TAP CHl NGHIEN CO-U Y HpC -
PHAN TiCH TRiNH TLF VUNG GEN TY THE HV1 VA HV2 TREN DAN TOC CHAM VIET NAM
Tran Thj Thuy Hang, Trdn Huy Thjnh, Tran Van Khanh Tnmng Dai hQC YHd N6i Nghien ciru du-oc thi/c hi$n v&i mijc ti§u xac <^nh trinh fy va phan tich tinh da hinh nucleotid tJon (Single nucleotid polymorphisms • SNP) vOng gen ty the HV1, HV2 tren dan tdc Cham Vi$t Nam. Ky thu$t giai trinh ti^ gen du^yc duuc ap dung de phan tich 100 mSu DNA cua ngu^i dan tgc ChSm, k4t qua duvc so sinh vol trinh tir chuan rCRS. tif do phan tich linh da hinh nucleotid don (SNP) cOa viing gen HV1 va HV2.
Ket qua cho thiy da xac dinh diroc trinh fy hoan chinh viing gen HV1, HV2 va phat hien dur?c nhieu SNP o cAc miu nghien cdu. Xac <Snh diK?c ty 1$ mOt so SNP hay g$p SNP A73G va A263G, 315msC g^p o 100% miu nghien cdu, 309insC la 64%. T16189C la 56%. A16183C la 44%, C16223T la 37%. G16129A la 23%, T16304C la 21%. C16261T la 20%; C150T la 20%; A210G la 20%. Day la s6 lieu dau tien, hoin chinh vS trinh li/ vung gen ty thi HV1 va HV2 cua dan t0c Cham voi s6 luvng miu tuong doi Ion Tfl khoa: DNAty th4, genHV1,gen HV2, dan toe Cham.
I. DAT VAN D^
Vung gen ty the HV1 va HV2 duocAnderson vd cijng sy xac dmh ndm 1981 ndm trong vung dieu khien D-loop cua DNA ty t h i duoc goi Id vCing sidu biln 1 (HV1- Hypervanable region 1) ed kich thu'flc 359bp a vi tri 16024 -16383 vd vung sieu biln 2 (HV2 - Hypervanable region 2) c6 kich thuac 315bp ndm a vi tri 57 - 372 [1]. Ddy Id vung c6 t i n s i ddt biln cao nhit trong hd gen ty t h i ngyfli [2]
Cung gilng nhu- he gen trong nhan, hd gen ty t h i mang nhflng trinh tu- da hinh. Da hinh id sy khde biet v l ehuli DNA gifla nhflng cd the, cdc nhdm, hoac ede quan the Nd bao g i m da hlnh nucleotid dan (SNP), cac chuli Idp, them doan, mdt doan vd tdi to hpp. Da hinh gen cd Dja Chilian he: Trin Van Khanh, Trung tam Nghien cdu Gen - Protein, Truung Dai hoc Y Ha NQI Email: khanhvan7364@gmail com Ngiy nh$n: 02/03/2017 Ngiy dwoc chip nhan. 26/6/2017
the Id k i t qua cua qud trinh t i l n hda hoacdLfoc tao ndn bai y l u t l bdn ngoai (nhu virus ho^c chilu xa), Ngu'fli ta cho ring da hlnh giCip ty the it nhay cam vfli su' thay d l i nang lupng, do do tao nhilu nhiet vd cdc goc ty do, giilp con ngu'fli thich flng khi di cu* tai nai cd khi hau lanh han nhy tfl chau Phi sang chau Au [3]
Cho d i n nay ngufli ta da thing ke dypc 623 kilu da hinh tren genom ty t h i ngyfli, trong dd tren gen HV1 cd 49 vj tri nucleotid thay doi, trdn gen HV2 cd 105 vi tri nucleotid thay doi dypc cdng bd trong (http://wv™ mitomap org) ndm 2013 [ 4 - 7 ]
Nhflng nghidn cflu g i n ddy, cho thiy da hinh tren gen HV1 vd HV2 cd lien quan den nhieu benh, cdc bdnh ty the thu'flng gdp nhu', hdi chflng Leigh, bdnh t h i n kinh Leber, bdnh LHON, hdi chflng Keams - Sayre. h|i chflng Pearson, hdi chflng NARP cdc benh lien quan den qud trinh chuyen hda, lao hda nhy Oai
thdo dyflng, Alzheimer, Pakinson; cac bdnh iiSn quan den ung thu [8 - 1 2 ]
D l tdi du-pc thyc hien vfli muc tieu, Xac djnh trinh t y va phdn tich tinh da hinh nucleotid dan vung gen ty t h i HV1, HV2 trdn ddn tdc Chdm Viet Nam.
II. D 6 | TU'gNG VA PHU-aNG PHAP
I . O S i tu'p'ng
100 mdu mau ngoai vi (chong ddng b i n g EDTA) cua ngu'fli binh thu'flng, khoe manh thudc dan tdc Cham Viet Nam (sdng fl tinh BinhThudn).
2. P h y a n g phap
DNA tpng s6 du-Pc tach chiet tfl cac mdu mdu toan phan theo phu'ong phap sfl dung en- zym protease K vd phenol.chlorofprm isoamyl alcohol.
Sfl'dyng cap mdi ddc hieu khuech dai vCing gen HV1 vd HV2
HV1F' 5'- CTC CAC CAT TAG CAC CCA AAG C -3'
HV1-R- 5'- CCT GAA GTA GGA AGO AGA T G - 3 '
HV2-F; 5'- GGT CTA TCA CCC TAT TAA CCA C -3'
T/iP C H I NGHIEN CCPO Y HpC HV2-R 5'- CTG TTA AAA GTG CAT ACC GCC A -3'
Thdnh p h i n eua phan flng PCR. 1X dem PCR; 2,5 mM dNTP; 0,2 p\ moi xuoi vd ngup'c;
0,5 unit Taq polymerase, 50ng DNA vd H20, tong t h i tich 20 pi
Chu trinh nhidtcua phan flng PCR. 94°C - a phiit; 30 ehu ky [94<'C- 30 giay, 54<'C - 30 gidy, 72°C - 30 gidy]; 72°C - 5 phut, bao quan mau fllS-'C.
Giai trinh t y gen theo qui trinh BigDye termi- natpr sequencing (Applied Bipsystems, Foster city, USA). T i l n hdnh tren mdy 3100-Avant Ge- netic Analyzer cua hang ABI-PRISM. So sdnh vfli trinh ty GeneBank d l xdc djnh da hinh K i t qua giai tnnh tu gen duac doi chilu vd so sdnh vfli trinh t y chuan J01415 trdn GeneBank (National center for biotechnology information, NCBI) phdn tich bdng p h i n mem CLC Main Workbench 6.01 d l xac dmh da hinh hai vung sieu bien HV1 vd HV2.
3. Dao dflc nghien c f l u
Doi tuang tham gia nghien cflu hoan todn tu- nguyen vd cd quyln rut khoi nghien cflu khi khdng ddng y tilp tuc tham gia Cdc thdng tin ca nhdn se duac dam bao bi mdt
III. K^T QUA
1. K h u l c h dai vung gen HV1 va HV2
DNA sau khi du-p-e fdeh chilt cd chdt lyang t6t se duoc sfl dung d l khuech dai gen HV1 vd HV2 b i n g cdc cap moi ddc hidu vfli d i l u kidn thdnh phan phan flng va chu trinh nhiet nhu- dd md ta a p h i n phLfong phdp.
U 1 2 S 4 5 « 7 a 5 10 -1 M 12 11 1* 14 18 17 « 1» M 21 22
422 bp
— M 3 b p
^BBHTtmri i fmr "IfiiP
(A) (B) Hinh 1. San phdm k h u l c h dai vung gen HV1 cua (A) va HV2 (B)
1-22:22 mau nghien cCfu ngutri dSn toe Cham. M. Thang chuSn 100bp
TCNCYH 108 (3)-2017
l a M H l NBHENiCU'U Y HQC-
San pham khuyech dai vdng gen HV1 vS HV2 06 chit \uqmg tot cW g6m mpt bSng a$c hi^u c6 kich tt\u6c 543 bp va 422 bp
2. KSt quS phan tich trinh tip vung gen HV1 va HV2 2.1. Trinh tip nucleotid vung gen ty the HV1
16024
'I'
TTCTTTC^ATG GGGAAGCAGA TTTGGGTACC ACCCAAGTAT TGACTGACCC 50 ATCAACAACC GCTATGTATT TCGTACATTA CTGCCAGCCA CCATGAATAT 100
T
TGTACGGTAC CATAAATACT TGACCACCTG TAGTACATAA AAACCCAATC 150 A G C CACATCAAAA CCCCCTCCCC ATGCTTACAA GCAAGTACAG CAATCAACCC 200
G
TCAACTATCA CACATCAACT GCAACTC CAA AGCCACCCCT "CACCCACTAG 260 C
GATACCAACA AACCTACCCA CCCTTAACAG TACATAGTAC ATAAAGCCAT 300 T T C l*)C (*)C
CC
TTACCGTACA TAGCACATTA CAGTCAAATC CCTTCTCGTC CC 343 16365
Hinh 2. Cac vj tri da hinh nucieotid den (SNP) cua vung gen HV1 du'O'C d6i chieu v 6 i trinh t y c h u t n do Anderson va cs cong b6 nam 1981 [1]
C1627ST C16291A T16297C Genebank | ^^ ^l'
J O U I S G A T A C C A A C A A A C C T A C C C A C C C T T A A C A G T A C A T A G T A C A T A A A G C C A l fContllct ,Car>flict ,Coiillict
x • >
Consensus 3 A T A T C A A C A A A C C T A C A C A C C C T C A A C A G T A C A T A G T A C A T A A A G C C A 1
Coverage J
MSIZ J J J ^
L M 0 3 2 5 0 2 H V S I - R 3 A T A T C A A C A A A C C T A C A C A C C C T C A A C A G T A C A T A G T A C A T A A A G C C A 1
Hinh 3. Hinh anh giai trinh t y mot sd SNP v i m g gen ty t h i HV1 cua m l u nghien c f l u ma s6 12 (MS12)
(SNP: C16278T; C16291A, T16297C)
- T i p CHi RGHliN CO'UJiJteCi
2.2. Trinh tir nucleotid vung gen ty the HV2 73
ATGCACGCGA TAGCATTGCG AGACGCTGGA GCCGGAGCAC CCTATGTCGC 50 G G C
AGTATCTGTC TTTGATTCCT GCCTCATCCT ATTATTTATC GCACCTACGT 100 G A C TTC G
TCAATATTAC AGGCGAACAT ACTTACTAAA GTGTGTTAAT TAATTAATGC 150 C C C G
TTGTAGGACA TAATAATAAC AATTGAATGT CTGCACAGCC ACTTTCCACA 200 G
CAGACATCAT AACAAAAAAT TTCCACCAAA CCCCCCCTCC CCCGCTTCTG 250 ( t ) C (+)C
CC CCC
GCCACAGCAC TTAAACAC 268
t
340
Hinh 4. Cac v i t r i da hinh nucieotid do'n (SNP) cua vOng gen HV2 du'O'C d d i c h i g u v 6 i trinh tir c h u i n do Anderson va cs cong bd nam 1981 [1]
249DelA A263G Cenebank \ i
J 0 1 4 1 5 ; A T A A T A A T A A C A A T T G A A T G T C T G C A C A G C C A C T T T C C A C A C A G A C A T I
Consensus:ATAATAATAACAATTGA.TGTCTGCACAGCCOCTTTCCACACAGACATi Coverage
MS15 J J
)7662_KN710_HVSJi;ArAAT'AAtAACAATTSA.IGIC i j . A C A O C C G C l 1 fCCACACAGACAT'
Hinh 5. Hinh anh giai trinh ty? m9t s6 SNP vung gen ty the HV2 cua m l u nghien c f l u ma s 6 15 (IVIS15)
(SNP: 249DelA. A263G)
TCNCYH 108 (3)-2017
TAP CHf NGHIEN CLPU Y HQC - 2.3. Da hinh nuleotid dcyn (SNP) vung gen ty the HV1 va HV2
K i t qua giai trinh tu gen HV1 vd HV2 cua 100 mau nghien cflu du'pc tien hanh so sanh vfli trinh tu- chuan, eho thiy co kha nhilu vi tri da hinh so vfli trinh tir chuan M l u co it vi tri da hlnh nhat Id 7, trong khi m l u ed nhieu vi tri da hlnh nhit Id 19. Chung tdi cQng nhdn thiy ring, cdc vi tri da hlnh tap trung nhilu han a gen HV1
Cac VI tri da hinh hay gap fl cdc mau nghidn
cflu Id 309insC Id 64%, T16189C la 56%, A16183C la 44%, C16223T la 37%, G16129A Id 23%, T16304C la 2 1 % , C16261T Id 20%, C150T Id 20%, A210G la 20%. dac biet Id ba vj tri da hinh A73G, A263G vd 315insC gapd 100% mau nghidn eflu Cdc dOt biln thay the IS pho biln nhdt, cdc nucleotid them vdo thu'flng la C trong khi cac nucleotide m i t thuang la A, Cdc k i t qua thu du-ac v l cdc vj tri da hlnh trgn hai vung gen HV1 vd HV2 ph^n anh toe do dot bien r i t cao cua vung gen ndy
Bang 1. Bang cac vt tri da hinh thu'flng gap tren gen HV1 va HV2 Gen HV1
T16189C A16183C C16223T G16129A T16304C C15261T
T y l e 56/100 44/100 37/100 23/100 21/100 20/100
Ty ie % 56%
44%
37%
23%
2 1 % 20%
Gen HV2 A73G 315insC
A263G 309insC A210G C150T
T y i 6 IOC/100 100/100 100/100 64/100 20/100 20/100
T y l e % 100%
100%
100%
64%
20%
20%
IV. BAN LUAN
Trinh tu- vung gen ty the HV1 vd HV2 da phdn tich du'oc c6 kich thuoc tuang flng Id 543bp vd 422bp theo trinh tu chudn CRS/
rCRS cua gen ty the Trong thuc te, cdc tai lieu khac nhau xac djnh do dai vDng gen HV1 vd HV2 la rdt khac nhau Vung gen HV1 eua trinh ti^ chuan CRS cd kich thu'flc 359bp (tfl vi tri 16024 - 16383) vd vijng gen HV2 cd kich thuac 325bp (tfl vi tri 057 - 372) [1] Theo TWGDAM (Technical Working Groups for DNA Analysis Methods) cho ring trinh tu t i i thieu diFpe chap nhan cho viing gen HV1 ia tfl vi tri 16024 - 16365 vd cho vung gen HV2 la tfl vi tri 073 - 340 Trinh tu' chuin Cambridge sau khi chinh sfla (rCRS) cho thiy vung dilu khiln D-loop n i m tfl vi tri 16104 d i n 16569 va tfl 1 den 191 va khdng chi rd vi tri viing gen ty the
HV1 va HV2 [4], Nhu" vay, trong nghien cdu nay chiing tdi dd xac dmh duac trinh tu' hoan chjnh ciia vung gen HV1 vd HV2.
Trinh tu eua ede mdu nghien cflu duoc doi chilu'vd so sdnh vfli trinh tu- chuin, kit qua cho thdy cd rat nhilu da hinh nucleotid don (cd tfl 7 d i n 19 SNP a m5i m l u nghien ciiu), Nghien cflu ndm 1999, trdn 50 ngu'fli Viet Nam da phat hien dupe 20 da hinh vi tri cdc enzym cdt tren DNA ty the, trong dd 3 vi tri cOa enzym Haell - 13Viet, Mspl - 19Viet, Mspl - 20Vietla cac da hlnh mfli [5], Khi giai trinh tu" 2 vimg gen HV1 vd HV2 tren ^ m l u ddn toe Kinh, 2 mau dan tdc Tay va mpt mdu ddn tdc H'Mdng, i3§
phdt hidn 26 vi trl da hinh trdn HV1 vd 5 vi tri da hinh tren HV2 [6], SM' da hinh tren hai viing sieu biln HV1 va HV2 h l u h i t Id cdc da hinh do'n 12
nucleotid (SNP). Cac vi tri da hlnh cd t h i Id cdc dot b i l n dong hodn (transition) - dot b i l n thay t h i nucleotid cd ciing g l c purin (A - G) hoac pyrimidin (C - T); hoac la cdc dot b i l n di hodn (transversion) - ddt b i l n thay t h i nucleotid cd goc purin thdnh pynmidin hoac ngu-pc lai (A - T, C - G). Cdc da hlnh hay gap nhit la su" thay t h i nucleptid, cac nucleotid chen vao thu'flng Id C trong khi cac nucleotid bi mat thu'flng Id A, [7]
Nghien eflu cua chung tdi cung nhdn thay cdc da hlnh thijang gap Id su' thay t h i nucleotid C.
Dgng dj hap l f l cua vung lap Cystein (C-stretch) trong trinh t y HV1 vd HV2 dd dupe nghien cflu nhilu vd ty Id da hlnh viing ndy ed sy khde biet ed y nghTa thing ke gifla ede chung tdc ngu'fli khde nhau. Vi vdy, da dang dl truyen trong viing C-stretch co y nghTa quan trpng trong giam dinh hinh su' gen vd nghidn cflu di truyln q u i n t h i . Nam 2009, mot nghidn cflu v l da dang di truyln eua viing C-stretch tren 919 ngu'fli Trung Qudc, thupc 8 chung tdc khac nhau Ket qua cho thiy, haplogroup AAAACCCCTCCCCC va AACCCCCCTCCCCC Id cdc haplogroup dac tru'ng cho qudn t h i ngufli dan tdc Tibetan va Salar cua Trung qudc vd tyle da hinhT16189C thudc vung lap nucleotide Cystein cua gen HV1 Id 25,14% [13], Ty Id da hinh nuleotid dan T16189C trdn dan tdc Cham trong nghidn cflu cua chCing tdi la 56%, c i n cd them nghien cflu trdn cdc dan toe khac de tim ra cdc haplogroup dae tryng cho tflng dan tdc
Nghidn cflu eung da thing ke ra mdt sd V! tri da hinh nucleotid don hay gap tren gen HV1 va HV2 cua DNA ty t h i 309insC Id 64%, T16189C Id 56%. A16183C la 44%. C16223T Id 37%, G16129A la 23%, T16304C Id 2 1 % , C16261T la 20%, C150T la 20%, A210G la 20%, 6$c biet Id ba vi tri da hinh A73G, A263G va 315ins (C hoac CC) gap fl 100% m l u nghien cflu. Ket qua nay cung tu'ang dong vfli
TfyP CHI NGHIEN CLPU Y HQC k i t qua trong nghien cflu CLJa cac tdc gia (ndm 2008) khi nghidn cflu trdn 78 ngyfli Viet Nam da thong kd du'pc 10 vj tr( da hlnh hay gap nhu' T16172C (22/78), A16183C (23/78), T16189C (31/78), C16223T (26/78), T16304 (26/78), C150T (20/78), 249DelA (25/78). A263G (75/78), 315C (24/78), A73G (78/78) trong dd V! tri A73G cung g d p f l 100% mdu nghien cflu, [7]
Tinh da hlnh/ddt b i l n cua DNA ty t h i cd the hen quan d i n cac loai benh khac nhau trong dd cd ede benh v l chuyen hda, Ido hda nhu' Odi thao dyflng, Alzheimer, Pakinson; cdc benh hen quan d i n ung thy [9 - 12],.. VI vdy, cdc phdt hien v l da hinh/ddt b i l n cua DNA ty the cd vai trd rat quan trong trong viec tim hieu ve phat smh benh a ngu'fli. Cdc sd lieu thu du'oc vltrinhtu'DNAty the ciing vfli cac VI tri da hinh DNA ty the dd phat hidn Id nhflng tu lidu cdn thilt cho nhflng nghien cflu t i l p theo v l bdnh ly di truyln lien quan d i n DNA ty t h i ngu'fli fl Viet Nam. Nhu vdy, nghidn cflu da xdc dmh duae trinh t y hodn chmh vung gen HV1 vd HV2, xdc dinh duac cdc vi tri da hinh trdn gen HV1, HV2. Ddy Id sd lidu dau tien hodn chinh v l trinh t y gen HV1 va HV2 cua ddn tdc Chdm Viet Nam vfli sd lupng mau tuang d l i Ifln.
V. K^T LUAN
Nghien cflu da xdc dinh dupe trinh t u hoan chinh viing gen HV1 vd HV2 cua 100 mau ngufli dan tdc Chdm Viet Nam Ty le mdt so SNP hay gap la 309insC Id 64%, T16189C Id 56%, A16183C id 44%, C16223T la 37%, G16129A Id 23%, T16304C Id 2 1 % , C16261T Ia20%, C150Tla20%, A210G ld20%, ddcbiet Id ba VI tri da hinh A73G, A263G va 315insC gdp fl 100% mdu nghien cflu.
Lai cam a n
Dd tdi duoc thuc hien vfli sy hd trp kmh phi cua de tdi nhanh cap nhd nuflc "Nghien TCNCYH 108 (3)-2017
J A t G H l NGHIEN CLFU Y HQ^Q, cflu m0t sc chl sp sinh hpc, trinh t y gen ty the ngyfli Viet Nam Iryflng thdnh vd ddt biln gen gdy benh thalassemia" thudc de tai nhiem vu Quy gen "Ddnh gid dac d i l m di truyln ngyfli Vidt Nam"
TAI LIEU THAM KHAO 1. Anderson A, Bankier AT, Barrel BG et
• al (1981), Sequence and organisation of the human mitochondnal genome. Nature 290, 457 - 465
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ngufli Vidt Nam. Tap chl Cong nghe Sinh hgc.
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7. Nguyin Dang Tdn, Nguyin thj Tji Linh, Vu Hai Chi, (2008) Da hinh don bOi DNA ty t h i cua cac cd the ngufli Vidt Nam.
Tgp chl COng nghB sinh hoc. 6, 579 - 590 8. Davidzon, et al (2005). Mitochondrial DNA and disease. Annals of Medicine 37,222 -232,
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Summary
SEQUENCING ANALYSIS OF mtDNA HYPERVARIABLE 1 AND HYPERVARIABLE 2 OF CHAM ETHIC IN VIETNAM
The objective cf this study was to perform the sequencing analysis of mtDNA HV1, HV2 and to identify single nucleotide polymorphisms (SNP) of the HV1 and HV2 on Cham people in Vietnam, Sequencing method is used to analyze 100 DNA samples of Cham. The results were compared with the rCRS sequence, reported in the Human mitochondrial database, and then classified those sequences into the mitochondnal DNA haplogroups. We have performed sequencing analysis of
TAP CHl K l G H r a r a M Y H Q C the HV1. HV2 and discovered many SNPs in tlie sample, it was found ttiat ttie frequencies of SNP A73G and A263G are 100%. 315insC is 100%, 309insC is 64%, T16189C is 56%, A16183C is 44%, C16223T is 37%, G16129A is 23%, T16304C is 2 1 % , C16261T is 20%, C150T is 20%, A210G Is 20%. In conciusion, we have assessed the single nucleotide polymorphisms of mitochondrial DNA HV1 and HV2 of 100 Cham ethnic samples
Key words: mitochondrial DNA, HV1 gene, HV2 gene, Cham ethnic.
TCNCYH 108 (3)-2017
