Content uploaded by Sayyed Mohammad Hossein Ghaderian
Author content
All content in this area was uploaded by Sayyed Mohammad Hossein Ghaderian on Dec 05, 2015
Content may be subject to copyright.
I
IJ
JM
MC
CM
M Original Article
S
Su
um
mm
me
er
r
2
20
01
15
5,
,
V
Vo
ol
l
4
4,
,
N
No
o
3
3
Analysis of rs6725887 in the WD Repeat Protein 12 in Association
with Coronary Artery Disease in Iranian Patients
Mohammad Piryaei
1
, Sayyed Mohammad Hossein Ghaderian
1∗
, Hossein Vakili
2
, Hooshang Zaimkohan
3
Nastaran Mohammadi Ghahhari
4
, Maryam Mafi Golchin
5
1. Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences,
Tehran, Iran.
2. Cardiovascular Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
3. Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
4. Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran.
5. Department of Genetics and Anatomy, Babol University of Medical Sciences, Babol, Iran.
Although genetic variants that affect susceptibility to coronary artery disease (CAD) have been greatly known, a
number of these single nucleotide polymorphisms (SNPs) remain to be analyzed in populations with different
ethnicities. CAD is influenced by numerous genetic, environmental, and lifestyle factors, and is an important
reason for mortality around the globe. In this study, a novel SNP (rs6725887) in the WD Repeat Protein 12
(WDR12) gene was selected to be examined in Iranian patients with CAD. Ninety eigth healthy controls and one
hundred and one CAD patients were enrolled from Iranian population, and their clinical data were collected for
further comparisons. After DNA extraction from each sample, genotypes were characterized by Taq Man probe
real- time PCR assay. Statistical analyses were performed to evaluate genotype and allele frequencies and
compared the values with clinical variables. Body mass index, blood pressure, fasting blood sugar, LDL, HDL,
cholesterol, and triglyceride significantly differed in CAD and control groups. Genotype and allele frequencies
of rs6725887 in CAD patients and controls showed no significant association in the distribution. However,
clinical parameters of CAD patients like HDL, LDL, FBS, TG, DBP and SBP had significantly (P<0.05) higher
levels compared to control group. The rs6725887 polymorphism is unlikely to play a key role in CAD risk in our
population. Further additional samples are required for better appreciation of the influence of WDR12 SNP on
CAD occurrence.
Key words: Atherosclerosis, WDR12 gene, polymorphism, risk factor, Iranian
∗
Corresponding author: Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran,
Iran. E-mails: sghaderian@sbmu.ac.ir; sghaderian@yahoo.co.uk
Submmited 20 June 2015; Accepted 25 July 2015; Published 10 August 2015
Polymorphism Analysis of WDR12 Gene in Association with CAD in Iran
161 Int J Mol Cell Med Summer 2015; Vol 4 No 3
oronary artery disease (CAD) and its main
complication, myocardial infarction (MI), are
the driving causes of morbidity worldwide. CAD
results from atherosclerosis, which is the blockage
of major cardiac vessels supplying blood to the
heart. The interplay between genetic and
environmental determinants and other common risk
factors such as cigarette smoking, high blood
pressure, diabetes and hypercholesterolemia lead to
the pathogenesis of CAD (1-3). Previous studies
indicated that genetic factors and biomarkers
significantly contribute to the development of
CAD. Several comprehensive studies with
advanced genome detection techniques such as
genome-wide linkage analysis and genome-wide
association studies (GWAS) have been successful
in providing data on new gene loci that are
important in developing CAD (4-6). These
techniques may assist scientists to explore and
analyze new signaling pathways that are associated
with atherosclerosis and the disease involving
potential risk (5). Due to differences in
geographical or ancestral origin of samples, the
association studies encompass different
duplicability. Consequently, to improve our
understanding about the function of genes in
pathology of CAD, apart from variations in
ethnicity, genetic and non-genetic factors
independent studies should be performed to validate
these observations (1, 7).
GWAS of CAD have resulted in the discovery
of a novel chromosome locus 2q33.1, being
significantly associated with early-onset MI.
Kathiresan et al. have recently introduced 2q33 as
an MI susceptibility locus that encompasses the
WDR12 gene (8).The gene is approximately 134 kb
in length and includes 13 exons and transcribes a
member of the WD repeat protein family. WD
repeat protein 12 (WDR12) is a ribosome
biogenesis protein with 40 amino acids and is
formed by gly-his and trp-asp (GH-WD). GH-WD
is necessary for proper configuration of heterotri-
meric or multiprotein complexes. Biological roles
of WDR12 in mammals are yet unknown, but
protein is functional in several processes in the cell,
such as cell division and proliferation, cell cycle
control, and ribosome biogenesis (9). Moreover, it
has been suggested that WDR12 is of great
importance in neovascularization and regeneration
of ischemic tissue and may regulate lipid level (10).
A cluster of single nucleotide polymorphisms
(SNP) on 2q33 locus are notably linked to CAD, of
which intronic SNP, rs6725887 near WDR12 gene
is significantly associated with early-onset MI (8,
11-14). Therefore, it is an obligation to obtain a
comprehensive list of potential disease genes that
increase genetic susceptibility to MI and CAD.
Likewise because populations of diverse origin
show genetic heterogeneity, it is essential to test
each risk polymorphism and related genotypes in
different ethnic individuals. Generation of a
complete picture of susceptible genes in expressing
clinically significant phenotypes in different
populations remains a challenge for scientists. Most
of the large-scale association studies for classifying
risk loci in CAD analyzed populations with a
different ancestral background exclusive of Iran
(15). The present study aimed to examine the
association between rs6725887 polymorphism in
WDR12 gene with CAD and various paraclinical
indicators in Iranian individuals.
Materials and methods
Study population
The study population comprised of one
hundred ninety nine subjects in total consisting of
one hundred and one CAD patients diagnosed by
positive angiography, and the ninety-eigth remai-
ning healthy subjects from both genders. CAD
patients underwent angiography to detect luminal
narrowing and individuals with stenosis ≥50% were
included in the case group. Individuals with
negative angiography and cardiac ventriculography
were classified as control subjects if they also had
C
Piryaei M et al.
Int J Mol Cell Med Summer 2015; Vol 4 No 3 162
no history of MI, hypertension, cigarette smoking,
diabetes, obesity, and high level of cholesterol. The
study was approved by the local Ethics Committee
and Rasearch Council of the Shahid Beheshti
University of Medical Sciences. All cases submi-
tted signed an informed consent prior to study.
Analysis of rs6725887 genotype
Blood samples were obtained and stored in
EDTA-containing tubes. Genomic DNA was extr-
acted by High Pure PCR Template Preparation Kit
(Roche, Germany). DNA was analyzed for quantity
and quality by spectrophotometer (Nanodrop 1000,
Thermo Fisher Scientific, Wilmington, DE, USA)
and using gel electrophoresis. The rs6725887 C>T
was genotyped by TaqMan probe real-time PCR.
LightCycler 96 (Roche, Germany) was used to
determine the genotypes of rs6725887. Primer-
probe sets were designed and manufactured by
Applied Biosystem custom service.
Statistical analyzes
Using SPSS 21 (SPSS Inc, Chicago, IL,
USA), clinical data and CAD risk factors such as
body mass index (BMI), diabetes, hypertension,
LDL, HDL, and triglyceride (TG) were compared
between groups by t-test. The chi-square test was
utilized to analyze the differences between the
qualitative data and groups. One-way ANOVA test
was performed to compare the differences in the
mean across groups. The association between
rs6725887 genotypes and CAD was tested by
logistic regression. Stepwise multivariate regression
was completed to evaluate the effect of independent
variables such as gender and age on genotypes and
CAD. Chi-square test was used to test the genotype
and allele frequencies for the Hardy-Weinberg
equilibrium. A p-value of <0.05 was considered as
statistically significant.
Results
Clinical characteristics of the study population
In this study, ninety eight healthy individuals
and one hundred and one CAD patients were
recruited. The study groups were compared with
regard to clinical characteristics and biochemical
findings. A detailed description of the clinical
parameters and their comparison in the CAD and
control groups is shown in Table 1. The presence of
hypertension was significantly higher in the CAD
group when compared to the controls by T-test (P <
0.0001). The control group showed significantly
higher age (61.14 ± 11.05 years) but body mass
index (BMI) was higher (27.47 ±6.78) in CAD
group compared to those without CAD. Moreover,
TG level, and total cholesterol (TC) amount
increased considerably in CAD group. Although
LDL level was significantly higher in controls, the
level of HDL meaningfully increased in the CAD
group in comparison to the healthy subjects
(P<0.0001).
Genotype and allele frequencies in the CAD and
control groups
Both genotype and allele frequencies of the
rs6725887 C>T polymorphism were in the Hardy-
Weinberg equilibrium; however, the genotype and
Table1. Comparison of clinical characteristics of healthy (control) and angiography negative (CAD) groups
Characteristics Control (n=98) CAD (n=101) P values
Age (years) 61.14 ± 11.05 58.65 ± 8.89 0.18
Male 0.49 ± 0.57 0.57 ± 0.49 0.2
BMI (kg/m2) 26.01 ± 4.08 27.47 ± 6.78 0.002
SBP (mm HG) 123.67 ± 14.90 135.81 ± 26.58 <0.0001
DBP (mm HG) 74.35 ± 7.91 83.50 ± 12.75 <0.0001
TG (mg/dl) 116.10 ± 64.71 155.81 ± 68.72 <0.0001
TC (mg/dl) 165.42 ± 27.24 173.57 ± 32.60 <0.0001
FBS (mg/dl) 122.20 ± 39.62 139.05 ± 62.57 <0.0001
HDL (mg/dl) 37.01±5.83 39.16 ± 8.10 <0.0001
LDL (mg/dl) 86.88 ± 2.74 102.04 ± 24.53 <0.0001
Polymorphism Analysis of WDR12 Gene in Association with CAD in Iran
163 Int J Mol Cell Med Summer 2015; Vol 4 No 3
Values are represented as mean ± SD for all the variables. BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood
pressure; TG: triglyceride; TC: total cholesterol; FBS: fasting blood sugar; HDL: high density lipoprotein; LDL: low density lipoprotein
Table2. Genotype and allele frequencies for rs6725887 T > C
OR (95% CI) P-value Control n (91) CAD n (101) Genotype 1 (Reference) - 74 (82%) 81 (80%)
CC 0.91 (0.42-1.91) 0.81 17 (18%) 17 (18%)
CT 6.39 (0.32 to 1.25) 0.22 0 (0%) 3 (2%)
TT 1 (Reference) - 165 (90.66%) 179 (88.61%)
C allele 1.24 (0.64 to 2.42) 0.51 17 (9.34%) 23 (11.39%)
T allele
Table3. One way ANOVA analysis of clinical parameters in association with genotypes
Variables Control (P value) CAD (P value)
Age (years) 0.89 0.98
Sex 0.24 0.10
BMI (kg/m
2
) 0.71 0.35
SBP (mm Hg) 0.64 0.90
DBP (mm Hg) 0.19 0.81
TG (mg/dl) 0.72 0.39
TC (mg/dl) 0.21 0.05
FBS (mg/dl) 0.60 0.85
HDL (mg/dl) 0.67 0.18
LDL (mg/dl) 0.84 0.07
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; TG: triglyceride; TC: total cholesterol; FBS:
fasting blood sugar; HDL: high density lipoprotein; LDL: low density lipoprotein.
allele frequencies of the SNP were not significantly
associated with the increased risk of CAD. There
was no difference in the frequencies between CAD
and control groups. The genotype distribution of
rs6725887 in the control group was CC = 74 (82%),
CT = 17 (18%), and TT = 0 (0%). In CAD group,
the genotype frequencies of the SNP was 81 (80%)
for CC, 17 (18%) for CT, and 3 (2%) for TT. For
the allele distributions, the frequencies of the T and
C alleles remained constant in both groups (Table
2). Overall, genotype and allele frequencies in the
CAD and control groups revealed no significant
association in the distribution.
Analysis of association between rs6725887
genotypes and CAD phenotypes
We performed a primary analysis for the
clinical variables in the control and CAD groups,
which showed that age, BMI, systolic blood
pressure (SBP), diastolic blood pressure (DBP), and
levels of TG, TC, FBS, HDL, and LDL were
significantly associated with CAD (Table 1). On the
other hand, genotype and allele frequencies were
not associated with CAD in the groups (Table 2).
To assess whether all these factors were associated
with CAD incidence in an independent manner, we
conducted one-way ANOVA test but none of the
factors were nominated as independent risk factors
for CAD in either patient group or control group.
Discussion
SNPs are among the genetic biomarkers,
which could greatly affect the susceptibility of
individuals to many diseases (16). Recent advances
in polymorphism screening technologies have made
possible a more comprehensive type of large scale
analysis through GWAS (17). Since CAD is a
complex genetic phenotype, it implicates the
interaction of a number of genes and environmental
elements to determine the incidence of the disease.
As of 30 January 2015, 2101 publications and
15268 SNPs discovered to be associated with CAD
from GWAS, were included in the catalog of the
National Human Genome Research Institute
database (18).Description of genes in these studies
Piryaei M et al.
Int J Mol Cell Med Summer 2015; Vol 4 No 3 164
attempts to collect potential correlated SNPs with
CAD, which will benefit the development of
personalized medicine in the near future (19-23).
However, it should be noted that in addition to their
effectiveness in providing detailed information on
SNPs, GWASs cannot offer data on all of the
polymorphisms in one experiment. The rs6725887
polymorphism in WDR12 gene has been included in
few GWAS analyses, but no report has
independently studied this SNP in single population
(8, 11, 24, 25). Therefore, our study aimed to
investigate this SNP on its intronic locus and
replicate the association of rs6725887 with CAD
and the correlated risk factors.
In the present study, however, no association
between the WDR12 rs6725887 SNP with the
presence of CAD and its risk factors such as
hypertension, and levels of FBS, HDL, LDL, and
TC could be observed. Although the statistical
analysis found a significant difference between the
means of variables of CAD risk factors in control
and CAD groups (Table 1), none of these elements
were associated with the genotype and allele
frequencies of the rs6725887 SNP. Furthermore,
this study could not detect any significant
differences between the genotype and allele
frequencies of the SNP in patients and controls in
the Iranian population. There is no single study to
compare the results of our analysis with; however,
GWAS reports on this locus on WDR12 gene are
indicative of an association between the SNP with
CAD and MI. For the first time, researchers in
Myocardial Infarction Genetics Consortium
succeeded in finding the C allele in WDR12
rs6725887 as a risk factor in association with CAD
and MI (P = 4 × 10
-4
, OR = 1.16, 95% CI = 1.10-
1.22) in 2,753 samples, but the present study was
unable to prove this relationship. In addition,
Kathiresan et al. could not reveal the association of
this SNP with LDL levels (8).This locus therefore,
requires replication in further samples. Later in
2011, O’Donnell etal. performed a meta-analysis of
GWASs from 5 cohorts for CAD in 9961 samples
with European origin where they found no
association between the WDR12 SNP with CAD/MI
(P= 0.09) (11). Therefore, it appears that the results
of our study are in accord with those observed by
O’Donnell analyses of CAD susceptibility loci.
Maouche and Schunkert in 2012 reported the
association of 2q33 locus containing rs6725887
with CAD, MI, osteoporosis, and Crohn’s disease
(OR= 1.14, 95% CI= 1.09–1.19) (15, 24). In 201,
Saade et al. performed a replication study on 2,002
patients and selected nine CAD risk loci including
WDR12 to identify genes predisposing to an
increased risk of CAD/MI occurrence.
Nevertheless, rs6725887 was not significantly
associated with CAD or MI, which is consistent
with our results (26). Recently, Blattmann et al.
attempted to test the pathogenic effects of genes
including WDR12, which were previously shown in
GWASs to be in association with CAD. They could
successfully appoint WDR12 with a possible role in
the regulation of lipid homeostasis as its siRNA-
mediated inhibition led to reduced levels of free
cholesterol (10). This could suggest the importance
of testing candidate genes from GWASs in
independent studies with large sample numbers and
in people from various ethnic origins.
The main limitation of this study is its small
sample size and lack of the functional analysis of
the WDR gene in our population. In the present
study, we analyzed only a single polymorphism but
haplotype analysis may better uncover the genetic
basis of CAD in our population. Also, the result of
the method may be confirmed by using sequencing
test.
In conclusion, similar cohort studies with
more subjects of both CAD patients and controls
will definitely lead to clear results. Additional
examination of diverse geographical regions,
especially in populations of non-European origin
could generalize the results through other ethnic
groups.
Polymorphism Analysis of WDR12 Gene in Association with CAD in Iran
165 Int J Mol Cell Med Summer 2015; Vol 4 No 3
Conflict of interests
The authors declared no conflict of interests.
Rferences
1. Hinohara K, Nakajima T, Takahashi M, et al. Replication of
the association between a chromosome 9p21 polymorphism and
coronary artery disease in Japanese and Korean populations. J
Hum Genet 2008;53:357-9.
2. Hirashiki A, Yamada Y, Murase Y, et al. Association of gene
polymorphisms with coronary artery disease in low- or high-risk
subjects defined by conventional risk factors. J Am Coll Cardiol
2003;42:1429-37.
3. Erbilgin A, Civelek M, Romanoski CE, et al. Identification of
CAD candidate genes in GWAS loci and their expression in
vascular cells. Journal of lipid research 2013;54:1894-905.
4. Ogawa N, Imai Y, Morita H, et al. Genome-Wide Association
Study of Coronary Artery Disease. International Journal of
Hypertension 2010;2010.
5. Davies RW, Wells GA, Stewart AF, et al. A genome-wide
association study for coronary artery disease identifies a novel
susceptibility locus in the major histocompatibility complex.
Circulation Cardiovascular genetics 2012;5:217-25.
6. Samani NJ, Erdmann J, Hall AS, et al. Genomewide
association analysis of coronary artery disease. The New
England journal of medicine 2007;357:443-53.
7. Morgan TM, Krumholz HM, Lifton RP, et al. Nonvalidation
of reported genetic risk factors for acute coronary syndrome in a
large-scale replication study. Jama 2007;297:1551-61.
8. Kathiresan S, Voight BF, Purcell S, et al. Genome-wide
association of early-onset myocardial infarction with single
nucleotide polymorphisms and copy number variants. Nat Genet
2009;41:334-41.
9. Holzel M, Rohrmoser M, Schlee M, et al. Mammalian
WDR12 is a novel member of the Pes1-Bop1 complex and is
required for ribosome biogenesis and cell proliferation. The
Journal of cell biology 2005;170:367-78.
10. Blattmann P, Schuberth C, Pepperkok R, et al. RNAi–Based
Functional Profiling of Loci from Blood Lipid Genome-Wide
Association Studies Identifies Genes with Cholesterol-
Regulatory Function. PLoS genetics 2013;9:e1003338.
11. O'Donnell CJ, Kavousi M, Smith AV, et al. Genome-wide
association study for coronary artery calcification with follow-up
in myocardial infarction. Circulation 2011;124:2855-64.
12. Schunkert H, Konig IR, Kathiresan S, et al. Large-scale
association analysis identifies 13 new susceptibility loci for
coronary artery disease. Nature genetics 2011;43:333-8.
13. Gamazon ER, Nicolae DL, Cox NJ. A study of CNVs as
trait-associated polymorphisms and as expression quantitative
trait loci. PLoS Genet 2011;7:e1001292.
14. Saade S, Cazier JB, Ghassibe-Sabbagh M, et al. Large scale
association analysis identifies three susceptibility loci for
coronary artery disease. PloS one 2011;6:e29427.
15. Reilly MP, Li M, He J, et al. Identification of ADAMTS7 as
a novel locus for coronary atherosclerosis and association of
ABO with myocardial infarction in the presence of coronary
atherosclerosis: two genome-wide association studies. Lancet
2011;377:383-92.
16. Barreiro LB, Laval G, Quach H, et al. Natural selection has
driven population differentiation in modern humans. Nat Genet
2008;40:340-5.
17. Baudhuin LM. Genetics of coronary artery disease: focus on
genome-wide association studies. American Journal of
Translational Research 2009;1:221-34.
18. Hindorff LA MJEBI, Morales J (European Bioinformatics
Institute), Junkins HA, Hall PN, Klemm AK, and Manolio TA.
A Catalog of Published Genome-Wide Association Studies.
Available at: www.genome.gov/gwastudies. Accessed
[02/02/2015].
19. Feng L, Nian SY, Hao YL, et al. A single nucleotide
polymorphism in the stromal cell-derived factor 1 gene is
associated with coronary heart disease in Chinese patients.
International journal of molecular sciences 2014;15:11054-63.
20. Camici PG, Crea F. Coronary microvascular dysfunction.
The New England journal of medicine 2007;356:830-40.
21. Fedele F, Mancone M, Chilian WM, et al. Role of genetic
polymorphisms of ion channels in the pathophysiology of
coronary microvascular dysfunction and ischemic heart disease.
Basic research in cardiology 2013;108:387.
22. Liu YH, Zhou YW, Yang JA, et al. Gene polymorphisms
associated with susceptibility to coronary artery disease in Han
Chinese people. Genetics and molecular research : GMR
2014;13:2619-27.
23. Chen L, Zhao S, Cheng G, et al. Meta-analysis of
myeloperoxidase gene polymorphism and coronary artery
Piryaei M et al.
Int J Mol Cell Med Summer 2015; Vol 4 No 3 166
disease susceptibility. Zhong nan da xue xue bao Yi xue ban =
Journal of Central South University Medical sciences
2014;39:217-31.
24. Maouche S, Schunkert H. Strategies beyond genome-wide
association studies for atherosclerosis. Arteriosclerosis,
thrombosis, and vascular biology 2012;32:170-81.
25. Girelli D, Martinelli N, Peyvandi F, et al. Genetic
architecture of coronary artery disease in the genome-wide era:
implications for the emerging "golden dozen" loci. Seminars in
thrombosis and hemostasis 2009;35:671-82.
26. Saade S, Cazier J-B, Ghassibe-Sabbagh M, et al. Large Scale
Association Analysis Identifies Three Susceptibility Loci for
Coronary Artery Disease. PLoS ONE 2011;6:e29427.2.
