Vietnam Journal of Physiology 13(3), 12/2009 ISSN

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Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1 8 5 9 - 2 3 7 6

significantly in the AD patients compared with that in the age-matched controls.

There was a significant correlation between hippocampal volumes and intracranial volumes but not with brain volumes in the controls. However, in the AD patients hippocampal volumes did not correlate with intracranial volumes but correlated with brain volumes. In those patients, hippocampal volumes also had positive

correlations with psychological tests, and showed a high sensitivity and specificity in discriminating between AD patients and controls. The results suggest that the hippocampal volume can be used as a good parameter in diagnosing and studying Alzheimer's disease.

Key words: Alzheimer's disease, hippocampus, MRI

PHAN TICH TRINH T V EXON 17 CUA GEN APP TREN BENH NHAN ALZHEIMER Nguyin Duy Bic, Trin Hai Anh, Le Thj Kim Dung,

Hoang Van Liro'ng, 0 6 Minh Trung Hoc vien Quan y

Nghien cii'u nay nham muc tieu xae dinh cac diim da hinh gen APP tren cac benh nhan Alzheimer (AD) d Viet Nam. Mau nghien cwu gdm 30 mau benh nhan AD va 15 mau chwng binh thwdng. Cac miu nay dwgc liy tO' Vien Lao khoa cua cac benh nhan tinh nguyen tham gia nghien cuv. Trinh tw doan exon 17 cua gen APP dwgc xae djnh bang phwang phap doc trinh tw trwc tiip tren may doc trinh tw ABI 3130 xl. Phan tich trinh tw cua toan bq exon 17 vdi chiiu dai 147 bp cua gen APP d 30 mau benh AD va 15 miu chwng, chiing tdi khdng tim thiy diim da hinh hay dot biin nao, ma chf xao djnh dwgc 1 alen dj hgp d vj tri 114 d 3 mau benh nhan 3, 19, 23. Day cd thi la mdt dac diim vi gen cua benh Alzheimer rai rac khdng mang tinh gia dinh d ngwdi Viet Nam.

TCF khoa: Benh Alzheimer, amyloid precursor protein, hai ma.

I.DATVANDg

Benh Alzheimer (Alzheimer's disease, AD) dai dien cho chirng sa sut tri tue la mgt trong nhQ'ng rdi loan thoai hda t h i n kinh pho b i i n n h i t cua tudi gia [7], [10]. Tuy k i l u hinh benh AD cd tfnh khdng ddng nhit, cac nghien cu'u lien quan d i n di truyin cho t h i y r i n g benh AD khdng phai la loai roi loan di truyin thuin nhit. Cac dot

b i i n d i i m cua cac gen khae nhau cd lien quan d i n benh AD [2], [3], [4], [5], [8], [9].

Sy thilu hut di truyin lien quan d i n benh AD gia dinh nam d gen APP (amyloid precursor protein) cua nhilm sac t h i 21 [4], [8], [10]. Trong benh AD gia dinh khdi phat sdm (eariy-onset familial AD, EOAD), 55%

sd benh nhan cd dot biin d gen presenilin 1 (PSEN 1) n i m tren nhiSm sac t h i 14 [1],

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[3], [5], [10] va 1 dot biin cua gen PSEN 2 tren nhilm s i c t h i 1. Sy cd mat cua gen alen apolipoprotein E (APOE) E4 tren nhiem s i c t h i 19 cd t h i lien quan vdi y l u td nguy ca cao cua phat triln benh AD khdi phat mudn (late-onset AD, LOAD) [10].

Ca s d di truyin cua benh AD d benh nhan AD cua Viet Nam cdn chua cd nhilu tac gia quan tam nghien ciru. D i u hieu diln hinh cua benh AD la tfch luy chudi peptid beta-amyloid trong cac mang bam ngoai bao, dugc tao ra tir qua trinh xue ly phan c l t protein eua beta-APP [2]. X u i t phat tir yeu c i u dd, d l tai nghien ciru cua ehiing tdi nham mdt muc tieu la phan tich trinh t u exon 17 cua gen APP cua cac benh nhan AD d Viet Nam.

2. D 6 | TU'gNG VA PHU'ONG PHAP NGHIEN CU'U

2.1. Ddi tu'O'ng nghien ciJU

T i t ca cac benh nhan dat dugc tieu chf chin doan theo Sd tay thing ke va c h i n doan I n ban thir 4 (Diagnostic and Statistical Manual IV revision - DSM-IV- TR) va tieu chi cua Vien qude gia v l cac rdi loan t h i n kinh va giao t i l p , dot quy va benh Alzheimer (National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association) (NINCDS-ADRDA) ap dung cho c h i n doan lam sang kha t h i benh AD [6], [7] dugc mdi tham gia vao nghien cu'u tai Vien lao khoa Qude gia. Nhdm chu'ng l i y tu" so benh nhan kham ngoai trii bao gdm 15

ngudi khoe manh va phii hgp ve do tudi vdi nhdm benh.

T i t ca cac ddi tugng dugc danh gia chirc nang nhan thurc va hanh ddng theo thang t r i m cam lao khoa GDS (geriatric depression scale) va chup cgng hudng tu' hat nhan (MRI). Tieu chf loai tru' gdm cac xet nghiem danh gia roi loan chire nang tuyin giap, thilu mau, thilu 812, tien sir cd diing ma tiiy, t i l n sir cd dot quy, hinh anh MRI cd tang ap lyc ngi sg hay t i e nao thit.

2.2. Phu'O'ng phap nghien ciru

2.2.1. Tach chilt ADN va nhan doan exon 17 cua gen APP

Tach chilt ADN tir t l bao bach c i u mau ngoai vi. M i u ADN tong so dugc luu tru d - 80°C cho d i n khi su' dung.

Cac hoa c h i t sir dung cho cdng viec nghien ciru dugc mua cua cac hang khae nhau va dugc trinh bay tren bang 1.

Su dung hai cap m i l xudi va ngugc dugc thilt k l cd trinh t u nhu sau: APP_F:

5' - CCCACCACTCACC/VV\TGTTCC - 3' va APP_R: 5' - TCAAGTTTACCTACCTC- CACCACAC - 3'.

Vi tri cua moi tren gen APP (ma sd D87675 tren ngan hang Genbank), vj tri cua moi xudi la 284.043 - 284.064 (tdng s l 22 bp), vj tri cua mdi ngugc la 284.564 - 284.540. Cac mdi d l nhan cac doan gen dugc dat t i n g hgp tai Hang Invitrogen.

Chilu dai doan gen dugc nhan len la 522 bp. San p h i m PCR dugc k i l m tra tren gel agarose 2%.

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Bang 1. Cac hoa chit sir dung trong thi nghiem Ten hoa chit

Protease K Ribonuclease Agarose

Thang chuin ADN EDTA

Taq ADN polymerase

Hoa chit tinh sach san p h i m PCR

BigDye® Terminator v3.1 Cycle Sequencing Kit

Cac hing cung c i p Sigma (My)

Sigma (My)

Gibco BRL Life technology (My) Fermentas (My)

Applied BioSciences (My) MBI Fermentas (My) Promega (My)

Applied BioSystems (My)

2.2.2. Doc va phin tich trinh t y gen APP Dgc trinh ty true t i l p gen APP tir cac san phim PCR cua benh nhan AD va nhdm chu'ng. Moi cua phan u'ng PCR dugc sir dung d l dpc trinh t u true tilp tren may ABI 3130 xl sir dung bd kit Bigdye v3.1 cua hang ABI.

Phan tich trinh t u exon 17 cua gen APP thu dugc bang phin m i m Sequencing analysis, Cluxtal, Bioedit... sir dung trinh t y chuin tren ngan hang gen GenBank (Ma sd D87675).

3. K^T QUA VA BAN LUAN

3.1. Nhin doan exon 17 cua gen APP bang ky thuit PCR

Vdi san p h i m ADN tdng so sau khi tach tu' m l u mau tuai, chung tdi tiln hanh toi uu hda phan u'ng PCR d l nhan dac hieu dugc exon 17 cua gen APP bing ky thuat PCR. Mdt phan irng PCR thanh cdng can phai cd thanh p h i n phan irng va chu trinh nhiet thfch hgp.

Sau khi tdi uu hda, chung tdi thu dugc thanh p h i n phan u'ng PCR nhan doan exon 17 cua gen APP nhu tren bang 2.

Bang 2. Thanh phan cac chit cho phan u'ng PCR nhan doan exon 17 gen APP TT

1 2 3 4 5 6 7 8

Thanh phin H2O

Buffer MgCb dNTP Primer F Primer R Taq polymease DNA (20ng) Tong t h i tich

T h i tich (Ml) 6,4 1,5 1 0,3 1,5 1,5 0,3 2,5 15

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Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1859-2376

Thanh phin quan trgng cua PCR la mdi. Viec d i l u chinh nhiet do g i n mdi va nong dp mdi thich hgp trong ky thuat PCR quylt djnh lugng san p h i m thu dugc.

Ndng do mdi cd lien quan tdi chilu dai doan ADN c i n nhan ban. Trong thi nghiem nay chiing tdi da sir dung nong do mdi loai moi la 10 pM. Cac dNTP la nguyen lieu cho qua trinh tong hgp sgi ADN mdi tir khudn.

Nong do ciia mdi loai dNTP phai bang nhau va phu thudc vao kich thudc cua doan ADN c i n nhan len. Ndng do cao dNTP khdng tdt cho phan irng vi nd se cd lap ion Mg^'^. Qua thir nghiem, chung tdi thiy nong do dNTP 1 mM la phu hgp.

Khudn cua PCR la ADN tdng sd da duge tach chilt va tinh sach d tren vdi ham lugng tu-10 - 100 ng ADN/phan u'ng. PCR dugc tiln hanh trong mdi trudng dem thich hgp cho hoat ddng cua enzym. Thanh phin cua dem cd t h i thay ddi tiiy theo tu'ng loai

enzym dugc su' dung trong phan irng.

Trong thf nghiem nay chung tdi sir dung dem Dream Taq Buffer cua hang Fermentas. Dem nay cho phep thich u'ng vdi mgt khoang b i i n thien rgng ve ndng do eua MgCl2 va cho san p h i m cd chit lugng tot han loai khdng chua (NH4)2S04. Dem nay cQng r i t phu hgp vdi boat ddng cua Taq ADN polymerase.

Odi vdi ky thuat PCR, thilt lap dugc mgt chu trinh nhiet phii hgp la y l u to quan trgng, dam bao cho sy gian xoan ciia phan tCr ADN cQng nhu viec g i n mdi va keo dai chudi trong mdi chu ky. N l u thay ddi khdng phil hgp mdt y l u to nao dd trong chu trinh nhiet cd t h i se thu dugc san phim PCR khdng nhu mong mudn. Sau khi d i l u chinh nhiet do g i n moi va nhiet do keo dai chudi d r i t nhilu nhiet do khae nhau tir dai 51°C d i n 94°C, chung tdi thu dugc chu trinh nhiet dp thfch hgp nhu tren bang 3.

Bang 3. Chu trinh nhiet tdi uu hda cho phan irng PCR nhan doan exon 17 gen APP Nhiet dp (°C)

Thdi gian

94 5' Ichu ky

94 30"

57 45"

72 45"

30 chu ky

72 7' 1 chu ky

4

+ M

Nham tach hoan toan cac sgi ADN kep thanh sgi dan, nhiet do phan irng duge tang len 94°C trong 4 phut. Giai doan chfnh cua phan u'ng PCR la 30 chu ky nhiet sau dd. D i u tien, nhiet do v i n dugc giQ' d 94°C trong 30 giay d l cho ADN mach kep tach hoan toan thanh mach dan. Sau khi tinh dugc nhiet do ndng chay theo ly thuylt, chung tdi tiln hanh PCR d cac nhiet dp

khae nhau va da chpn dugc nhiet do gan mdi tdi uu ddi vdi cap moi APP la 57°C trong thdi gian 45 giay.

Sau dd, nhiet do cua phan irng dugc dua v l 72°C. Day la nhiet do thfch hgp cho Taq ADN polymerase tdng hgp keo dai chudi. Do dai cua giai doan nay cQng phu thudc nhilu vao chilu dai cua doan gen c i n nhan len. Vdi tdc do tdng hgp trung

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Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1859-2376

binh cua Taq ADN polymerase la 1500 bp/phiit d 72°C, chiing tdi chgn 72°C, 45 giay ddi vdi cap moi APP F/R d l dam bao cho viec keo dai dugc xay ra hoan toan.

Sau khi kit thuc 30 chu ky, chung tdi dat nhiet do 72°C trong 6 phut d l dam bao sy tdng hgp dugc kit thuc hoan toan. San

30 29 28. 27 0 Au . z."

pham PCR dugc bao quan d nhiet do 4°C sau khi dugc kiem tra tren gel agarose 2%.

Vdi chu trinh nhiet do tdi uu dd, chiing tdi da thu dugc cac san phim PCR nhan doan exon 17 cua gen APP vdi chit lugng tdt, dugc t h i hien tren hinh anh dien di san phim PCR (hinh 1).

Hinh 1. Anh dien di san pham PCR vdi cap mdi APP F/R tren gel agarose 2%.

Mlu 17-30: san phim PCR miu benh nhan AD; M: thang ADN chuin lOObp.

Kit qua cho thiy san phim PCR la nhung bang sang dam net, tap trung cd kich thudc khoang tren 500 bp, phii hgp vdi tfnh toan ly thuylt (kfch thudc theo tfnh toan ly thuylt la 522 bp). Trong dd exon 17 cd 147 bp, tfnh tir vj tri 195 din 431.

3.2. K i t qua trinh t y exon 17 gen APP

Bang phuang phap giai trinh ty true tilp san phim PCR, chung tdi da dgc va phan tfch dugc toan bg exon 17 vdi tdng sd 147 bp d 30 mlu benh nhan AD va 15 mlu chirng. Trinh ty so sanh cua mdt so mlu benh nhan dugc trinh bay tren hinh 2.

E17_

Mau Mau Mau Mau Mau Mau Mau Mau Mau Mau

.Ref 1 7 . 1 8 . 1 9 . 2 0 . 2 1 . 2 2 . 2 3 . 2 4 . 2 5 . 2 6 .

. t t t t t t t t t t

rprnmii III IIIII iiiprprp rprri rn rp rri rp rp rp IT! rri rprp rp ITT rp rp rp rp rp IT!

rprprnfprpi pf p n i r p / p ipniiMii irprprprprprp m r p iTi rpiTiiTi rp rp r p rp rprp rp fp rp rpfp rp rp rp f^fpj MM ifprprprp rprp rpryi rn ip rp rp fp rp rp rp iTUp rp rp rp m i p rn rp m

Hinh 2.

t t t t t t a a a g TTTTTTASAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG TTTTTTAAAG

Trinh tu exon

taagcatcaa a t a t t t g a c c

TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC TAAGCATCAA ATATTTGACC

17 ciia gen APP d mdt s l

aaccagttgg AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG AACCAGTTGG

miu benh AD

gcagg GCAGS GCAGS GCAGB GCAGffl GCAGS GCAGS GCAGS GCAGS GCAGS GCAGS

aata AATA AATA AATA AATA AATA AATA AATA AATA AATA AATA

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Vietnam Journal of Physiology 13(3), 12/2009 ISSN: 1859-2376

Chung tdi khdng thiy cd diim da hinh hinh 3. Cd thi day la mdt dac diim cua hay dot biin nao tren cac mau da phan benh Alzheimer rai rac khdng mang tfnh gia tich. Tuy nhien, xult hien alen dj hgp d vj dinh tren nhdm benh nhan nay.

tri 114 d 3 mlu 3, 19, 23. Diem nay dugc thi hien d 2 dinh nhgn d cung vj tri tren

91 101 111 12J

t c a c c 1 1 g g t g a t cr c t g a a Cf a a g a a a c a q t a c g

I T L V H L K K K Q Y "

l l l l l l l l l l l I I J l l l l l l l l l l l . l

H

I l l l l l l l l l l » I I . B l l l l l l l l _ | | l l a l ,

T C A C C T T G G T G A T G C T G A A G A A G A A M C A G T A C i i

1 1 - I II • l l i l l l l l _ l l i l . l

. . H H

Il-llllillllillwllll|||||_||||.|

T C H C C T T G G T G A T G C T G A A G A A G A A E C A G T A C

l l l l l l . l l i l l l l . l l l l l l l l l . l l 1 1 1

H ;

Hinh 3. Hai alen khae nhau ciia gen APP d mgt s l miu benh AD

4. KtJ LUAN

- Da thilt k l dugc cap moi dac hieu d l nhan exon 17 cua gen APP.

- Da nhan thanh cdng doan ADN cd kfch thudc 522 bp chu'a exon 17 cua gen APP eua 30 mlu benh AD va 15 miu chirng.

- Da xae djnh dugc trinh ty exon 17 cua gen APP bang phuang phap dgc tryc tilp tu' san phinn PCR ciia 45 mlu.

- Phan tfch trinh ty cua toan bd exon 17 vdi chilu dai 147 bp cua gen APP, chung tdi khdng tim dugc diim da hinh hay dot biin nao. Tuy nhien, xuit hien alen dj hgp d vj tri 114 d 3 mlu 3, 23, 19. Day cd thi la mdt dac diim v l gen cua benh Alzheimer rai rac khdng mang tfnh gia dinh d mdt sd ngudi Viet Nam.

TAI LIEU THAM KHAO

1. Chartier-Hariin M.C. et al., (1991), Eariy-onset Alzheimer's disease

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caused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature, 353(6347): 844-846.

2. Citron M. et al., (1992), Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production. Nature, 360(6405): 6 7 2 - 6 7 4 .

3. Cruts M., Van Broeckhoven C.

(1998), Presenilin mutations in Alzheimer's disease. Hum Mutat, 11(3):

183-190.

4. Goate A. et al., (1991), Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature, 349(6311): 704-706.

5. Janssen J.C. et al., (2000), Alzheimer's disease due to an intronic presenilin-1 (PSEN1 intron 4) mutation:

A clinicopathological study. Brain, 123 (Pt 5): 894-907.

6. Morris J . C , (1997), Clinical assessment of Alzheimer's disease.

Neurology, 49(3 Suppl 3): S7-10.

7. Morris J.C. et al., (1989), The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part I.

Clinical and neuropsychological assessment of Alzheimer's disease.

Neurology, 39(9): 1159-1165.

8. Pasalar P. et al., (2002), An Iranian family with Alzheimer's disease caused by a novel APP mutation (Thr714Ala), Neurology, 58(10): 1574-1575.

9. Saunders A.M., Roses A.D., (1993), Apolipoprotein E4 allele frequency, ischemic cerebrovascular disease, and Alzheimer's disease. Stroke, 24(9):

1416-1417.

10. van Duijn C M . et a!., (1994), A population-based study of familial Alzheimer disease: linkage to chromosomes 14, 19, and 2 1 , Am J Hum Genet, 55(4): 714-727.

SUMMARY

Analysing sequence of exon 17 on APP gene in Alzheimer's disease patients

We aimed to determine if the amyloid precursor protein (APP) gene polymorphism is present in Vietnamese patients with Alzheimer's disease (AD). The subjects were 30 AD patients and 15 unaffected controls. Participants were recruited from Vietnam National Institute of Gerontology. Sequence of exon 17 on APP gene was analysed by direct sequencing on a sequencer ABI 3130 xl. After analysing exon 17 with the total lenght of 147 bp in 30 AD samples and 15 control samples, neither polymorphisms nor mutations were found, except only 1 heterozygous allele at nucleotide 114 in 3 samples numbered 3, 19, and 23. This finding may be a genetic characteristic of Vietnamese non-familial sporadic AD.

Keywords: Alzheimer's disease, amyloid precursor protein, hippocampus