trỡ s n nh mng, bo v t bo c b tn thng quỏ trỡnh co c [4, 5] Hin nay, bn t bin gen dystrophin gn nh ó c xỏc nh Dng t bin xúa on gen l ph bin nht, chim 60% Dng t bin im ng th hai v mc thng gp, chim 20-30% Dng t bin lp on chim khong 10-15% [5] Nhiu cụng trỡnh nghiờn cu cho thy, t bin xúa on gen thng trung vo hai vựng trng im (hotspot): vựng trung tõm (exon 43-60) v vựng tn (exon 1-19) [3, 6] t bin im v t bin lp on nm ri rỏc trờn chiu di gen [5] Hin cha cú phng phỏp iu tr c hiu Vi s phỏt trin ca ngnh sinh hc phõn t nhng nm gn õy, liu phỏp iu tr gen i vi bnh nhõn DMD ó em li hy vng to ln cho ngi bnh Tuy nhiờn vi mi dng t bin gen dystrophin khỏc s cú liu phỏp iu tr gen khỏc Vỡ vy, vic nghiờn cu xỏc nh t bin gen cho bnh nhõn l tin quan trng la chn liu phỏp iu tr gen hiu qu nht Ngoi ra, vic xỏc nh chớnh xỏc dng t bin gen cũn giỳp cho chn oỏn ngi lnh mang bnh, chn oỏn trc sinh t di truyn nhm gim t l mc bnh, gim hu qu ca bnh gõy cho gia ỡnh bnh nhõn v xó hi Nhng nm gn õy, nhiu nh khoa hc nc ngoi cng nh nc ó tin hnh nghiờn cu v phỏt hin t bin gen dystrophin cỏc bnh nhõn DMD Vit Nam ó cú nhiu cụng trỡnh nghiờn cu v t bin gen dystrophin Tuy nhiờn, cỏc cụng trỡnh ch yu mi ch dng phỏt hin t bin xúa on v trung vo vựng t bin trng im, cha cú nghiờn cu no tin hnh phỏt hin ton din cỏc dng t bin xúa on, t bin lp on v t bin im trờn ton b 79 exon ca gen dystrophin Xut phỏt t thc tin ú, ti Nghiờn cu xỏc nh t bin v lp bn t bin gen dystrophin trờn bnh nhõn lon dng c Duchenne Vit Nam c tin hnh vi cỏc mc tiờu sau: Xỏc nh t bin xúa on, lp on v t bin im ca gen dystrophin trờn bnh nhõn DMD/BMD Bc u xõy dng bn t bin gen dystrophin trờn bnh nhõn DMD/BMD Vit Nam CHNG TNG QUAN TI LIU 1.1 c im ca bnh DMD 1.1.1 Lch s phỏt hin bnh Nm 1851, Meryon ghi nhn cú tr gia ỡnh mc cựng mt loi bnh vi biu hin yu c dn dn v dn n t vong thi niờn thiu m ụng cho rng thiu dinh dng gõy nờn, ụng nhn thy bnh ch gp tr trai v ty khụng b tn thng, vỡ th bnh c t tờn l bnh Meryon [7] Nm 1861, bỏc s ngi Phỏp Guillaume Benjamin Amand Duchenne (1806 1875) ó mụ t chi tit dng gi phỡ i c trai mc bnh lon dng c quyn sỏch ca ụng mang tờn "Paraplộgie Hypertrophique de lenfance de cause cộrộbrale", xut bn nm 1861 n nm 1868, Duchenne tin hnh mt nghiờn cu quy mụ trờn 13 tr cú biu hin lit c gi phỡ i (cũn gi l nhc c) ễng l ngi u tiờn ó tin hnh sinh thit c trờn ngi sng lm xột nghim mụ hc v nhn thy cú s thay th mụ c bng mụ x hoc mụ liờn kt trờn tiờu bn, triu chng cú ci thin vi bnh nhõn iu tr bng thy tr liu kt hp xoa búp T ú ụng a tiờu chun chn oỏn bnh da trờn kt qu gii phu bnh v ghi in c, vỡ th, bnh c t tờn l bnh lon dng c Duchenne [8] Nm 1879, Gower cng nhn thy nhng biu hin yu c ch ghi nhn tr nam v l bnh di truyn thụng qua ngi m ễng ghi nhn cỏc tr bnh cú c im ging thay i t th t nm sang ngi hay t ngi sang ng v du hiu ny c t tờn l du hiu Gower, rt c trng cho bnh lon dng c Duchenne [9] Trong thi gian di sau ú, cỏc nghiờn cu hu nh ch dng li mc ỏnh giỏ cỏc biu hin lõm sng v iu tr h tr lm gim cỏc triu chng ca bnh Nm 1950, Peter Emil Becker mt bỏc s ngi c ó mụ t mt nhng dng ca bnh DMD, th bnh ny cú c ch bnh sinh ging bnh DMD nhng bnh cnh lõm sng thng nh hn Th bnh ny ó c mang tờn ụng Becker muscular dystrophy (BMD) Nm 1981, Zatz M v CS quan sỏt nhng tr gỏi mc bnh v phỏt hin gen dystrophin nm v trớ Xp21 trờn nhim sc th (NST) X, nhng tr gỏi ny mang chuyn on gia NST X v NST thng [10] Nm 1985, Kukel v CS ó phõn lp c DNA t mt bnh nhõn nam mc bnh DMD v thy cú xúa on ln NST X ca bnh nhõn ny Nhng nm sau ú, nh s phỏt hin gen dystrophin v sn phm protein tng ng vo nm 1987, cựng vi cỏc k thut di truyn phõn t phỏt trin mnh ó giỳp phỏt hin c t bin trờn gen dystrophin gõy bnh lon dng c Duchenne v phỏt hin ngi m mang gen bnh [11, 12] n nm 1993 ngi ta ó phỏt hin c y cỏc thnh phn v cu trỳc ca gen dystrophin gm 79 exon vi promoter khỏc nhau, mó húa cho 14kb mRNA, ú hn 99% l intron Ngy nay, c ch phõn t ca bnh ngy cng c hiu rừ ó giỳp hon thin thờm cỏc k thut chn oỏn bnh, t di truyn, tm soỏt trc sinh, cng nh bc u ng dng liu phỏp gen iu tr bnh DMD, nhm nõng cao cht lng cuc sng cho bnh nhõn v lm gim gỏnh nng cho gia ỡnh v xó hi 1.1.2 Biu hin lõm sng ca DMD Tr trai him biu hin cỏc triu chng lỳc sinh hoc nhng nm u ca thi k u nhi mc dự mt s tr ó cú biu hin gim trng lc c kớn ỏo Cỏc k nng ng thụ sm nh lt, ngi v ng thng phự hp vi la tui hoc cú th hi chm Du hiu sm nht ca yu c cú th l tr gi c kộm hn Nhng triu chng thng bt u t 3-5 tui, u bi s khú i li, khú khn leo cu thang v ng, chõn bnh nhõn thng dng cho chc chn, lng n bự tr cho c mụng b yu Du hiu Gower sm cú th thy rừ vo lỳc tui v biu hin y vo lỳc tr c n tui: Khi ngi bnh ang ngi xm phi ng lờn hoc ang nm phi ngi dy thỡ ngi bnh phi quay ngi sang mt bờn, gp u gi vo mụng, hai tay chng nng ly thõn gi t th qu bn, sau ú, bng cỏch tỡ hai tay ln lt lờn cng chõn, u gi v ựi, ngi bnh y cho thõn thng dy S tip ni cỏc ng tỏc nh vy c xem l c hiu cho bnh lon dng c tun tin Dỏng i Trendelenberg hay hụng lc l cng xut hin vo thi im ny [13] Hỡnh 1.1 Du hiu Gower (ngun http://www.alison-burke.com/works-medicine.html ) Thi gian bnh nhi cũn trỡ c kh nng di chuyn thay i rt ln Mt s bnh nhõn phi s dng xe ln lỳc tui, ú mt s bnh nhõn khỏc cú th i li mc dự cú khú khn n nm 10 tui m khụng cn cú bt k mt can thip chnh hỡnh no Bng vic s dng cỏc khung nõng , vt lý tr liu v ụi bng nhng can thip phu thut nh (kộo di gõn Achilles), hu ht tr mc DMD cú th i li n nm 12 tui Di chuyn khụng ch cú ý ngha quan trng vic lm chm li quỏ trỡnh c ch tõm thn thng i kốm vi vic ngi bnh mt i s c lp cỏ nhõn m nú cũn cú tỏc dng phũng nga chng vo ct sng cỏc bnh nhõn ny Chng vo ct sng cú th phũng nga c c bnh nhõn ch cũn trỡ c quỏ trỡnh di chuyn 1gi/ngy Ngc li, bnh nhõn ph thuc vo xe ln thỡ chng vo ct sng s tin trin rt nhanh chúng [14] Quỏ trỡnh yu c khụng th khng ch c tip tc tin trin thp niờn th hai ca i sng Chc nng ca cỏc c xa cũn c bo tn tng i tt ú tr cú th s dng c cỏc dng c n ung, cm bỳt v s dng bn phớm vi tớnh Yu c hụ hp biu hin bng ho yu v khụng hiu qu, thng xuyờn nhim khun hụ hp v gim dung tớch phi Yu c vựng hu hng cú th to iu kin gõy nờn hớt nhm thc n vo ng hụ hp, tro ngc dch qua mi v tr cú ging mi Chc nng ca cỏc c nhón cũn c bo tn tng i tt i tiu tin khụng t ch yu c vũng hu mụn cng nh c vũng niu o thng ớt gp v ch xy vo giai on mun ca bnh Co rỳt c thng gp mt cỏ, u gi, khp hỏng v khuu tay Chng vo ct sng thng gp Bin dng lng ngc lm nng thờm s suy gim dung tớch phi v gõy chốn ộp tim ụi vo ct sng cng gõy khú chu v au n 53 Torella, Annalaura, et al (2010), "One hundred twenty-one dystrophin point mutations detected from stored DNA samples by combinatorial denaturing high-performance liquid chromatography", The Journal of Molecular Diagnostics 12(1), pp 65-73 54 Prior, Thomas W, et al (1995), "Spectrum of small mutations in the dystrophin coding region", American journal of human genetics 57(1), p 22 55 Takeshima, Yasuhiro, et al (2010), "Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center", Journal of human genetics 55(6), pp 379-388 56 Barton-Davis, E R., et al (1999), "Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice", J Clin Invest 104, pp 375-381 57 Nguyn Th Trang, Nguyn Th Phng (1996), Giỏ tr ca CK chn oỏn v phỏt hin d hp t ca bnh nhõn lon dng c Duchenne, Di truyn hc v ng dng, Vol 2, Hi di truyn hc Vit Nam xut bn 58 Nguyn Th Phng, V Chớ Dng (2002), "Kt qu bc u phỏt hin t bin gen gõy bnh DMD Vit Nam", p ch Nhi khoa 10(10), pp 521-6 59 Trn Võn Khỏnh v cng s (2004), "Chn oỏn 85 bnh nhõn Vit Nam mc bnh nhc c Duchenne/Becker bng phng phỏp polymerase chain reaction", p ch Y hc Vit Nam 12, pp 33-8 60 Nguyn Th Trang, Hong Hnh Phỳc v cng s (2004), "nh lng creatine kinase phi hp vi lõm sng v phõn tớch ph h gúp phn chn oỏn mt s bnh c di truyn", p ch Nghiờn cu y hc 32(6), pp 133-9 61 Nguyn Th Trang, Nguyn Th Hon (2005), Phỏt hin t bin mt on gen dystrophin mt s bnh nhõn lon dng c Duchenne bng k thut Multiplex PCR, Nhng v n nghiờn cu c bn khoa hc s s ng, Bỏo cỏo hi ngh ton quc 3/11/2005, pp 1435-7 62 Nguyn Th Trang v cng s (2006), "Phỏt hin t bin mt on exon 46 v 51 gen dystrophin mt s bnh nhõn DMD bng k thut PCR", p ch nghiờn cu y hc 45(5), pp 18-23 63 Nguyn Th Phng Mai, V Chớ Dng, Hong Th Thanh v cng s (2007), p dng k thut multiplex PCR thay th PCR c in phõn tớch gen dystrophin cỏc bnh nhõn lon dng c Duchenne, Hi ngh nhi khoa Vit c, Editor Editors, Bnh vin Nhi ng TP HCM, p 111 64 Nguyn Khc Hõn Hoan, Phm Ngc Khụi v cng s (2009), "Phỏt hin t bin mt on gen gõy bnh lon dng c Duchene v Becker bng k thut MLPA", Y hc P H Ch Minh 13(2), pp 169 175 65 Nguyn Th Bng Sng, Trn Võn Khỏnh v cng s (2009), "Xỏc nh t bin mt on gen dystrophin mc mRNA", p ch Y hc PHCM 13(2), pp 98 104 66 Rappolee, Daniel A, et al (1989), "Novel method for studying mRNA phenotypes in single or small numbers of cells", Journal of cellular biochemistry 39(1), pp 1-11 67 Bennett, R R., et al (2001), "Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing", BMC Genet 2, p 17 68 Roberts, Roland G, et al (1993), "Exon structure of the human dystrophin gene", Genomics 16(2), pp 536-538 69 Ahn, Andrew H and Kunkel, Louis M (1993), "The structural and functional diversity of dystrophin", Nature genetics 3(4), pp 283-291 70 Adeli, K and Ogbonna, G (1990), "Rapid purification of human DNA from whole blood for potential application in clinical chemistry laboratories", Clinical chemistry 36(2), pp 261-264 71 Tay, Stacey Kiat Hong, et al (2006), "Diagnostic strategy for the detection of dystrophin gene mutations in asớan patients and carriers using immortalized cell lines", Journal of child neurology 21(2), pp 150-155 72 Armour, JAL, et al (2002), "The detection of large deletions or duplications in genomic DNA", Human mutation 20(5), pp 325-337 73 Den Dunnen, JT, et al (1989), "Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications", American journal of human genetics 45(6), p 835 74 Stuppia, Liborio, et al (2012), "Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases", International journal of molecular sciences 13(3), pp 32453276 75 Lalic, Tanja, et al (2005), "Deletion and duplication screening in the DMD gene using MLPA", European journal of human genetics 13(11), pp 1231-1234 76 Koenig, M., Monaco, A.P., and Kunkel, L M (1988), "The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein", Cell 53, pp 219-226 77 Zimowski, J G., et al (2014), "MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy", Neurol Neurochir Pol 48(6), pp 416-22 78 Carsana, Antonella, et al (2010), "A 15-year molecular analysis of DMD/BMD: genetic features in a large cohort", Front Biosci (Elite Ed) 2, pp 547-58 79 Stockley, Tracy L, et al (2006), "Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies", Genetic testing 10(4), pp 229-243 80 Hiraishi, Yoshiyuki, et al (1992), "Quantitative Southern blot analysis in the dystrophin gene of Japanese patients with Duchenne or Becker muscular dystrophy: a high frequency of duplications", Journal of medical genetics 29(12), pp 897-901 81 Baranov, VS, et al (1993), "Dystrophin gene analysis and prenatal diagnosis of Duchenne muscular dystrophy in Russia", Prenatal diagnosis 13(5), pp 323-333 82 Shomrat, Ruth, et al (1994), "Relatively low proportion of dystrophin gene deletions in Israeli Duchenne and Becker muscular dystrophy patients", American journal of medical genetics 49(4), pp 369-373 83 Florentin, Lina, et al (1995), "Deletion patterns of Duchenne and Becker muscular dystrophies in Greece", Journal of medical genetics 32(1), pp 48-51 84 Patiủo, Ana, Narbona, Juan, and GarcớaDelgado, Marina (1995), "Molecular analysis of the Duchenne muscular dystrophy gene in Spanish individuals: Deletion detection and familial diagnosis", American journal of medical genetics 59(2), pp 182-187 85 Singh, Vinita, et al (1997), "Proportion and pattern of dystrophin gene deletions in north Indian Duchenne and Becker muscular dystrophy patients", Human genetics 99(2), pp 206-208 86 Chaudhary, Adeel G, et al (2008), "Mutation analysis in Saudi Duchenne and Becker muscular dystrophy patients using multiplex PCR", ARCHIVES OF MEDICAL SCIENCE 4(1), p 16 87 Soong, BingWen, et al (1991), "DNA polymorphisms and deletion analysis of the DuchenneBecker muscular dystrophy gene in the chinese", American journal of medical genetics 38(4), pp 593-600 88 Niemann-Seyde, Susanne, et al (1992), "Molecular genetic analysis of 67 patients with Duchenne/Becker muscular dystrophy", Human genetics 90(1-2), pp 65-70 89 Gửkgửz, Nalan, et al (1993), "Screening of deletions and RFLP analysis in Turkish DMD/BMD families by PCR", Clinical genetics 43(5), pp 261-266 90 Trimarco, Amelia, et al (2008), "Log-PCR: A New Tool for Immediate and Cost-Effective Diagnosis of up to 85% of Dystrophin Gene Mutations", Clinical Chemistry 54(6), pp 973-981 91 Zeng, Fanyi, et al (2008), "ArrayMLPA: comprehensive detection of deletions and duplications and its application to DMD patients", Human mutation 29(1), pp 190-197 92 Wang, Xiaozhu, et al (2008), "Similarity of DMD gene deletion and duplication in the Chinese patients compared to global populations", Behav Brain Funct 4(20), pp 1-9 93 Gaudio, Daniela del, et al (2008), "Molecular diagnosis of Duchenne/Becker muscular dystrophy: enhanced detection dystrophin gene rearrangements by oligonucleotide array comparative genomic hybridization", Human mutation 29(9), pp 1100-1107 94 Flanigan, Kevin M, et al (2009), "Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort", Human mutation 30(12), pp 1657-1666 95 White, Stefan, et al (2002), "Comprehensive Detection of Genomic Duplications and Deletions in the< i> DMD Gene, by Use of Multiplex Amplifiable Probe Hybridization", The American Journal of Human Genetics 71(2), pp 365-374 96 Hegde, Madhuri R, et al (2008), "Microarraybased mutation detection in the dystrophin gene", Human mutation 29(9), pp 1091-1099 97 Hwa, Hsiao-Lin, et al (2007), "Multiplex ligation-dependent probe amplification identification of deletions and duplications of the Duchenne muscular dystrophy gene in Taiwanese subjects", Journal of the Formosan Medical Association 106(5), pp 339-346 of 98 Sherratt, T G., et al (1993), "Exon skipping and translation in patients with frameshift deletions in the dystrophin gene", Am J Hum Genet 53, pp 1007-1015 99 Welch, Ellen M, et al (2007), "PTC124 targets genetic disorders caused by nonsense mutations", Nature 447(7140), pp 87-91 100 Cirak, Sebahattin, et al (2011), "Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study", The Lancet 378(9791), pp 595-605 101 Bộroud, Christophe, et al (2007), "Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy", Human mutation 28(2), pp 196-202 LI CM N Li u tiờn cho phộp tụi xin by t lũng tri õn sõu sc ti TS Trn Võn Khỏnh, Phú Giỏm c Trung tõm Nghiờn cu Gen - Protein, Trng i hc Y H Ni l ngi hng dn khoa hc, ngi Thy ó ht lũng giỳp , dỡu dt tụi quỏ trỡnh hc tp, nghiờn cu, truyn t kin thc v nhng kinh nghim quý bỏu, cng nh to mi iu kin thun li giỳp tụi hon thnh lun ỏn ny Tụi xin by t lũng bit n sõu sc n GS.TS T Thnh Vn, Phú hiu trng, Giỏm c Trung tõm Nghiờn cu Gen - Protein, Trng i hc Y H Ni, ngi Thy ó nh hng, trc tip hng dn tụi thc hin nghiờn cu v giỳp tụi sut quỏ trỡnh hc cng nh to mi thun li cho tụi quỏ trỡnh thc hin ti v hon thnh lun ỏn ngy hụm Tụi xin by t lũng bit n sõu sc ti PGS.TS Nguyn Th H, nguyờn Phú Hiu trng Trng i hc Y H Ni, ngi Thy ó giỳp v truyn cho tụi nhng kinh nghim quý bỏu hc v nghiờn cu khoa hc Tụi xin by t lũng bit n chõn thnh n: PGS.TS Phm Thin Ngc, Trng B mụn Húa sinh, Trng i hc Y H Ni, Trng khoa Sinh húa, Bnh vin Bch Mai ó to mi iu kin, giỳp tụi quỏ trỡnh hc v thc hin lun ỏn Cỏc Nghiờn cu viờn ti Trung tõm nghiờn cu Gen-Protein, Trng i hc Y H Ni ó giỳp , to iu kin, chia s nhng kinh nghim quý bỏu tụi thc hin cỏc k thut nghiờn cu v hon thnh ti ny Cỏc Thy, Cụ B mụn Húa sinh, Trng i hc Y H Ni ó dy d v truyn t nhng kin thc quý bỏu quỏ trỡnh hc v nghiờn cu Cỏc Bỏc s, y tỏ Bnh Vin Nhi Trung ng ó trc tip giỳp tụi vic thu thp mu nghiờn cu Tụi xin gi li cm n chõn thnh n: - Ban Giỏm hiu, Phũng o to Sau i hc, Trng i hc Y H Ni - Ban Giỏm c, cỏc Phũng ban chc nng, cỏc Cỏn b viờn chc Khoa Sinh húa Bnh vin Hu Ngh Vit Tip Hi Phũng ó to iu kin cho tụi sut quỏ trỡnh hc v nghiờn cu - Xin c gi li cm n n cỏc bnh nhõn ó giỳp tụi cú c s liu lun ỏn ny - Xin cm n bn bố, ng nghip ó giỳp , ng viờn tụi rt nhiu quỏ trỡnh hc Cui cựng, tụi xin ghi nh cụng n sinh thnh, nuụi dng v tỡnh yờu thng ca Cha m tụi, cựng s ng h, giỳp , ng viờn ca v, trai, nhng ngi ó luụn bờn tụi, l ch da vng chc tụi yờn tõm hc v hon thnh lun ỏn H N, ngy thỏng 11 nm 2015 Ngc Hi MC LC T VN CHNG 1: TNG QUAN TI LIU 1.1 c im ca bnh DMD 1.1.1 Lch s phỏt hin bnh 1.1.2 Biu hin lõm sng ca DMD 1.1.3 Xột nghim cn lõm sng 1.1.4 iu tr bnh DMD 11 1.1.5 Di truyn hc ca bnh DMD 20 1.1.6 Bnh hc phõn t bnh DMD 22 1.2 Cỏc dng t bin cu trỳc ca gen dystrophin 27 1.2.1 t bin xúa on gen 27 1.2.2 t bin lp on gen 29 1.2.3 t bin im v nhng t bin nh khỏc 29 1.3 Cỏc k thut phỏt hin t bin gen dystrophin 30 1.3.1 K thut PCR 30 1.3.2 K thut FISH 31 1.3.3 K thut Southern blot 32 1.3.4 K thut MLPA 33 1.3.5 K thut gii trỡnh t gen bng mỏy t ng 38 1.4 Tỡnh hỡnh nghiờn cu bnh DMD 39 1.4.1 Trờn th gii 39 1.4.2 Ti Vit Nam 42 CHNG 2: I TNG V PHNG PHP NGHIấN CU 45 2.1 i tng nghiờn cu 45 2.2 Phng phỏp nghiờn cu 45 2.3 Dng c, trang thit b v húa cht nghiờn cu 46 2.3.1 Dng c 46 2.3.2 Hoỏ cht 46 2.4 Quy trỡnh nghiờn cu 49 2.4.1 Ly mu 49 2.4.2 a im nghiờn cu 49 2.4.3 Quy trỡnh 49 2.4.4 K thut gii trỡnh t gen xỏc nh t bin im 57 2.5 Vn o c nghiờn cu 59 Chng 3: KT QU NGHIấN CU 60 3.1 Kt qu tỏch chit DNA, RNA, tng hp cDNA 60 3.2 Kt qu xỏc nh t bin gen dystrophin 61 3.2.1 Kt qu xỏc nh t bin bng k thut MLPA 61 3.2.2 Kt qu xỏc nh t bin bng k thut gii trỡnh t gen 74 3.3 Bn t bin gen dystrophin trờn bnh nhõn DMD/BMD Vit Nam 78 3.3.1 Phõn b cỏc dng t bin gen dystrophin 78 3.3.2 Phõn b cỏc dng t bin xoỏ on gen dystrophin 81 3.3.3 Phõn b cỏc dng t bin lp on gen dystrophin 82 3.3.4 Phõn b cỏc dng t bin im gen dystrophin 84 Chng 4: BN LUN 87 4.1 Quy trỡnh tỏch chit DNA, RNA v tng hp cDNA 88 4.2 Xỏc nh t bin gen dystrophin 90 4.2.1 Xỏc nh t bin bng k thut MLPA 90 4.2.2 t bin im bng k thut gii trỡnh t gen 105 4.3 Mt s ng dng ca bn t bin iu tr v chn oỏn ngi mang gen 107 KT LUN 110 KIN NGH 111 MT S CễNG TRèNH CễNG B LIấN QUAN N LUN N TI LIU THAM KHO PH LC DANH MC BNG Bng 2.1 Kiu gen b m v t l b bnh th h 21 Bng 3.1 Kt qu phỏt hin t bin xúa on gen dystrophin 68 Bng 3.2 Kt qu phỏt hin t bin lp on trờn gen dystrophin 72 Bng 3.3 Kt qu t bin im trờn gen dystrophin 77 Bng 3.4 T l phõn b cỏc dng t bin gen dystrophin 78 Bng 3.5 T l phõn b cỏc dng t bin gen trờn bnh nhõn DMD v BMD 79 Bng 3.6 T l cỏc dng t xúa on 81 Bng 3.7 T l phõn b vựng t bin xoỏ on 82 Bng 3.8 T l cỏc dng t bin lp on 82 Bng 3.9 T l phõn b vựng t bin lp on 83 Bng 3.10 T l phõn b cỏc dng t bin im 84 Bng 4.1 u v nhc im ca k thut MLPA so vi mt s k thut sinh hc phõn t thụng dng khỏc 93 Bng 4.2 So sỏnh vựng t bin xúa on vi cỏc nghiờn cu khỏc 102 Bng 4.3 So sỏnh t l t bin lp on v xúa on 104 DANH MC BIU Biu 3.1 Cỏc dng t bin gen dystrophin trờn bnh nhõn DMD 79 Biu 3.2 Cỏc dng t bin gen dystrophin trờn bnh nhõn BMD 80 Biu 3.3 T l cỏc dng t bin xúa on 81 Biu 3.4 T l cỏc dng t bin lp on 83 Biu 3.5 Cỏc dng t bin im trờn gen dystrophin 84 DANH MC HèNH Hỡnh 1.1 Du hiu Gower Hỡnh 1.2 Hỡnh nh húa mụ dch ca protein dystrophin nhum tiờu bn sinh thit c 10 Hỡnh 1.3 Cu trỳc phõn t mRNA dystrophin trng thnh v cỏc mụ hỡnh micro v mini dystrophin 15 Hỡnh 1.4 Gentamicin giỳp b mỏy dch mó ca t bo vt qua mó dng t bin bng vic s dng axit amin glutamin cho mó dng UAG 17 Hỡnh 1.5 Vai trũ ca vựng ESE quỏ trỡnh ct ni exon-intron 18 Hỡnh 1.6 Mụ hỡnh liu phỏp s dng AON gõy xúa exon khụi phc li khung dch mó dystrophin 19 Hỡnh 1.7 V trớ ca gen DMD trờn NST X 23 Hỡnh 1.8 Cu trỳc ca gen Dystrophin 24 Hỡnh 1.9 Cỏc vựng chc nng ca protein dystrophin 25 Hỡnh 1.10 C ch bnh sinh ca DMD thiu ht dystrophin 26 Hỡnh 1.11 Gi thuyt v khung dch mó bnh Becker v Duchenne 28 Hỡnh 1.12 Xúa on vựng trung tõm gen DMD 28 Hỡnh 1.13 Phõn b t bin lp on gen ca bnh DMD/BMD bng k thut Southern blot 29 Hỡnh 1.14 Nguyờn tc ca k thut multiplex PCR 30 Hỡnh 1.15 Hỡnh nh xỏc nh t bin gen dystrophin bng k thut FISH 32 Hỡnh 1.16 K thut Southern blot xỏc nh t bin gen dystrophin 33 Hỡnh 1.17 Nguyờn tc ca k thut MLPA 35 Hỡnh 1.18 Kt qu MLPA phỏt hin xúa on gen DMD exon 48-50 37 Hỡnh 1.19 Kt qu MLPA phỏt hin t bin lp on gen 37 Hỡnh 1.20 Nguyờn tc gii trỡnh t gen theo Sanger v CS 38 Hỡnh 1.21 Mụ hỡnh gõy skip exon 19 chuyn t th bnh DMD 42 Hỡnh 2.1 Hỡnh nh minh cỏch tớnh toỏn exon lp on tng ng vi phn mm GeneMarker 56 Hỡnh 3.1 Kt qu o nng DNA bng mỏy Nanodrop ca bnh nhõn MS33 60 Hỡnh 3.2 Kt qu o nng RNA v cDNA bng mỏy Nanodrop ca bnh nhõn MS28 61 Hỡnh 3.3 Kt qu MLPA ca bnh nhõn MS1 62 Hỡnh 3.4 Kt qu MLPA ca bnh nhõn MS4 63 Hỡnh 3.5 Kt qu MLPA ca bnh nhõn MS33 64 Hỡnh 3.6 Kt qu MLPA ca bnh nhõn MS24 65 Hỡnh 3.7 Kt qu MLPA ca bnh nhõn MS53 66 Hỡnh 3.8 Kt qu in di sn phm PCR ca DNA bnh nhõn MS53 vi cp mi 44,45 (-)i chng õm, (+)i chng dng, (P) mu bnh nhõn 66 Hỡnh 3.9 Kt qu MLPA ca bnh nhõn MS3 67 Hỡnh 3.10 Kt qu MLPA ca bnh nhõn MS10 70 Hỡnh 3.11 Kt qu MLPA ca bnh nhõn mó s MS120 71 Hỡnh 3.12 Kt qu xỏc nh t bin gen ca bnh nhõn mó s MS28 73 Hỡnh 3.13 Kt qu phõn tớch cDNA ca bnh nhõn mó s MS123 75 Hỡnh 3.14 Kt qu phõn tớch cDNA ca bnh nhõn MS128 76 Hỡnh 3.15 Kt qu phõn tớch cDNA ca bnh nhõn MS121 76 Hỡnh 3.16 Bn t bin xúa on gen dystrophin trờn bnh nhõn DMD/BMD Vit Nam 85 Hỡnh 3.17 Bn t bin lp on, t bin im gen dystrophin trờn bnh nhõn DMD/BMD Vit Nam 86 5,10,15,17,18,19,23,24,25,28,32,33,35,37,38,45,60,61,62,63,64,65,66,67,70,71,73,75,76,79,80,81, 83,84,86 2-4,6-9,11-14,16,20-22,26,27,29-31,34,36,39-44,46-59,68-69,72,74,77,78,82,87-