Xác định đột biến trên một số exon trọng điểm của gen RB1 ở bệnh nhân u nguyên bào võng mạc

B GIO DC V O TO B Y T TRNG I HC Y H NI -*** - VU C ANH Xỏc nh t bin trờn mt s exon trng im ca gen RB1 bnh nhõn u nguyờn bo vừng mc Chuyờn ngnh : Hoa sinh y hoc Mó s : 62720401 LUN VN THC S Y HC Ngi hng dn khoa hoc: TS TRN HUY THINH H NI - 2017 LI CM N Vi lũng kớnh trng v bit n sõu sc, tụi xin gi li cm n chõn thnh ti: TS.BS Trn Huy Thnh, Phú Trng B mụn Hoỏ sinh, Trng i hc Y H Ni, ngi hng dn khoa hc, ngi Thy ó trc tip ch bo, tn tỡnh truyn t nhng kin thc, kinh nghim quý bỏu v to mi iu kin thun li sut quỏ trỡnh hc v nghiờn cu tụi cú th hon thnh lun ny GS.TS.BS T Thnh Vn, Phú Hiu trng Trng i hc Y H Ni, Trng B mụn Hoỏ sinh, Giỏm c Trung tõm nghiờn cu Gen- Protein, Trng i hc Y H Ni ó giỳp v to mi iu kin thun li cho tụi sut quỏ trỡnh hc ti b mụn PGS.TS.BS Phm Trng Vn, Trng B mụn Mt, Trng i hc Y H Ni, Trng Khoa To hỡnh- Thm m Bnh vin Mt Trung ng, ch nhim ti cp b Nghiờn cu c im lõm sng, cn lõm sng bnh U nguyờn bo vừng mc v xỏc nh yu t di truyn bng k thut sinh hc phõn t ó giỳp v to mi iu kin thun li cho tụi sut quỏ trỡnh thc hin ti ny Tụi xin gi li cm n sõu sc ti cỏc Thy Cụ, cỏn b ging dy, k thut viờn v y cụng B mụn Hoỏ sinh Trng i hc Y H Ni, ó ch bo, giỳp tụi sut quỏ trỡnh hc ti b mụn Tụi xin gi li cm n chõn thnh ti PGS.TS BS Trn Võn Khỏnh, Phú Giỏm c Trung tõm nghiờn cu GenProtein, Trng i hc Y H Ni cựng ton th cỏn b Trung tõm nghiờn cu Gen- Protein, Trng i hc Y H Ni ó tn tỡnh giỳp tụi quỏ trỡnh hc v 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 ó to iu kin cho tụi c hc v nghiờn cu Tụi xin gi li cm n sõu sc n cỏc bnh nhõn v ngi tỡnh nguyn ó giỳp tụi cú th thc hin c nghiờn cu ny Cui cựng tụi xin ghi nh cụng n sinh thnh, nuụi dng v tỡnh yờu thng ca Cha M tụi, cng nh s giỳp ca cỏc anh em gia ỡnh, nhng ngi ó luụn bờn tụi, l ch da vng chc cho tụi yờn tõm hc v hon thnh lun H Ni, ngy thỏng nm Bỏc s ni trỳ V C ANH LI CAM OAN Tụi l: V c Anh, hoc viờn Bỏc s ni trỳ khoa 40 Trng i hoc Y H Ni, chuyờn ngnh Hoa Sinh, xin cam oan: õy l lun bn thõn tụi trc tip thc hin di s hng dn ca Thy: TS.BS Trn Huy Thnh 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 16 thỏng nm 2017 V C ANH MC LC DANH MC CC CH VIT TT UNBVM U nguyờn bo vừng mc ddNTP Dideoxyribonucleotide triphosphate EMQN DNA European Molecular Genetics Quality Network (Mng li Cht lng Phõn t chõu u) Deoxyribonucleic acid dNTP Deoxyribonucleotid-5-triphosphate RNA Ribonucleic acid IARC International Agency for Research on Cancer (T chc nghiờn cu ung th quc t) Polymerase chain reaction (phn ng khuch i chui gen) Union for International Cancer Control (Liờn minh Kim soỏt Ung th Quc t ) World health organization (t chc y t th gii) PCR UICC WHO DANH MC CC BNG Bng 1.1 Kh nng di truyn ca UNBVM 12 Bng 1.2 Xỏc xut proband mang t bin gen RB1 t bo mm 12 Bng 2.1 Thnh phn v iu kin phn ng PCR 35 Bng 2.2 Thnh phn ca phn ng PCR 37 Bng 2.3 Chu trỡnh nhit phn ng PCR 38 Bng 3.1 c im v nhom tui thi im phỏt hin bnh ca bnh nhõn UNBVM 41 Bng 3.2 c im v th bnh ca bnh nhõn UNBVM 42 Bng 3.3 Kt qu o nng v tinh sch ca cỏc mu DNA sau tỏch chit t mỏu ngoi vi ca bnh nhõn 44 Bng 3.4 Bng tng hp cỏc t bin ti exon 10, 14 v 20 50 Bng 4.1 Cỏc phng phỏp xỏc nh t bin gen RB1 53 Bng 4.2 T l phỏt hin t bin trờn gen RB1 cỏc bnh nhõn UNBVM c bỏo cỏo t cỏc quc gia khỏc 54 DANH MC CC HèNH Hỡnh 1.1 ng t mu trng UNBVM 10 Hỡnh 1.2 T bo th phm gõy u nguyờn bo vừng mc 19 Hỡnh 1.3 Hỡnh nh hin vi in t UNBVM 19 Hỡnh 1.4 Phõn tớch cỏc dũng t bo UNBVM giai on sm 20 Hỡnh 1.5 Cỏc s kin t bin gen RB1 quỏ trỡnh phỏt sinh UNBVM 21 Hỡnh 1.6 Gen RB1 21 Hỡnh 1.7 Chc nng ca protein RB 22 Hỡnh 1.8 Cỏc bc c bn ca phn ng PCR 28 Hỡnh 3.1 c im v gii ca nhom bnh nhõn UNBVM 41 Hỡnh 3.2 Kt qu o quang nng DNA ca bnh nhõn mó s (MS) RB75 40 Hỡnh 3.3 Hỡnh nh in di sn phm PCR exon 10 ca gen RB1 42 Hỡnh 3.4 Gii trỡnh t exon 10 gen RB1 ca bnh nhõn RB79 43 Hỡnh 3.5 Gii trỡnh t exon 14 gen RB1 ca bnh nhõn RB57 44 Hỡnh 3.6 Gii trỡnh t exon 20 gen RB1 ca bnh nhõn RB29 44 Hỡnh 3.7 Gii trỡnh t exon 20 gen RB1 ca bnh nhõn RB68 45 Hỡnh 3.8 S lng t bin c phỏt hin trờn cỏc exon 10, 14, 20 46 Hỡnh 3.9 Phõn b cỏc dng t bin trờn cỏc exon 10, 14, 20 59 thp Nu cỏc nh b nhiu, cỏc tớn hiu nn cao kho xỏc nh c t bin, chng t mu PCR o co ln nhiu sn phm ph buc phi tin hnh li Nghiờn cu ca chỳng tụi, cỏc hỡnh nh gii trỡnh t bng mỏy t ng u rừ nột, rt ớt nhiu chng t quy trỡnh chun hoa ó ti u Bng 4.1 Cỏc phng phỏp xỏc nh t bin gen RB1 [7] K thut FISH Loi t bin phỏt hin T l phỏt hin Mt on ln (chuyn on nu > 8% s dng u dũ phự hp) QM-PCR Mt on, thờm on, tỏi sp xp MLPA Mt on, thờm on v tỏi sp xp Sng loc tng exon tỡm t t bin on nh, thay th bin bng gii trỡnh t nucleotid Phõn tớch t bin s dng cỏc Cỏc t bin im c bit hay ớch (Allele-specific PCR) lp li PCR methyl hoa c thự Methyl hoa quỏ mc gen ng Phõn tớch RNA t mỏu ngoi vi (phõn tớch sõu cỏc intron) phỏt hin cỏc t bin v trớ ni, cỏc on tỏi sp xp ln 37% 16% 70-75% 30% 10-12% mụ cha t bo UNBVM < 5% cỏc cỏ nhõn khụng xỏc nh c t bin trờn DNA Da vo cỏc nghiờn cu trờn th gii (bng 4.1), nghiờn cu ca chỳng tụi ó s dng phng phỏp gii trỡnh t gen phõn tớch cỏc gen t bin õy l mt phng phỏp c in c s dng rng rói phỏt hin nhng t bin ó c cụng b v tỡm nhng t bin mi cha c cụng b u im ca phng phỏp ny: t l phỏt hin t bin cao nht 70-75%, giỏ thnh r [7] Tuy nhiờn k thut ny cn thc hin qua nhiu bc, thi gian thc hin mu mt khong 3-4 ngy sau mi co kt qu 4.3 Kt qu xỏc nh t bin T l phỏt hin t bin nghiờn cu ca chỳng tụi trờn exon gm: exon 10, exon 14, exon 20 ch chim 10,2 % (5/49) thp hn so vi 60 mt s nghiờn cu trờn th gii Thp hn mt s nc chõu nh Trung Quc 19% [60] n 66% [65] Bng 4.2 T l phỏt hin t bin trờn gen RB1 cỏc bnh nhõn UNBVM c bỏo cỏo t cỏc quc gia khỏc Quc gia Nht Bn c Tõy Ban Nha, Colombia, Cuba Trung Quc Phng phỏp nghiờn C mu cu SSCP, DHPLC, FISH 51 SSCP, HDA, gii trỡnh 71 t Gii trỡnh t, Microsatellite marker SSCP SSCP, gii trỡnh t, í RT-PCR Gii trỡnh t, MLPA, New Zeland FISH, phng phỏp bisulphite chõu u, Bc QM-PCR, gii trỡnh t, M, chõu AS-PCR DHPLC, gii trỡnh t, Thy in STR marker QM-PCR RFLP, FG, n gii trỡnh t Phỏp DHPLC, QMPSF Mexico SSCP-gii trỡnh t Argentina Gii trỡnh t Gii trỡnh t,RT, Bc M QSBA, LOH Tõy Ban Nha Gii trỡnh t, RT-PCR T l t Trớch bin dn 39% [57] 72% [58] 107 50% [59] 42 19% [60] 35 37% [61] 20 50% [62] 1020 49% [63] 65 45% [64] 74 66% [65] 192 48 21 46% 19% 24% [66] [67] [68] 180 50% [69] 43 67% [70] Trong nghiờn cu ca chỳng tụi, gii trỡnh t phỏt hin co mt t bin mt on c.1033delCT dn ti lch ton b trỡnh t khung sau t bin ti exon 10 t bin ti v trớ ny lm thay i acid amin th 345 t Threonin thnh Arginin v khung oc dn ti to mó kt thỳc sm (stop codon) sau codon k tip Trong nghiờn cu cha co iu kin tin hnh kim tra mc gõy bnh ca t bin mt nucleotid, nhiờn cỏc 61 nghiờn cu trc õy ó chng mỡnh rng, cỏc t bin mt nucleotid u s dn ti dch khung v to mó kt thỳc sm Nhng t bin ny ó c chng minh l lm thay i v lm mt chc nng ca protein RB, l nhng t bin gõy bnh [59], [63], [69] Cũn li ch yu gp cỏc t bin sai ngha (missense) chim t l 83,3%, o co 03 t bin d hp t trờn cựng exon 14 C 02 t bin ny u l dng t bin sai ngha t bin ti v trớ c.1357 A>T lm bin i acid amin Tyrosin v trớ 452 thnh Phenylalanin gp bnh nhõn, t bin ti v trớ c.1366 T>A lm thay i acid amin Valin v trớ 455 thnh Glutamat, t bin c.2041 G>T trờn exon 20 thay lm thay i b ba mó hoa cho acid amin Tryptophan tr thnh b ba Cystein t bin trờn exon 20 c.2077_2079 AAC>TTA lm thay i acid amin lin k: Glutamat thnh Valin v Leucin thnh Isoleucin ti v trớ 692 v 693 tng ng Cỏc t bin ny u l t bin mi v cha tng c cụng b trc õy Trong o a phn cỏc nghiờn cu v t bin RB1 thy t bin vụ ngha (nonsense) xut hin vi t l nhiu hn [4],[52] Mc dự nghiờn cu ny, cỏc t bin sai ngha mi phỏt hin cha c kim chng thc nghim mc gõy bnh trờn invo, nhiờn cỏc nghiờn cu trc õy ó chng minh rng cỏc t bin sai ngha ti cỏc v trớ khỏc co th lm nh hng ti chc nng ca cỏc tỳi A, B (pocket A, B), cỏc cu trỳc helix lm nh hng ti chc nng ca protein RB [52], [59] Co s khỏc bit nh trờn l mu nghiờn cu ca chỳng tụi, ch chon ngu nhiờn 03 s cỏc exon khụng phi l hotspot nhng gp tn sut t bin cao v co th cỏc nghiờn cu ti cỏc quc gia khỏc nhau, s dng phng phỏp nghiờn cu khỏc 62 KT LUN Qua nghiờn cu xỏc nh t bin gen trờn exon 10, 14, 20 49 bnh nhõn UNBVM chỳng tụi rỳt mt s kt lun nh sau: Xỏc nh c 5/49 (10,2%) bnh nhõn UNBVM co t bin d hp t gm: c.2041 G>T, t bin c.1357 A>T, t bin c.1366 T>A, c.2077_2079 AAC>TTA, c.1033delCT õy l cỏc t bin mi cha c cụng b T l t bin nhom nghiờn cu cao nht gp exon 14 (3 t bin), sau o l exon 20 (2 t bin) v exon 10 (1 t bin) Trong o co 5/6 l t bin sai ngha (missense mutaion), v 1/6 l t bin xoa on (deletion mutation) TI LIU THAM KHO Bishop JO, Madson EC (1975) Retinoblastoma: Review of the current status Surv Ophthalmol, 19, 342-66 < https://ghr.nlm.nih.gov/condition/retinoblastoma > oc ngy 25/09/2016 Shields 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mutations by 67 DHPLC and QMPSF Hum Mutat 23, 193-202 Macias M, Dean M, Atkinson A et al (2008) Spectrum of RB1 gene mutations and loss of hererozygosity in Mexican patients with retinoblastoma : indentification of six New mutations Cancer 68 Biomark 4, 93-9 Dalamon V, Surace E, Giliberto F et al (2004) Detection of germline mutations in Argentine retinoblastoma patients: low low and full penetrance retinoblastoma caused by the same germline truncating 69 mutation J Biochem Mol Biol 37,246-53 Nichols KE, Houseknecht MD, Godmilow L, et al (2005) Sensitive multistep clinical molecular screening of 180 unrelated individuals with retinoblastoma detects 36 New mutations in the RB1 gene 70 Hum Mutat 25, 566-74 Alonso J, Garcớa-Miguel P, Abelairas J, et al (2001) Spectrum of germline RB1 gene mutations in Spanish retinoblastoma patients: Phenotypic and molecular epidemiological implications Hum Mutat 17, 412-22 PH LC PHIU THễNG TIN BNH NHN Thụng tin chung Ho v tờn: Nam N Nm sinh: a ch: Ho v tờn b: Tui: Ho v tờn m: Tui: Ho v tờn anh/ch/em rut: Tui: Liờn h: Mó s h s: Tin s Tin s bn thõn: Tin s gia ỡnh: Tỡnh trng bnh UNBVM ti thi im nghiờn cu Thi im phỏt hin bnh: Kt qu gii phu bnh: Xột nghim t bin gen Ngy nhn mu: Tỡnh trng bnh phm: Kt qu xỏc nh t bin gen RB1 trờn 03 exon Exon 10: t bin Khụng t bin Exon 14: t bin Khụng t bin Exon 20: t bin Khụng t bin DANH SCH BNH NHN U NGUYấN BO VếNG MC LM XẫT NGHIM GEN TI TRUNG TM NGHIấN CU GEN PROTEIN TRNG I HC Y H NI STT 10 11 Mó bnh nhõn R01 R03 R04 R05 R06 R08 R09 R10 R11 R17 R19 H v tờn Hong Phng Th Phm Tun Nguyn Danh Kh ng c Th Nguyn Minh Kh H A Lờ Ngoc L Nguyn Tr M Nguyn Th Võn Kh H V D Ngụ Bo Ch Tui thi im phỏt hin bnh 36 thỏng 30 thỏng 48 thỏng 14 thỏng 36 thỏng 36 thỏng 48 thỏng 10 thỏng 29 thỏng 24 thỏng 24 thỏng Gii N Nam Nam Nam Nam Nam N N N Nam N Th bnh mt mt mt mt mt mt mt mt mt mt mt 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 R21 R25 R26 R27 R29 R30 R31 R41 R43 R50 R51 R52 R53 R54 R55 R56 R57 R58 R59 R60 R61 R63 R64 R65 R66 R67 R68 R69 R70 R71 R72 R73 R74 R75 R76 R77 R78 R79 Trung Nguyn Th Tr G Nguyn Quang Kh Ma Th Qunh Th Nguyn Danh D Phm Tun V Nguyn Th S Hong Chớ D Nguyn Hong A Vit L Bựi Hu A Phm Tun V Hong Chớ D Phm Duy Ph Trn Thy T Phm Ngc T Mai Th Anh Th Chu Anh Q Nguyn Bo L Phan Ngc H Bựi Phng Th Lờ Vn Th Giang Gia H Nguyn Anh Trn Minh T V Thanh H Phm Minh Ng Phm Minh Th Nguyn Thanh Ph Nguyn Th Bo Tr Nguyn Tun K V Vit Ph V Linh Ch Th Phm T N Nguyn Ngc B Nguyn Th Ngc A Bựi Trng G 36 thỏng 23 thỏng 28 thỏng 13 thỏng 20 thỏng 14 thỏng 48 thỏng 12 thỏng thỏng thỏng 36 thỏng 15 thỏng 12 thỏng 48 thỏng thỏng tui 24 thỏng tui 20 thỏng 36 thỏng thỏng thỏng 19 thỏng thỏng thỏng thỏng 10 thỏng 4,5 thỏng 20 thỏng 16 thỏng 11 thỏng thỏng 12 thỏng 48 thỏng 12 thỏng 42 thỏng 19 thỏng 03 thỏng Nam N Nam N Nam Nam N Nam Nam Nam Nam Nam Nam Nam N Nam N Nam N Nam N Nam Nam Nam Nam Nam N Nam Nam N Nam Nam N Nam Nam N N Nam mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt mt H Ni, ngy thỏng nm 2017 Xỏc nhn ca Xỏc nhn ca trng Ngi hng dn khoa hoc i hoc Y H Ni ... võng mạc tiến hành với mục ti u: Xác định đột biến số exon trọng điểm gen RB1 bệnh nhân u nguyên bào võng mạc kỹ thuật giải trình tự gen 13 Đánh giá phân bố, tỷ lệ dạng đột biến số exon trọng điểm. .. trọng điểm gen RB1 bệnh nhân u nguyên bào võng mạc CHƯƠNG TỔNG QUAN 1.1 Đặc điểm bệnh u nguyên bào võng mạc 1.1.1 Lịch sử nghiên c u bệnh u nguyên bào võng mạc Năm 1657, Peter Pawlus Amsterdam... nên số lượng nghiên c u gen liên quan tới UNBVM chưa nhi u, thông tin hạn chế Xuất phát từ thực tiễn đó, Đề tài nghiên c u Xác định đột biến số exon trọng điểm gen RB1 bệnh nhân u nguyên bào võng