Journal of Life Sciences Volume 7 Number (1)
J LS
Journal of Life Sciences
Volume 7, Number 4, April 2013 (Serial Number 60)
Contents
Molecular Biology and Bioinformatics
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
Ke Xu, Yi Tang, Jia Lai, Ze-Sheng Yan, Qian Luo and Huan-Xiu Li 341
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension Related LVH (Left Ventricular Hypertrophy) Patients in Malaysia
Julia Ashazila Mat Jusoh, Norlaila Danuri, Fadhlina Abdul Majid, Hoh Boon Peng and Khalid Yusoff
348 Charaterization of Citrus Hybrid “Huangguogan” through the Combination of Morphological and Molecular Markers
Xue-Fei Wang, Xi-Rui Xiong, Xue-Li Pu, Qiao-Qiao Yan, Bo Xiong, Feng-Ling Liao, Qian-Qian Fan and Zhi-Hui Wang
A Test for Stabilization of an Oligomeric Protein by Introduction of Aromatic Residues into the Interface
Yuho Mano, Ayako Shiota, Kotaro Hara, Azumi Hirata, Masayuki Oda and Kazufumi Takano
358 Automated Classification of Segmented Cancerous Cells in Multispectral Images
Alaa Hilal, Jamal Charara, Ali Al Houseini, Walid Hassan and Mohamad Nassreddine 363
Biphasic Response of Mouse Ileal Smooth Muscles to Aspirin in a Dose Response Manner
Ismail Salih Kakey and Sundus Majeed Hamza 370
The Effect of Mercury on Lipid Peroxidation and Its Relation with Vitamin (A, E) and Essential Elements in Dentals Serum
Jaffer Hashim Mohsen, Hanan Fadel Abbas and Kasim Kadhim Alasedi
Microbiology and Biological Engineering
377 Production of Aflatoxins from Aspergillus flavus in Liquid Medium
Maria Edite Bezerra da Rocha, Francisco das Chagas Oliveira Freire, Ícaro Gusmão Pinto Vieira, José Maria dos Santos Filho, Fábio Erlan Feitosa Maia, Maria Izabel Florindo Guedes, Davide Rondina
Numerical Classification of Brevibacterium and Related Genera Using Linocin M18 Bacteriocin
Essra Gh. Al-Sammak 390 Incidence of Coagulase Positive Staphylococcus aureus in Raw Cow Milk Produced by Cattle
Farms in Fieri Region in Albania
Kapllan Sulaj, Jorida Terpollari, Renata Kongoli, Kastriot Korro, Sokol Duro, Fejzo Selami, Ilirian Kumbe and Bejo Bizhga
Botany and Zoology
Artificial Pollination and Seed Germination of Dendrobium candidum Wall. ex Lindl
Ke Xu, Yi Tang, Jia Lai, Ze-Sheng Yan, Qian Luo and Huan-Xiu Li 400 The Quality of Sour Cherry Maidens Fertilized with Various Biopreparations in an Organic
Nursery
Zygmunt Stanis ław Grzyb, Wojciech Piotrowski, Lidia Sas Paszt and Paweł Bielicki 410
Note on the Orchids of the Moutas Hunting Reserve—Tlemcen (Western Algeria)
Brahim Babali, Abderrahmane Hasnaoui and Mohammed Bouazza 416
Study of Camelina Biodiversity in Southwestern of Algeria
Cherifi Youcef Amine, Gaouar Souheil Bachir Samir, Moussi Nasreddine, Tabet Aoul Nacera and Saïdi-Mehtar Nadhira
428 Antioxidant Properties, Polyphenol Content and Colorimetric Characteristics of Different Floral Origin Honeys from Different Areas of Southern Italy
Annamaria Perna, Amalia Simonetti, Immacolata Intaglietta and Emilio Gambacorta 437
Investigation of Biochemical Properties and Fractional Composition of Amaranth Oil
Raushan Uazhanova, Mariam Alimardanova and Maigul Kizatova
Apr. 2013, Vol. 7, No. 4, pp. 333-340 Journal of Life Sciences, ISSN 1934-7391, USA
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
Ke Xu, Yi Tang, Jia Lai, Ze-Sheng Yan, Qian Luo and Huan-Xiu Li College of Horticulture, Sichuan Agricultural University, Ya’An 62504, Sichuan, China
Received: April 2, 2013 / Accepted: April 17, 2013 / Published: April 30, 2013.
Abstract: Many studies suggest that ethylene plays an important role in regulating metabolite synthesis. Dendrobium plants are traditional Chinese medicine and nowadays its medicinal components are known to be secondary metabolites. In present study, a homolog of ACC oxidase (ACO) gene was isolated from flowers of Dendrobium officinale Kimura et Migo by PCR-method. The obtained cDNA of DoACO is 970 bp long and contains an open reading frame (ORF) encoding a protein with 314 amino acid residues. The DoACO shows high identity to its homologues from other plant species, that has 94.8% closest amino acid sequence of related protein with the ACO from Dendrobium hybrid cultivar. The putative ORF of the obtained sequence could encode a proper
protein in respect of molecular weight under T 7 Lac promoter in E. coli.
Key words: Dendrobium officinale , ACC oxidase gene, gene clone, recombinant protein, heterologous expression.
1. Introduction effects [11]. Therefore, it is extremely important to improve the alkaloids contents.
Dendrobium plants are important ornamental and Ethylene can induce enzyme involved in metabolite medicine plants. There are 74 Dendrobium species
biosynthesis such as phenylalanine ammonia-lyase reported in China and among them D. officinale, D. (PAL), peroxidase, polyphenol oxidase and chitinase chrysanthum , D. fimbriatum, D. loddigesii and D. [12-15]. Among these enzymes, PAL plays an nobile are listed in the Chinese Phrmacopoeia [1, 2]. especially role in phenylpropanoids synthesis since it Dendrobium officinale Kimura et Migo is the orchid is positioned at the first step of phenylpropanoid Dendrobium perennial herbaceous plants [3]. pathway and controls the metabolite flux into this Dendrobium has been known for its amazing curative secondary pathway [16, 17]. Some researchers effects, such as enhancing the body immunity, reported a significant correlation between reducing blood sugar level and clearing away toxic Dendrobium’s alkaloids content and PAL activity, materials accumulated in human body [4-7]. The indicating PAL is the key factor for the synthesis of medicinal components of Dendrobium have been Dendrobium alkaloids [18-21]. In the process of plant identified to be alkaloid [8, 9]. As early as 1932, growth and development, PAL can be accumulated Suzuki [10] reported that alkaloid from Dendrobium along with endogenous ethylene synthesis [22]. was the major alkaloid of the Chinese herbal medicine. Rickey [23], Chen [24], Wang [25], and Liu [26] The total content of alkaloids is rather low in provided evidences that ethylene is likely to be the Dendrobium plants and it is only approximately endogenous signal molecules which was able to 0.02% despite its determinant of Dendrobium curative
induce PAL expression.
In the pathway leading to ethylene, ACC oxidase
Corresponding author: Huan-Xiu Li, professor, research
fields: application of biological technology in horticulture plant. (ACO, also known as EFE) catalyzes the last step that E-mail: [email protected].
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
ACC converts to ethylene [27]. It is proposed that
5 ′-TCAAGCAGTAGGAATCGGCTGA-3′ (P2). A 50 ACO controls ethylene synthesis rate [28]. ACO gene
L PCR mixture consists of Taq DNA Polymerase (5 has been isolated and characterized in various plant
U/ L), 5 L 10×LA PCR buffer, 8 L dNTP (2.5 species like Wheat, Cabbage, etc.. These studies show
mM), 1 L upstream primer, 1 L downstream primer that ACO are highly conserved among higher plants in
(10 M) and 1 L reverse transcription product. The amino acid sequence and usually several ACO genes
reaction condition is as following: 35 cycles of 1-min constitute a small gene family in each plant species
denaturation (94 °C), 1 min annealing (56 °C), and [29]. The expression pattern of ACO in Dendrobium
1 min extension (72 °C); a final extension was has not yet been well revealed. In flower organs, Ketsa
conducted at 72 °C for 5 min.
[30] found ethylene yield increased significantly 9 h PCR products were segregated by 1% agarose gel. after the Dendrobium pollination. This is quite The target single band was cut and subsequently to be agreement with the profiles of ethylene synthesis during
recovered by using DNA Gel Extraction Kit (Takara). flower development in many plant species [31, 32].
The recovered fragments were then inserted into Recently, many studies of Dendrobium concentrate
pMD19-T vector and the plasmid DNA was extracted on its micropropagation via tissue culture. Quite a few
by using a plasmid Miniprep kit (Takara) and then studies are about the biosynthesis of medicinal
sent to the Invitrogen Trading Co., Ltd. for metabolites. In present study, we isolated the cDNA
sequencing.
of ACO from Dendrobium officinale. This lays the foundation to study the ethylene biosynthesis and has 2.4 Sequence Analysis
A Blast search (NCBI) was completed to identify future.
an implication to improve the alkaloids content in the
genes showing a high identity of amino acid sequence
2. Materials and Methods
(Table 1). Multiple alignment analysis was done using MEGA 4.0 software and the phylogenetic tree was
2.1 Plant Materials
made.
Dendrobium officinale Kimura et Migo was used in
2.5 Construction of the Expression Vector this study, this plant material was collected from
Yunnan province and cultured in incubator. To construct the expression vector of DoACO, primers P3 (CCCATATGATGGA-GCTTCTTG
2.2 RNA Extraction AGGGTT) and P4 (CCCAAGCTTTCAAGCAGTAG
Flower petals of 9 h after pollination were collected GAATCGG) were designed that carried Nde I and and used to RNA extraction. The RNA extraction is
Hind III site respectively and amplified the putative done by CTAB method described by Liu [33].
ORF of DoACO. The PCR-amplified fragment inserted into pMD19-T and the recombinant plasmids
2.3 Gene Fragment Isolation were transformed into E. coli DH5 α competent cells. First strand cDNA was synthesized using Super
For extracting pMD19-T-ACC and pET-28a (+) Script III reverse transcriptase (Formentas) and poly
plasmids, double digest with restriction enzyme T-adaptor primer. Specific primers were designed
Hind Ⅲ and Nde Ⅰ at 37 °C for 2 h and then subject based on the conserved region among ACO from
agarose gel electrophoresis were carried out. For Dianthus caryophyllus , Cattleya hybrida, Paeonia
recovering ACO gene fragment and pET-28a(+) DNA suffruticosa and Lilium brownii. The primers are
large fragment separately, the recovered targeted
5 ′-GCAGAAGCTTCCC TGTGA-TTAA-3′ (P1) and fragments were ligated into vector, and the ligation
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
Table 1 Reference plants sequence information.
No. Name of the plant
Genbank accession 1 Dendrobium hybrid cultivar
Place of origin
Thailand EF487342 2 Dendrobium cv. 'Sonia'
Thailand EF061081 3 Dendrobium hybrid cultivar Sonia 'Earsakul'
Thailand HQ186252 4 Dendrobium hybrid cultivar
Thailand EU151724 5 Dendrobium hybrid cultivar Anna
Thailand GQ332400 6 Dendrobium crumenatum
Singapore AF038840 7 Cattleya bicolor
AY598793 8 Cattleya intermedia
Taiwan
AY598794 9 Laelia anceps
Taiwan
Taiwan AY598795 10 Cymbidium hybrid cultivar
Japan AB257311 11 Phalaenopsis sp. 'True Lady'
Taiwan AF004662 12 X Doritaenopsis sp.
DORACCOXID 13 X Sophrolaeliocattleya 'Love Castle'
Unkown
EU363762 14 X Brassolaeliocattleya 'Sung Ya Green'
China
EU363763 15 x Doritaenopsis sp.
China
DORCAROXI 16 Papilionanthe hookeriana x Papilionanthe teres
Unkown
Thailand GQ140315 17 Oncidium hybrid cultivar Kutoo
China JN997419 18 Vitis vinifera clone SS0AFA20YK24
France FQ394455 19 Cucumis sativus
Israel AF033582 20 Musa acuminata
Singapore AF081917 21 Lilium hybrid cultivar Polyanna
China EU296623 22 Manihot esculenta
EF035079 23 Hevea brasiliensis
U.K.
France AM743172 24 Siraitia grosvenorii
China HQ141614 25 Elaeis guineensis putative
Thailand
JN203256
mixtures were transformed into E. coli DH5 α. induction at 37 °C for 4 h. The different concentration Individual colonies were picked from plate, and -1 of IPTG (0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mmol·L )
inoculated into 3 mL LB liquid medium which were studied in protein expression. The null vector containing Amp (100 g/mL) at 37 °C for 12-16 h
and pET-28a-ACC without IPTG-induction was used shaking cultivation. The plasmids were extracted and
as control. The induction time was set for 0 h, 1 h, 2 h, identified by enzyme cut assay with Hind Ⅲ and
3 h, 4 h and 5 h. All the treatments were examined by Nde Ⅰ .
SDS-PAGE protein electrophoresis.
2.6 Heterologous Expression Analysis
3. Results
The pET-28a-ACC plasmid was transformed into
3.1 Sequence Analysis
BL21 (DE3) cells and then cultured the cells on LB The obtained ACO fragment was 970 bp in length plate at 4 °C overnight. Positive colony was identified
as expected. The sequence has been submitted to by PCR method and then was used to inoculate liquid
Genebank database, and the accession number is LB medium containing Amp (100 g/mL). Then the
JX679494. DoACO display high identity with
E. coli culture was used to inoculate fresh liquid LB Orchidaceae plants after blast search and the most medium with the ratio of 1:100. When the cell
closed protein is that from Dendrobium crumenatum. concentration reached 0.4-0.6 (OD 600 ), IPTG was
The result of homologous analysis is shown in Table 2. added to a final concentration of 1 mmol/L for
There was basically 49.4%~98.9% identity on the
336
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
nucleotide sequence compared with the relevant Ⅲ . Two fragments in length of approximately 5,900 fragments of 25 plants, during which there was 98.9%
bp and 970 bp were obtained (Fig. 3), which indicates identity between Dendrobium cv “sonic” and D.
that the ACO gene has been successfully cloned in the hybrid cultivar, there was 94.8% identity between
expression vector.
JX679494 (D. officinale) and D. hybrid cultivar The constructed pET-28a-ACC was transformed (GeneBank: EF487342). A phylogenetic tree, drawn
into E. coli BL21 (DE3). After induction with IPTG by using truncated nucleotide sequences, showed that
for 4 h, the products were analyzed by SDS-PAGE. A DoACO formed a cluster with those from Orchidaceae
specific protein band with a molecular weight of about plants (Fig.1).
55 kDa was presented and that was in line with expectation (Figs. 4-6). The authors tested the
3.2 Heterologous Expression of DoACO in E. coli expression level of DoACO in different IPTG Cells concentration in E. coli, the highest expression level
In the construction of the expression vector, primers occurred 4 h after IPTG-induction when the IPTG P3 and P4 amplify a fragment of 970 bp as shown in
concentration was 0.4 mmol/L.
Fig. 2. After digesting T-clone with EcoR Ⅰ and
4. Discussion
Xho Ⅰ , a fragment of the same length was resulted in, which indicated that the PCR-amplified DoACO has
This is the first report about cloning the ACO gene been linked to the empty vector in a right way. The
from Dendrobium officinale Kimura et Migo. We recombinant plasmids pET-28a-ACO was identified
successfully constructed the prokaryotic expression by restriction enzyme cleaving with Nde Ⅰ and Hind
vector, and then optimized the protein expression
Table 2 Homogeneous analysis of ACO gene.
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
5.Dendrobium_hybrid_cultivar_Anna.seq 3.Dendrobium_hybrid_cultivar_Sonia_Earsakul.seq
4.Dendrobium_hybrid_cultivar.seq 2.Dendrobium_cv._Sonia.seq 1.Dendrobium_hybrid_cultivar.seq JX679494.seq 6.Dendrobium_crumenatum.seq 9.Laelia_anceps.seq 8.Cattleya_intermedia.seq 7.Cattleya_bicolor.seq
14.X_Brassolaeliocattleya_Sung_Ya_Green.seq 13.X_Sophrolaeliocattleya_Love_Castle.seq
16.Papilionanthe_hookeriana_x_Papilionanthe_teres.seq
12.X_Doritaenopsis_sp..seq 15.Doritaenopsis_sp..seq 11.Phalaenopsis_sp._True_Lady.seq 10.Cymbidium_hybrid_cultivar.seq
17.Oncidium_hybrid_cultivar_Kutoo.seq 25.Elaeis_guineensis_putative.seq 20.Musa_acuminata.seq
21.Lilium_hybrid_cultivar_Polyanna.seq 19.Cucumis_sativus.seq 24.Siraitia_grosvenorii.seq 18.Vitis_vinifera_clone_SS0AFA20YK24.seq
23.Hevea_brasiliensis.seq 22.Manihot_esculenta.seq
Fig. 1 Phylogenetic tree of ACO gene.
factors, all of which would play a fundamental role in the future’s research about the expression of the ACO gene and production of the ethylene in Dendrobium officinale .
The precursor of ethylene was ACC (aminocyciopropane-1-carboxyiicacid, ACC), that converts to ethylene under the catalysis of ACO [34]. Previous study suggests a correlation between Alkaloids synthesis and PAL activity [35-37].
However, only in correspondence to the expression of 1: Result of PCR amplification of expressed gene; 2: Result of
Fig. 2 Electrophoregram of PCR products of ACO gene.
biosynthesized ethylene gene the PAL gene can be restriction enzyme after T clone.
largely expressed [38]. Hughes [39] used the inducer
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
Fig. 6 Effect of the induction time on protein expression.
1-6: Induction time was 0, 1, 2, 3, 4, 5 h.
Fig. 3 The electrophoresis map of enzyme cleave of
recombinant expression plasmids pET-28a-ACC.
Many factors affect the exogenous gene expression
efficiency when using the E. coli. For instance, the varieties of E. coli strains, density of inducer, inducing temperature and the inducing time are all playing significant role in the gene expression [40]. In this study, BL21/pET-28a (+) were selected to express the exogenous gene. Firstly, BL21 is an E. coli strain which has an advantage that the gene expression products will not be degraded by the OmpT (outer membrane protein), for BL21 is lack of the protein.
Fig. 4 SDS-PAGE identification of the expression products of
Secondly, pET-28a (+) is a reliable vector which is
recombined protein.
usually used to express the exogenous gene. And the 1: Uninduced recombinant protein; 2: Induced recombinant protein;
vector owns the MCS (Multiple Cloning Site), making 3: Uninduced empty vector; 4: Induced empty vector. the exogenous gene easier to insert in. Additionally,
the T 7 Lac promoter of the vector has a notable characteristic that the up-stream Lacl gene is able to encode enough Lac repressor which will block the
producing of T 7 RNA polymerase and then decrease transcription of the exogenous gene. This characteristic makes the basic transcription level be the lowest, and that will finally conduce to the
Fig. 5 Effect of the concentration of IPTG on protein
stability of the recombinant vector [41] What’s more,
expression.
IPTG induces the expression of the TTRNA 1-7: Concentration of IPTG was 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2
polymerase which will promote T 7 Lac promoter to mmol·L -1 . transcript and translate the exogenous gene [42].
coming from the Colletotrichum lindemuthianum to Moreover, the inducing density and time of using react with the leaves of Phaseolus vulgaris L., ACC
IPTG which could affect the expression of the and the ethylene will soon be synthesized. Because of
exogenous gene has been studied. the ACC and the ethylene, PAL will not be suppressed
As the result illustrated, the density of IPTG ranges by AVG (Aminoethoxyvinylglycine), which has the
between 0.2-1.2 mmol·L -1 would make the Lac ability to make PAL suppression.
promoter effectively promote the expression reaction.
Cloning of ACC Oxidase (ACO) Gene from Dendrobium officinale
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vitro and vivo antitumoral phenanthrenes from the aerial by IPTG, 0.8 mmol·L would perfectly induce the parts of Dendrobium nobile, Planta Med. 61 (1995) 178. expression. Then the inducing time has been designed
H. Suzuki, I. Keimatsu, Alkaloid of the Chinese drug, from 1 h to 5 h. From 1 h to 4 h, as time went by, the
“Chin-Shin-Hu”. II. Dendrobine, J. Pharm. Soc. Jpn. 52 expression products had increased rapidly. However,
(1932) 1049-1060.
[11] from 4 h to 5 h, the products remained steady, without Y.F. Li, X.H. Zhang, G.M. Sun, Determination of alkaloids and polysaccharide in Herba dendrobii, Chinese
any significant changes. Notwithstanding that BL21 Pharmaceutical Affairs 16 (7) (2002) 426-428. (DE3) is an E. coli strain lacking of the OmpT (outer
[12] S.Y. Cheng, Y. Wang, W.H. Liu, H.W. Du, K.S. Chen, membrane protein), the increasing expression products
Effects of plant growth regulators on phenylalanine may also be degraded by other proteases in the E. coli. ammonia-lyase (PAL) activities in leaves of Ginkgo biloba in vitro, Journal of Plant Resources and
So the inducing time was controlled 4 h after adding Environment 14 (1) (2005) 20-22. the IPTG to express the exogenous gene.
C.F. Zhou, Y.R. Li, L.T. Yang, Effects of ethephon sprayed at early tillering stage on the activities of
Acknowledgments
peroxidase, IAA oxidase and acid invertase in sugarcane in correlation to tillering, Guihaia 27 (4) (2007) 649-652.
This work was supported by Sichuan Agricultural [14] Z.T. Ying, S.X. Li, Relation of sec expression to ethylene University “Shuang-Zhi Plan”.
evolution and oxidase activity in Lagenaria leucantha and Cucumis sativus, Acta Horticulturae Sinica 17 (1)
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Apr. 2013, Vol. 7, No. 4, pp. 341-347 Journal of Life Sciences, ISSN 1934-7391, USA
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension Related LVH (Left Ventricular Hypertrophy) Patients in Malaysia
Julia Ashazila Mat Jusoh 2 , Norlaila Danuri , Fadhlina Abdul Majid , Hoh Boon Peng and Khalid Yusoff
1. Institute of Medical Molecular Biotechnology, Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Malaysia 2. Faculty of Medicine, Universiti Teknologi MARA, Batu Caves 68100, Selangor, Malaysia
Received: September 24, 2012 / Accepted: December 07, 2012 / Published: April 30, 2013.
Abstract: LVH (Left ventricular hypertrophy) is an independent risk factor for the development of heart failure, cardiac arrhythmias and stroke. A recent genome-wide association study reported the involvement of a candidate gene namely KCNB1 in mechanism for development of LVH in hypertension. This study aimed to replicate the finding by investigating the genetic association of KCNB1 gene among the hypertensive LVH patients from Malaysia. We genotyped a SNP (single nucleotide polymorphism) located in KCNB1 namely, rs6063397 among 200 subjects consisting of 61 LVH and 139 non LVH patients using Sanger sequencing method. Statistical analysis revealed no significant association between the LVH susceptibility between the allele and genotype frequencies (P = 0.2719 and 0.4768, respectively). This finding suggests that KCNB1 may not play a role in LVH susceptibility in hypertensive patients in Southeast Asian populations.
Key words: Left ventricular hypertrophy, KCNB1, SNP (single nucleotide polymorphism), Malaysia.
1. Introduction blocker like losartan has been shown to improve the reversal effect [4-6]. However, it is unknown whether
LVH (Left ventricular hypertrophy) is an using this anti-HT agent alone would be useful in independent risk factor for the development of heart preventing LVH. Hence, identifying HT patients with failure, cardiac arrhythmias and stroke. It develops as the risk of LVH may allow this hypothesis to be tested,
a result of hemodynamic overload, for instance, and if successful, would lead to the prevention, hypertension [1, 2]. Blood pressure is an important treatment and improvement of prognosis of LVH. determinant of LVH, and a significant proportion of The normal distribution of LV mass (as an indicator patients with essential hypertension develops this of LVH) in the population indicates the involvement complication. However, this condition varies in a wide of complex and multiple genetic factors to the trait. range of phenotypes, and studies had shown that Various genetic studies had been carried out patients with LVH may have near-normal blood extensively, reporting mainly on the candidate genes pressure, suggesting that development of LVH may be like ACE (angiotensin converting enzyme), guanine due to a genetic factor independent of
nucleotide-binding protein gene (GNB3), IGF-1 hypertension [3]. (insulin-like growth factor), AGT II (angiotensin II), LVH can be reversed with anti-HT AGTRs (angiotensin receptors) [7-12] etc., but no (anti-hypertensive) agents. Angiotensinogen receptor
conclusive result was obtained.
A recent genome-wide association study (GWAS) Corresponding author: Khalid Yusoff, professor, FRCP, research field: cardiology. E-mail: [email protected].
carried out by the HyperGEN study [13] reported the
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension Related Left Ventricular Hypertrophy (LVH) Patients in Malaysia
involvement of a candidate gene KCNB1 located at thickness. Subjects were re-consented before further chromosome 20q13.2. The protein produced by this
experimental procedure was carried out. gene is dephosphorylated by calcineurin, which is
2.2 Echocardiography
well known to be associated with LVH [14], reflecting
a unique mechanism for development of LVH in Echocardiography measurements were made using hypertension. However, whether the finding is the Echo Pac in our Non Invasive Cardiac Laboratory. relevant to the Southeast Asia populations or not
Doppler, two-dimensional (2D), and M-mode remains to be validated, as the study was carried out in
(2D-guided) echocardiograms were performed the Western populations, of which the allele following a standardized protocol. Measurements
frequencies and the structure of LD (linkage were made using M-Mode at the PSAX (Parasternal disequilibrium) are known to be different between the
Short Axis view) using a computerized review station two major continents.
equipped with a digitizing tablet and monitor overlay This proposed study hence, attempts to verify the
used for calibration and quantification. study by replicating the finding in our population, i.e.,
Transthoracic echocardiogram criteria for LV mass studying the SNPs of the KCNB1 gene among the
index used the formula, LV mass index = (0.8 (1:04 hypertensive patients with LVH.
3 ([LVIDD PWTD IVSTD] 3 -[LVIDD] ) (0.6
2. Materials and Methods
g/heigh ) > 110 g/m in female and > 125 g/m in male (Devereux Criteria).
2.1 Sample Recruitment
2.3 DNA Extraction
The study was approved by UiTM Research and Ethics Committee, as well as ethics committee of
Genomic DNA was extracted from whole blood Ministry of Health. A total of 200 blood samples (61
from the recruited subjects using commercially LVH and 139 non LVH) were recruited from the
available kit namely QIAamp DNA Blood Midi Kit PURE (Prospective Urban-Rural Epidemiologic). (Qiagen, Inc., Valencia, CA.). Study from 2007 to 2010. General consent was
2.4 Genotyping
obtained earlier. We defined the control group as those hypertensive patients without LVH; whilst the
2.4.1 PCR (Polymerase Chain Reaction) case group as those hypertensive patients with LVH.
PCR amplification was performed on a total The following criteria were followed during sample
reaction volume of 20 µL. Each PCR mixture recruitment:
contained 60 ng of sample DNA, 1 × PCR buffer (10 (1) Age 30-60 years old;
mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.3), 0.5 mM (2) Hypertension, defined as: systolic blood dNTP, 1.5 mM MgCl 2 , 5 pmol of each primer
pressure ≥ 140 mmHg and/or diastolic blood pressure (Forward: 5’-AAAGAGCTTGCTTGAGAGTGAA-3’ ≥ 90 mmHg;
and Reverse: 5’-CTCTAGCTTGGGCAATAG (3) Have not received any anti-hypertensive therapy
AACA-3’), and 0.5 unit of Taq DNA polymerase during the participation of the study;
(Promega, Madison, WI). The cycling conditions were initial denaturation at 95 (4) Non-smoker; o
C for 5 min followed by 35
(5) Non-alchohol takers. o cycles at 95 C for 30 s, 58 C for 20 s and 72
C for 30 To enhance the potential genetic differences and
s with an additional extension after the last cycle for 5 statistical power, we selected the highest and lowest o min at 72
C. The PCR products were purified using a deciles for LVMI followed by ventricular wall
QIAquick column and subjected to sequencing.
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension
Related Left Ventricular Hypertrophy (LVH) Patients in Malaysia
2.4.2 Sequencing allele (T/T genotype plus T/C genotype) and those DNA sequencing of the KCNB1 gene SNP was
with the C/C genotype; P values for C allele positivity performed with a CEQ dye terminator cycle were obtained in a similar way. Two-sided P values sequencing Quick Start kit (Beckman Coulter, were calculated; P values less than 0.05 were Fullerton, CA) according to the manufacturer’s
considered significant. OR (Odds ratio) and a recommendations. Sequencing analyses were done
Cornfield’s 95% confidence interval (95% CI) were with the CEQ 8000 genetic analysis system software
calculated.
(Beckman Coulter, Fullerton, CA) followed by BioEdit sequence alignment editor (Ibis Therapeutics,
3. Results and Discussion
Carlsbad, CA).
3.1 Demographic and Clinical Characteristics
2.5 Statistical Analysis Of the 200 hypertensive subjects recruited, 139 The genotype (C/C, C/T and T/T) and allele (C or T)
were non LVH and the remaining had LVH. The frequencies, as well as the allele carriage frequencies
demographic and clinical characteristics of these (the percentage of individuals carrying at least 1 copy
subjects are summarized in Table 1. There was no of the T or C allele) were determined by direct
significant difference between the case and control counting. Chi-square test with 2 degrees of freedom
groups with regards to their ethnicity, weight, height, was used to determine the significance of the
systolic and diastolic blood pressure and IVSD. difference in genotype distributions. The chi-square
However, male was found to be significantly higher in test with Yates’ correction and Fisher’s exact test for
case group (P = 0.011). This data agrees with previous ≥ 1 cell with < 5 counts were used to test the
research reported that the incidence of LVH were significance of differences in 2 × 2 contingency tables.
different between males and females suggesting P values for T allele positivity were obtained from the
gender differences in the pathogenesis of the comparison between individuals with at least 1 T
condition [15].
Table 1 Demographic and clinical characteristics of case-control cohort.
Characteristics
P value Mean weight (kg)
Case (n = 61)
Control (n = 139)
0.640 Mean height (cm)
0.602 Mean systolic blood pressure (mmHg)
0.083 Mean diastolic blood pressure (mmHg)
0.275 Mean IVSD
0.463 Mean LV mass
< 0.0001* Mean LVMI
Ethnicity Indian 1 5 0.744 Aborigine 0
Others 2
Mean age
Gender 0.011 Female 10
*IVSD, interventricular septal defect; LV, left ventricular; LVMI, left ventricular mass index.
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension Related Left Ventricular Hypertrophy (LVH) Patients in Malaysia
3.2 Allele and Genotype Frequencies (Fig. 1) indicating that the SNPs were highly associated with each other and contained in a single
Genotype analysis of the SNP rs6063397 revealed LD block [20]. In other words, significant association that the distribution of the CC, CT and TT genotypes in of rs756529 found in the previous research in the both groups was 50%, 27.5% and 22.5% principle should be replicable by rs6063397 [10].
respectively; and were in Hardy-Weinberg equilibrium. Indeed, these two variants are physically located close
Interestingly, the overall MAF (minor allele frequency) to each other (~ 5,000 bp). To further confirm the
observed in this study was 47.5% for C allele. This is different from those reported amongst Asian population
linkage disequilibrium between the two variants, we in 1000 Genomes study (http://www. sequenced the SNP rs756529 in 50 samples selected 2
ncbi.nlm.nih.gov/variation/tools/1000genomes/) with randomly in this study and observed that r and D’ an overall frequency of 43% for the T allele in Table 2. values were 1. The genotype and allele frequencies in both case and control groups are shown in Table 3.
3.3 Genetic Association Analysis Fisher exact test revealed no significant association Genetic variants in specific genes or regions of the
observed between both allele and genotypes of human genome are known to be responsible for a
rs6063397 with LVH susceptibility. Permutation test variety of phenotypes such as disease risk or variable
was run (1,000 ×) but the result remained not drug response [16-19]. These variants can be significant (P = 0.264). Therefore we suspect that investigated either directly, or through an indirect
KCNB1 may not play a role in LVH susceptibility in method via their non-random associations with the
hypertensive patients in Southeast Asian populations neighboring markers called, the LD (linkage (in particular Malaysian population). disequilibrium). Previous study found that SNP
Ethnic differences may lead to the conflicting (rs756529) located in KCNB1 gene were associated
results in genetic association study [21]. Replication with LV mass in the European population [13].
in other populations with different ethnicities allows Protein product by KCNB1 gene was us to know whether a reported association signal is dephosphorylated by calcineurin, a protein known to
operative across populations, but it also provides
be associated with LVH in both animal model and valuable insights into the disease network or human [14].
mechanism which may be different among ethnicities In the current study, we tested the association of
[16] if different association signals were observed. SNP rs6063397 with LVH susceptibility in Hilgado et al [16] showed that the disease case-control samples of Malay and Chinese origins.
comorbidities of hypertension and ischemic heart Analysis with Haploview on the HapMap CHB and
disease were different between black and white men JPT samples revealed that rs756529 and rs6063397
[16] suggesting a different disease aetiology in were in full linkage disequilibrium (D’ = 1: r 2 = 1) different populations. In addition to that, environmental
Table 2 Allele frequencies distribution.
Minor allele (%) Arnett et al. (2009) [10]
SNP region
Chr position
SNP ID
Major allele (%)
A (47) Current study
T (43) Chr, Chromosome; SNP, Single Nucleotide Polymorphism.
1000 Genomes
C (57)
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension
Related Left Ventricular Hypertrophy (LVH) Patients in Malaysia
Fig. 1 LD block of KCNB1 gene. Haploview plot showing pairwise LD (D’ values). Each square plots the level of LD between a pair of SNPs; comparisons between neighboring SNPs located along the first line under the names of the SNPs. Numbers within squares indicate the D’ value expressed in percentile. Red squares indicate strong LD (D’ = 1) with LOD scores for LD ≥ 2, pink squares indicates LD < 1 with LOD ≥ 2, brighter pink indicates intermediate LD, and white indicate weak LD D’ < 1, and LOD score < 2.
Table 3 Frequency distribution of the KCNB1 polymorphism in control and case.
SNP ID No. (%) in control (n = 139) No. (%) in case (n = 61) P value
Genotype frequency C/T
Allele frequency
factors, especially nutrients, have to be precisely polymorphic locus and a disease has been suggested evaluated together with complex genotyping, in order
to yield an odds ratio between 1.1 and 1.5 [22, 23]. to establish their importance in masking or unmasking
Thus at least 1,000 subjects (statistical power > 80%) functional variants correlated to a specific genetic
would be required to detect this association, background.
depending on the prevalence of polymorphism. However, a relatively small number of samples
However, studies usually report sample sizes from recruited in this study may be a drawback in this study.
100 to 300 and rarely above 1,000 subjects [23].
A most realistic genetic association between a Haplotype analyses with multiple SNPs should be
Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension Related Left Ventricular Hypertrophy (LVH) Patients in Malaysia
considered in genetic association study, as it would detected left ventricular hypertrophy: Prevalence and risk factors, The Framingham Heart Study, Annals of Internal
increase chances of tagging the uncommon variants,
Medicine 108 (1988) 7-13.
A.H. Gradman, R.E. Schmieder, R.L. Lins, J. Nussberger, the non-significant finding is that rs756529 may play
often causative variants [17]. Another possibility of
Y. Chiang, M.P. Bedigian, Aliskiren, a novel orally an indirect role in the mechanisms of LVH
effective renin inhibitor, provides dose-dependent antihypertensive efficacy and placebo-like tolerability in
development, such as transcriptional factor regulation hypertensive patients, Circulation 111 (2005) 1012-1018.
etc. However, UCSC Genome Browser [5] J.M. Cruickshank, J. Lewis, V. Moore, C. Dodd, (http://genome.ucsc.edu/index.html) revealed no
Reversibility of left ventricular hypertrophy by differing apparent functional role played by this variant.
types of antihypertensive therapy, Jounal of Human Hypertension 6 (1992) 85-90.
[6] M.E. Safar, J.J. Toto-Moukouo, J.A. Bouthier, R.E. Asmar, J.A. Levenson, A.C. Simon, et al., Arterial In summary, our study observed no significant
4. Conclusion
dynamics, cardiac hypertrophy, and antihypertensive association between KCNB1 genetic variation and
treatment, Circulation 75 (1987) I156-161. [7] K. Lindpaintner, M. Lee, M.G. Larson, V.S. Rao, M.A.
hypertensive LVH. We suspect that the role of this Pfeffer, J.M. Ordovas, et al., Absence of association or
gene in LVH pathogenesis may be relatively modest genetic linkage between the and only associated with specific ethnic groups. Our
angiotensin-converting-enzyme gene and left ventricular study would be informative to the meta-analysis for
mass, New England Journal of Medicine 334 (1996) the studies of gene mapping of hypertensive LVH 1023-1028. [8] Z. Nagy, A. Busjahn, S. Bahring, H.D. Faulhaber, H.R.
with Asian origin. Further studies are crucial to Gohlke, H. Knoblauch, et al., Quantitative trait loci for improve the understanding of the disease aetiology of
blood pressure exist near the IGF-1, the Liddle syndrome, hypertensive LVH.
the angiotensin II-receptor gene and the renin loci in man, Jounal of the American of Nephrology 10 (1999)
Acknowledgments
1709-1716.
A. Semplicini, W. Siffert, M. Sartori, A. Monari, C. This study is supported by the Malaysian Society of
Naber, G. Frigo, et al., G protein beta3 subunit gene 825T Hypertension research grant (100-RMI/PRI 16/6/2
allele is associated with increased left ventricular mass in (74/2010)), and the Ministry of Higher Education young subjects with mild hypertension, American Journal
of Hypertension 14 (2001) 1191-1195.
FRGS (600-RMI/ST/FRGS 5/3/fst (61/2010)) grant.
A. Olszanecka, K. Kawecka-Jaszcz, T. Kuznetsova, K. The authors would like to thank staff and students of
Stolarz, E. Brand, A. Ryabikov, et al., Ambulatory blood Institute Medical Molecular Biotechnology, PURE
pressure and left ventricular structure and function in RUS team, staff of Clinical Teaching Center, Faculty relation to the G-protein beta3-subunit polymorphism C825T in White Europeans, Journal of Human
of Medicine, Universiti Teknologi MARA for their
Hypertension 17 (2003) 325-332.
G. Doolan, L. Nguyen, J. Chung, J. Ingles, C. Semsarian, subjects who had participated into this study.
help rendered in conducting this study, and the
Progression of left ventricular hypertrophy and the angiotensin-converting enzyme gene polymorphism in
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