gene variants in Vietnamese using RFLP-PCR

VNU Journal of Science, N a tu ral Sciences a n d Technology 25 (2009) 228-233

Assessment o f

^{O P R M l}

and

^{H R H 2}

gene variants in Vietnamese using RFLP-PCR

Dinh Doan Long*, Nguyen Thi Hong Van, Nguyen Anh Luong, Tran Thi Thuy Anh

Faculty' o f Biology'. C o lle g e o f Scien ce, VNU, Ĩ 3 4 N gu yen Trai, H an oi, lleĩnci}}!

Received 17 December 2009

Abstract. The uene encodinu the mu-opioid r c c c p t o r (O P R M Ỉ) has been reported 10 associate w it h a ra n ge o f s u b s t a n c e d e p e n d e n c e a n d ỈỈR ỊỈ2 is the ge n e c o d i n g h i s t a m i n e ỈỈ2 r e c c p t o r . the site o f a c t io n f o r v a r i o u s c o m p o u n d s , e.g. a m i t r i p t ih n e a n d m ia n s e r in , u s e d in the t r e a im e n t o f p s y c h i a t r i c d is o r d e rs . B o t h O PRM Ỉĩxná ỈỈ R ỈỈ2 ge n e s c o n t a i n s o m e s i n g le n u c l e o t i d e p o l y m o r p h i s m s ( S N P s ) , including +118A/G in O P R M Ị and +398T/C in HRH2, which were reported to change in protein s e q u e n c e s a n d r e s u lt in a f f e c t i n g the t h e r a p e u t ic re sp o n se . S o m e p r e v i o u s s t u d ie s r e v e a le d l in k a g e b e t w e e n these ra re a l le l e s a n d p r o p e n s i t y t o w a r d s d r u g d c p c n d e n c e . T h i s p a p e r r e p o rts the data o f an i n v e s t ig a t i o n o n f r e q u c n c i c s o f s e v e ra l S N P s at O P R M Ỉ a n d H R ỈỈ2 l o c i in a V i e t n a m e s e population by using RFLP-PCR technique. For O P R M l, the I V S 2 + 6 9 IG allele appeared wilh a f r e q u e n c y o f 2 5 % . T h e S N P ^ 5 4 3 G / A w e r e f o u n d at the H R ỈỈ2 lo c u s w i t h f r e q u e n c y o f 5 % . 1 ’hese frequencies seemed to concur with those found in previous studies in some other Asian populations, such as Japanese, Indian or Chinese but notably the frequencies o f these alleles found in Vietnamese appeared to dii'fer significantly from those found in European and African- A i i i c i i c a i i p u p u ì a ũ o n ^ dò i c v c a l c đ \t y p i c v i o u s i i i v c b l i i ; a U o n s .

K eyw ords: mu-opioid receptor, histamine H2 rcccptor, sinule nucleotide p o l y m o r p h i s m (SNP), PCR-RFLP.

1. In tro d u ctio n

distinct m echanism s, k n o w le d g e o f specific gcnctic prcdictors o f response could make it Phaưnacogenetic ap proach es provide a possible for

Ircatmcnl

to be m atched lo patients method for id entifyin g m ech a n ism s un d erlyin g so as to optim ize thcrapcutic response and interindividual response variation and, in m in im ize ad v erse effccts.

particular, identifying patients w h o have a C o m p o u n d s used to treat m an y d iseases

higher probability o f benefiting from a work by activating a rcccplor or inhibiting the

particular m edication [ 1 ] . B e c a u s e m edications action i f it is natural ligand. Variation in appear to exert their th erapeutic c f f e c t s through rcccp tors a m o n g s t the p o p u la lio n IS k n o w n to be caused b y allelic variation and this variation

'T’ 1 Í TOCC1-7ÍO can alter the response o f a disease to a drue

Coưcsponding author, Tcl.: S4-4-38584748 ^ t,

E-mail: longd d_ksh @ vnu.cd u.vn

amongst patients.

2 2 8

D.D. Loỉi*ị et aỉ. ỉ VhỉU Journal of Science, Natural Sciences and Technclo^y 25 (2009) 22S-233 2 2 9

Ihe mu-opioid rcccptor is implicated in the

rew ard , tolerance and w ith d raw al e ffe cts o f alco h o l and other drugs o f abuse. T h u s, the genetic loci that c o d e for these opioid receptors, O P R M l , O P R D l , and O P R K l , r e s p c c liv e ly , arc potential targets for pharm acogen etic studies o f naltrexone ( N T X ) treatment effects, '['he m u-opjoid reccp lo r p la y s a ccntra! role in m cd iatin c the c ffe c ts o f

morphine

and related opioid agonists. D cleciio n o f uenetic variation

affecting O PR M l expression or mu-opioid

rcccptor function w o u ld be an important step to w ard s understanding the origins o f intcr- individual

variation

in resp o n se to m u-opioid receptor liuands and in d is e a se s o f substance d ependence. T h e

O PR M I

c cn e cn c o d es the mu- o pioid receptor,

O PR M Ỉ

m ap s lo chrom o som e

6q24Hi25 [2].

Fo u r nuclcotide varian ts ( + 1 7 C / T , M 1 8 A / G . + 4 4 0 C / G (relative to the A T G ) and Ị V S 2 + 6 9 1 C / G ) w ere o b se rv ed [3]. One o b vio u s candidate variant for such an c ffec t is the A 1 1 8 C Ì single nucleotid e p o ly m o rp h is m ( S N P ) m exo n 1 o f O P R M l . T h is polym on'ihism c n co d cs an A s n 4 0 A s p a m m o acid substiluiion and IS reported to be iu n ctio n a lly important, but w h ile there are several reports s h o w in g that this variant ts functional, they are not e a s ily placcd in a consistent fram e w o rk regard in g the nature o f the functional e ffects [4]. A d d itio n ally, the C 1 7 T w hich e n co d cs a variant receptor with a V al at position 40 instead o f A la , have been found with varied frequen cies across populations. T h e s e fin d in g s d cm onslratcd that the opioid system is in v o lv e d in the reinforcing properties o f alcohol and that allclic variation at O P R M l is associated with differential response to m edications activc at the mu-rcceptor.

rh crclb re, genctic variation at loci cod in g for opiate system proteins, including O P R M 1 might be exp ected to affe c t risk for drug

d epen d ence and alco h o lism , and p o ss ib ly other addictive b eh av io rs as well.

Histamine is natural constituent o f many

o rgan s and tissues including the gastrointestinal tract, the im m une syste m and the brain [5]. It is a central n eu rotransm itter in the brain and is fo m ie d in the posterior hypothalam us from

cxogcneous histidine by histidine decarboxylasc (HDC). There are three known

histam ine rcccptors: H I , H2 and 1 1 3 , the latter functioning as an autoreceptor. 'I'hc IỈ2 receptor is a site o f action o f v ariou s com poun d s used m

the treatment o f psychiatric disorder, e.g.

amitriptiline and mianserin. Health et al. (2002)

have reported the su cccssfu l response o f patients with chronic, predom inantly negative t>pe schizophrenia, to the h igh ly specific H2 reccptor antagonist fam otidine [6]. ỈỈR II2 IS g e n e e n c o d i n g the hum an h ista m in e H2

receptor. The A649G SNP in this gene, leading

to am ino acid substitution o f an A sn at position 2 1 7 b y A s p w a s found to be associated with schizophrenia in B ritish C a u c a s ia n s [6].

In the present study, we applied PCR-RFLP

in investiíỉatiníỉ the frcqu cn cv o f such S N P s in V ic ln a m e se subjects as our prelim inary

assessment [7]. For O P R M I. Ihc 118G allele

appeared w ith a r e la tiv e ly h ig h freq uency o f

38%. Neither the 171' nor 649G allele 0ĨH R H 2

w ere found in the study, suggesting that these variants, i f p resent, IS u n c o m m o n in the V ietn am e se population. In this publication, \vc extended our assessm en t by an alyzin g the

frcqucncics o f IVS2 +C691G locus in intron 2 o f O PR M I gene and G543A SNP in ỈỈRỈỈ2 gene in Vietnamese, using PCR-RFLP method.

T h e s e results suíỉgest that natural variations that m ight affect function and/or be associated with psychiatric phen otyp es related to therapeutic response in m edication.

230

D.D. Loĩĩ^ et aỊ. I VNƯ louniíìỉ of Science, Naturai Scieĩices and Technoh^y 25 (2009) 22S-233

2. M aterials a n d m eth od s 2.1. Malerials

The Vietnamese population sample is composed o f 100 Vietnamese individuals and were collected randomly from patients in the Hanoi Huu Nghi Hospital and National Institute o f Hematology and Blood Transfusion, Vietnam. Venous blood samples were collected in vials containing EDTA and stored in -20‘’c for a week to a month.

2. /. M e íỉìo d s

D NA extraction. Genomic DNA was extracted from blood samples by using standard precipitation described by Sambrook eỉ a i (2001) [8] with some minor modifications. The exưacted DNA products were analyzed on a 0

.

8

% agarose gel and m easured at OD280 and OD260- OD260/OD280 w a s calculated to id en tify

the extraction efficacy and intactness o f the genomic DNA.

P C R -R F L P genotyping. For O P R M I, fragment 235bp was amplified by using the primers designed incorporating mismatched base (capitalized below) lo produce artificial restriction sites (bold) in order to analyze SNP IVS2+691C/G: forward primer: 5'-g e t ctg gtc aag get aaG a a t- 3 ’ (where a G was substituted for an A at position -4 to create a H injl site);

reverse primer: 5 ’ - g a t cat c a g tcc ata g c a cac

g g - 3 ’). PCR cycling parameters consist o f a 5min hold at 95^c, followed by 30 cycles o f 95"C (Imin), 6 r c (Im in) and 72"c (Im in).

Reaction mixture consists o f 0.4|iM each primer (Bioneer), 0.2mM dNTP, lu Taq polymerase/25|iL o f reaction and 4mM MgCK.

Following amplification, the reaction mixture was digested with Hinfl restriction

endonuclease (Fennentas) which

correspondingly cuts the 691C allele resulting in 20bp and 215bp fragments. Fragments were

resolv ed b y

3

% a g a ro s e gel elecừ op horesis.

For HRH2, the pnm crs included: forward

5 ’ -cca atg gca c a g cct c t t - 3 ’ and reversed 5 ' - g c a g ca gaa g a g ctg l t g - 3 ’ w a s u s e d to a m p lify

a 909 bp fragment, optim ized conditions for this PCR reaction arc 0.4ụM o f each primers, 0.4mM dNTP, 1 u Taq polymerase/25 |iL reaction and 3mM MgCb- PCR cycling parameters consist o f a 5-min hold at

followed by 30 cycles o f 9 6 T (Imin), 5 7 T (Im in) and IT 'C (Im in 20s). PCR products

w e r e s u b s e q u e n tly a d d e d to X m il restriction

endonuclease (Fermentas) which yield fragments o f 536bp and 373bp upon X m il digestion o f the variant PCR product containing allele (rare) 543A. The common allele 543G

has no X m il restriction site. Reaction products

were analyzed by electrophorcsis on the 1.5%

agarose gel.

The y j test was applied for verifying the allele frequency distribution o f the SNPs.

3. R esults a n d discussion

D N A extraction. Genomic DNA was extracted from blood samples anticoagulatcd

w ilii e ith e r E D T A b y u^iiig th e m c l h o d a

described by Sambrook et al (2001) [8],

Fig 1. Elecưophoresis on 1% agarose gel o f genomic D N A extracted from blood samples. M: Marker X

///rtdlll; E l - E8: Genomic D N A products.

D.D. Lon^ et al. / VNU Jounial of Science, Natural Sciences and Technoỉo<;Ịy 25 (2009) 228-233

231

In our study, the results sh o w ed that gen o m ic D N A w a s extracted s u c c e s sfu lly with this m ethod ( F i g . l ) . W hole gen o m ic D N A appears as a sharp, bright band in a g aro se gel o f electrophorcsis. O ptical density a s s a y show ed relativ e ly purified products o f O D

260'280

values ranging irom 1.6 to 2 and the concentration o f D N A w e re 3 0 - 4 0 0 |ig /m L . T h e D N A sam ples subsequently w ere diluted to concentration o f 50 |.ig/mL for further P C R experim ents.

Polym erase chain reactions. The optimizalion o f primer an nealing w a s perform ed on purified D N A sam ples. The anncalinu temperature w a s identified as

61°c

for the best result, l-'or a m p lificaiio n o f 2 3 5 b p fraumcnt to an alyze I V S 2 + 6 9 1 C / G S N P in O PRM Ỉ uene, PC'Rs w e re operated and w e have obtained specific D N A bands at the size o f 2 3 5 b p as exp ected accord ing to Its theoretical calculation (p'lg. 2).

235bp

Fig 2. Hlcclrophoresis o f PCR products O PR M Ỉ gene on agarose 1.5%, 60V. DC; neuative control;

lane 1-'^; PCR products; M: D N A marker Ikb.

F o r gcnot\piniĩ o f ỈỈRH 2, the am p lifv in g also obtained the 909b p fragm ent (nucleotide

^^8 to nucleotide + 9 1 6 ) , w h ich appeared as one sharp and specific band in the electrophoresis.

These results w ere illustrated in F ig u re 3.

9 0 9 b p

Fig 3. Electrophoresis o f PCR products HRH2 iiene on agarose 1.5%, 60V. DC; negative control; lane 1-

7: PCR products; M: D N A marker Ikb.

Genotyping an d data analysis. The P C R products frorri both o f O PR M I and IIR IỈ2 genes w e re used for digestion reaction with H inf[ and A>/h7 respectively. U p on I l i ỉì ĩ l digestion, in 100 sam ples m this study, frequen cy o f individuals with h o m o z y g o lic gen o typ e I V S 2 + 6 9 1 C / C w as 0 .56 , heterozygotic gen o typ e + 6 9 1 C /G w as 0 .3 7 and + 6 9 1 G / G w a s 0.07. T h e figure 4 b e lo w is illustration o f this result.

235bp 215bp 20bp

Fiíị4. Digestion o f PCR product W S l-O P R M ! gene with Ỉiiỉiữ for analysis o f IVS2 ^691C/G SNP. M;

marker Ikb; Lane 1, 6 and 7: homozygote CC; lane 3 and 4: GG; lane 2 and 5: heterozygote C/G.

From that, w e calculatcd allele frequencies o f ^ 6 9 1 C and ^ 6 9 I G as 0 .7 5 and 0.25.

2 3 2 D.D. Lon>^ et aỉ. / VNLỈ journal of Science, Natural Sciences and Technology 25 (2009) 228-233

respectively. T h ese results con firm ed b y

x~

(x'( 2 ) “ 0.098

lo w er than the

X'

value o f statistical significant at p = 0 .0 5 w hich is 5.99), sh o w in g that the frequen cies o f these a lle le s reached to balanced state and there w e re no deviations from H a rd y -W e in b c rg exp ectatio n s in the population. T h e c o m p ariso n s o f + 6 9 I G allele frequencies betw een V ietn am e se and other population indicated that, ihc frequen cy o f this rare allele V ietn am e se is o f av e rage to other A sia n populations and re la tiv e ly lo w c om pared to other populations w o rld -w id e.

G en otyp in g o f H RH 2 gene for a n aly sis o f allele frequencies o f + 5 4 3 G / A S N P w a s perform ed b y digestion reaction o f Xmi\.

B e c a u se o f the com m on allele + 5 4 3 G without restnction site and the rare (mutated) alle le + 5 4 3 A with one restriction site o f this en z y m e and result in two bands 5 3 6 and 3 7 3 bp, w e can identify the gen otype o f ind ividu als in study.

3 4 > 6 7 s

xh'

9 ũ y b ị j -

536bp- 3 7 3 b p -

tcst ( x ‘ (

2

) = 0 . 2 7 7 , lo w e r than the X ' value o f statistical sig n ific an t at p = 0 .0 5 w h ic h IS 5.99).

T h is result also indicated that there were no d eviation s from H a r d y -W c in b e rg expectations for distribution o f alleles + 5 4 3 G / A in this V ietn am ese population.

4. C onclusion

T h e a sse ssm e n t o f the frequen cies o f single nucleotide p o ly m o r p h ism s in O PR M Ì gene ( 1 V S 2 + 6 9 1 C / G ) and gen e c o d in g histamine Ỉ12 receptor ỈỈR H 2 ( + 5 4 3 G / A ) with 10 0 V ietn am ese in d iv id u als sh o w ed that, both o f these S N P S w e r e found in our subjects with identified frequ en cies. T h e + 6 9 I G variant in the O PR M I gene w a s at the fre q u en cy o f 0 .2 5 and + 5 4 3 A variant o f H R ỈỈ2 w a s 0 .0 5 . In the an alyzed loci, the frequen cies o f genot>T^cs are fo llo w ed H ard y - W e in b e r g exp e clatio n . T h is m eans that, the genetic c o m p o sitio n s o f these alleles are quite balanced , at least m our 10 0 individuals o f this study.

T h e c o m p a ris o n s o f the frequencies o f c om m on S N P s in O PRA ÍỈ and ỈỈR II2 gen es in

V ic ln a in c s c a p p e a r e d lu b e s iM iiia i lo litu s c p re v io u s ly found in A sia n s, e.g. Ja p an ese, Indian, C h in e s e etc. but sig n ific a n tly differed from those o f E u ro p ea n s and A frican - A m e ric a n s, like our ju dgưient in previous publication [7].

Fig 5. Digestion o f PCR product HRH2 gene with Aw/1 for analysis o f +543G /A SNP. M: D N A marker

Ikb; Lane land 3: heterozygote G/A; lane 2, 4 - 8:

homozygote GG.

A m o n g st o f 1 0 0 sam ples, 9 0 % are G G ho m o zygo us, 1 0 % are G A h eterozygous and there is no A A h o m o z y g o te in our study. T h e frequencies o f these allele s are 0.95 (G ) and 0.05 (A ). T h ese frequencies w e re tested u s i n g x “

A ckn ow ledg em ents

The authors w ould like to express sincere thanks to the Vietnam National University-Hanoi for funding the project (Research Grant No.

QG.08.09). We are also grateful to A sso c. Prof.

Trinli Dinh Dat from Faculty o f B io lo g y , Hanoi University o f Science, for his continuing expertise support and proof-reading the manuscript.

D.D. Long et al. / V N U journal of Science, Natural Sciences and Technology 25 (2009) 228-233

233

R eferences

[1] G. Joel, G. Ralitza, et al.y Opioid Receptor

Gene (O P R M l, O P R K l, and O P R D l) Variants and Response to Naltrexone Treatment for A lcoh ol Dependence, A lcoholism : C lin ical a n d experim ental research. Vol. 31, No. 4 (2007).

[2] J. Gelemter, H. Franzler, J. Cubells, Genetics

o f two mu-opioid receptor gene (O P R M l)

exon I polymoq)hisms: population studies,

and allele frequencies in alcohol- and drug-

dependent subjects, Molecular Psychiatry 4

(1 9 9 9 ) 4 7 6 .

[3] A. w . Bergen e t al.y ^ opioid receptor gene variants: lack o f association with alcohol

dependence,

M o lecu la r P sych ia try 2

(1997)

490.

[4] c. Bond, €t a l, SNP in the human mu-opioid

receptor gene alters P-endorphin binding and

activity. Proceedings o f the National Academy

o f Sciences USA 95 (1 9 9 8 ) 9608.

[5] Paul Roy Heath et a. I, Allenle of Human

histamine H2 receptor and methods o f

detection of H2 receptor variants. United states Patent, Patent No. u s 6,440,670 B1

(2002).

[6] P.R. Health, et aL, Allele of human histamine

H2 receptor and methods o f detection o f H2 receptor variants,

us Patent,

u s

6440670

(2002).

[7] Dinh Doan Long, Nghiem Phuong Le,

Nguyen Thi Hong Van, Hoang Thi Hoa, Single nucleotide polymorphism in |i-opioid

and histamine H2 receptor GENES WITHFN

Vietnamese population, VNU Journal of Science, Natural Sciences and Technology 24 (2008)

[8] Sambrook et al., M olecular cloning: A laboratory manual. Vol. 1. Cold Spring Harbor Laboratory Press (2001).

Đánh giá đa hình đơn nucleotit trong gen

^{O P R M l}

và

H R H 2 ở

người Việt Nam sử dụng RFLP-PCR

Đinh Đoàn Long, Nguyễn Thi Hồng Vân, Nguyễn Anh Lương, Trần Thị Thùy Anh

Khoa Sinh học, Trường Đ ại học Khoa học Tự nhiên, ĐHQGHN, 334 Nguyễn Trãi, Hà Nội, Việt Nam

Gen mã hóa thụ thể mu-opioid {O PR M I) được cho là có liên quan tới sự phụ thuộc thuốc và các

chất gây nghiện. HRH2 là gen mã hóa thụ thể histamine H

2

, vị trí hoạt động cùa nhiều hợp chất được

sử dụng trong điều trị các bệnh liên quan đến tâm thần (amitriptiline và mianserin). Cả hai gen này đều

chứa những vị trí đa hình đơn nucleotit - SNP (chẳng hạn, + 1 18A/G ở gen O P R M l, +398T/C ở gen

Ỉ-ĨRỈĨ2...) dẫn đến những thay đồi về trinh tự axit amin của protein và từ đỏ làm ành hường tới đáp

ứiig thuốc trong điều trị. Nhiều nghiên cứu trước đây đã đưa ra mối liên kết giữa các alen hiếm này và

xu hướng phụ thuộc thuốc. Trong nghiên cứu này, chúng tôi khảo sát các SNP IVS2+691C/G và

+543G/A ờ hai gen O P R M I và H RH 2 trong nhóm 100 cá thể người Việt Nam được thu thập ngẫu

nhiên từ Bệnh viện Hữu Nghị, Hà Nội và Viện Huyết học và Truyền máu Trung ương, Việt Nam,

bằng kỹ thuật RFLP-PCR. Kết quà cho thấy, với gen O P R M Ỉ, alen IVS2+691G xuất hiện với tần số

25%. Alen +543A ờ HRH2 được tìm thấy với tần số 5%. Các tần số này khá gần với tần số các alen

tương ứng trong các quần thể người ở Châu Á, như N hật Bàn, Ấn Độ, Trung Q uốc... nhưng khác biệt

so với tần số của các alen này ở người Châu Âu và người Mỹ gốc Phi.