A Single-Nucleotide Polymorphism in

C12orf43 Region is Associated with the

Risk of Coronary Artery Disease in a
Pakistani Cohort
Shafiqa Shahzadi, Shabana, Mamoonah
Chaudhry, Iqra Arooj & Shahida
Hasnain
Biochemical Genetics
ISSN 0006-2928
Volume 54
Number 5
Biochem Genet (2016) 54:676-684
DOI 10.1007/s10528-016-9746-9

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Biochem Genet (2016) 54:676684
DOI 10.1007/s10528-016-9746-9
ORIGINAL ARTICLE

A Single-Nucleotide Polymorphism in C12orf43 Region

is Associated with the Risk of Coronary Artery Disease
in a Pakistani Cohort
Shafiqa Shahzadi1 Shabana1 Mamoonah Chaudhry1
Iqra Arooj2 Shahida Hasnain1,2

Received: 20 January 2016 / Accepted: 27 May 2016 / Published online: 4 June 2016
Springer Science+Business Media New York 2016

Abstract Coronary artery disease (CAD) is one of the leading public health
problems associated with mortality and morbidity in the world. It is a complex
disorder influenced by both genetic and environmental factors. Atherosclerosis and
elevated levels of plasma cholesterol contribute to increased risk for CAD. Other
risk factors include age, hypertension, obesity, diabetes, smoking, and family history. Previous genetic studies have identified multiple polymorphisms in various
genes to be associated with the risk of CAD in different populations. We aimed to
examine the association of MRAS/rs9818870 and C12orf43/rs2258287 polymorphisms with the risk of CAD in a Pakistani sample. A total of 200 samples (100
cases and 100 controls) was analyzed by Allele-specific PCR. Genotypes were
determined by agarose gel electrophoresis. In the current study, locus C12orf43/
rs2258287 was found to be associated with the risk of CAD in the studied Pakistani
cohort (OR 0.18; CI 0.080.37; p = 0.0001) while no association was observed for
MRAS/rs9818870 (OR 1.34; CI 0.652.76; p = 0.42). In conclusion, the rs2258287
SNP may play an important role in the progression of CAD in the Pakistani subjects.
However, future studies should be done on a larger sample size to fully establish its
exact role in CAD.
Keywords Coronary artery disease (CAD)  Atherosclerosis  MRAS  C12orf43 
Pakistan

& Shabana
shabana.mmg@pu.edu.pk
1

Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore,

Pakistan

The Women University Multan, Multan, Pakistan

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Introduction
Coronary artery disease (CAD) is a major cause of mortality all over the world
(Braund 2015; Beaney et al. 2015). In the UK, 82,528 deaths from CAD accounted
for 1 in 7 deaths, while rate of premature CAD deaths was 1 in 3 deaths in the year
2009 (de Lemos et al. 2010). According to the World Health Organization (WHO),
7.3 million deaths occurred due to CAD in the year 2008 (Mendis et al. 2011). CAD
is also named as Atherosclerosis of coronary arteries. The walls of coronary arteries
start narrowing due to fatty plaques. These plaques gradually lead to ischemia or
atherothrombotic occlusion (Braund 2015). Ischemia is a phase in which blood
vessels start narrowing while atherothrombotic occlusion leads to inefficient supply
of nutrient and oxygen to the myocardium. CAD is an asymptomatic disease in most
of the individuals because it progresses silently (Braund 2015). It progresses in four
phases as angina pectoris, myocardial infarction, chronic CAD with heart failure,
and sudden cardiac death (Regieli et al. 2009). It is a multifactorial disease
influenced by both genetic and environmental contribution. The progression to CAD
is influenced by various modifiable as well as non-modifiable risk factors. The
modifiable risk factors are obesity, smoking, dyslipidemia, and diabetes while nonmodifiable risk factors as age, gender, and family history (Braund 2015). The
heritability of disease is more than 50 % reported by twin and family studies. These
studies proved that family history is an independent risk factor of CAD (Lusis et al.
2004). These loci associated with HDL-C and triglycerides also show association
with CAD (Waterworth et al. 2010). The first disease causing gene identified to be
associated with autosomal dominant CAD and MI was MEF2A (located on
Chromosome 15q26). It is involved in a signaling pathway in which development of
the plaques on endothelium are an early trigger for the disease. Major diseasesusceptibility genes located on 2q21.122, 3q13,16p13-ter, and Xq2326 causing
CAD have been reported in various populations (Waterworth et al. 2010). The first
CAD risk variant 9p21 was published in 2009 and within 2 years, 11 more genetic
regions were mapped depicting increased risk of disease in the same region
(Waterworth et al. 2010; Pais et al. 1996; Navarro-Lopez 2002). New susceptibility
loci for CAD have been identified in German population by GWAS. Such loci were
held on these candidate genes Muscle RAS oncogene homolog (MRAS) and
Hepatocyte Nuclear Factor 1 home box AChromosome 12 open reading frame 43
(HNF1A-C12orf43), respectively. MRAS and HNF1A-C12orf43 have been shown to
be involved in atherosclerosis, blood coagulation, and lipid metabolism (Erdmann
et al. 2009). These loci were also replicated in other Chinese and European
populations and results showed variation in different populations (Liu et al. 2015).
These SNPs were selected on the basic criteria: both SNPs having short DNA
sequence less than 25 kb and were also in strong Linkage Disequilibrium (LD)
block (Erdmann et al. 2009). In our work, we replicated these novel loci for CAD
cases in the Pakistani population. The aim of the study was to check the association
of MRAS/rs9818870 and C12orf43/rs2258287 with risk of CAD in the Pakistani
samples.

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Methods
Study Subjects
The study included 100 cardiac cases and 100 controls. Cardiac cases were recruited
from various hospitals of Gujranwala division (Jinnah District Head Quarter
Hospital, Siddique Sadiq Hospital, Cheema Heart Complex, and Social Security
Hospital) and Lahore division (Jinnah Hospital, Mayo Hospital, and Punjab Institute
of Cardiology) of Punjab. Inclusion criteria considered for CAD cases was based on
ECG, cardiac echo, radiologic, and troponine T/I data by the cardiologist. All cases
were newly diagnosed and were not taking any lipid lowering or anti-hypertensive
drugs. The exclusion criteria for CAD cases was the coexistence of any other
chronic disease like liver or kidney disease, cancer, or any ongoing acute infection.
Non-CAD controls were ethnicity matched healthy individuals without any history
of cardiovascular disease and were recruited from the general population. The
control subjects included employees in the Gujranwala Electric Power Company
(GEPCO), Wapda Hospital, and Electric Drive Option (EDO) office Gujranwala.
The control subjects having a family history of early CHD and the obese subjects
were excluded. All the subjects were pre-screened for the presence of hepatitis B
virus, hepatitis C virus, and human immune deficiency virus before starting
biochemical and genetic analysis. Seropositive subjects were excluded from the
study so as not to expose the handlers and environment to the infectious agents. A
formal consent was also signed by the subjects who agreed to participate in the
study. The age, sex, weight, height, body mass index, blood pressure, pulse rate, and
smoking status of cases and controls was also noted on specially designed form. The
study was approved by the institutional ethics committee and all procedures adopted
were in compliance with the Helsinki declaration.
Blood Sample Collection and DNA Extraction
5 ml of blood was collected from case and control subjects who consented to
participate in the study in sterile EDTA vials. Blood sample vials were properly
labeled and stored at 4 C for later genomic DNA isolation. Genomic DNA was
extracted from blood samples using Promega Wizard Genomic DNA purification
kit (Thermo Fisher Scientific, USA). All DNA samples were stored at -20 C.
Genotyping
Genotypes were determined by Allele Specific Polymerase Chain Reaction (ASPCR). The primers used were as follows: three primers were used for SNP MRAS/
rs9818870 as two forward primers F1A 50 -GCTGCTTGGTGCCTCTCTGATAC-30 ,
F1B 50 -GCTGCTTGGTGCCTCTCTGATAT-30 , and one common reverse primer
R1 50 -CGAGGTAGGAACACAGCAGCA-30 . Similarly, another set of three primers
were used for SNP C12orf43/rs2258287 as two forward primers F2A 50 -CGTCATG
AAGGAGGCTTGATAACG-30 , F2B 50 -CGTCATGAAGGAGGCTTGATAACT-30 ,

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and one common reverse primer R2 50 -ACTGCTTTGGCAACAACCT-30 . The

Allele-Specific-PCR reaction mixture contained 1 ll DNA, 5 ll/2X of Fermentas
#1081 PCR master mix, 3.4 ll of ampule water, 0.06 ll/1 lM of forward primer
(F1A), 0.06 ll/1 lM of reverse primer (R1), and 0.6 ll/3 % of DMSO (Dimethyle
sulfo-oxide) for in a total volume of 10 ll. The reaction conditions were as follows:
95 C for 2 min, 35 cycles of 95 C for 1 min, 61.2 C for 1 min, and 72 C for
1 min and a final extension at 72 C for 5 min.
Functional and Statistical analysis
All the statistical analyses were done by Statistical Package for the Social Sciences
(SPSS, IBM atatitics, USA). To determine whether the population is in HWE for
genotype distribution an online calculator was used (www.oege.org). Allele frequencies were also determined using this calculator. Odds ratio (OR) (www.
medcalc.org) calculator was used to check association between polymorphism and
the trait. A p value less than 0.05 was considered significant.

Results
Among 200 DNA samples, the genotyping success rate for two SNPs ranged from
8087 %. The characteristics of the cases and controls have been listed in Table 1.
There was no marked difference of mean height, weight, and BMI among cases and
controls. The proportion of CAD subjects with diabetes, hypertension, smoking, and
family history of CAD was higher in cases (30, 40, 63, and 39 %, respectively)
compared to controls (3, 2, 33, and 39 %, respectively). The current study included
both male and female subjects aged B65 years. In control subjects, 82 % were male
and 18 % were female while in cases, 81 % were male and 19 % were females. The
mean age of cases (53.59 10.99) was higher than the mean age for controls
(48.34 5.60). The mean age of male cases was 50.43 11.14 and mean age of
female cases was 49.6513.56.
The distribution of genotypes of both SNPs among cases and controls is shown in
Table 2. The genotype frequencies of the MRAS/rs9818870 C/T polymorphism
Table 1 Baseline characteristics of controls and cases
Characteristics

Cases (n = 100)

Controls (n = 100)

p value

Age (years)

53.59 10.99

48.35 5.60

3.4 9 10-5

Height (m)

1.57 0.15

1.59 0.24

Weight (kg)

71.75 16.52

74.35 13.48

0.224

BMI (kg/m2)

26.4 5.92

26.31 4.16

0.901

82.06 9.14

78.37 9.33

0.005

Pulse rate (beats/min)

0.481

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11

55

GT

18

CT

GG

CC

Controls

54

TT

13

TT

0.11

0.53

0.89

0.47

2.034

6.907

0.154

0.008

GG

21

CC

Cases

45

GT

40

CT

37

TT

19

TT

0.32

0.51

0.68

0.49

SNP single-nucleotide polymorphism, OR odds ratio, 95 %CI 95 % confidence interval, v Chi-square value

Rs2258287

Rs9818870

SNP

Table 2 SNPs loci allelic and genotypic frequency distribution and relation with CAD

3.353

3.128

0.067

0.99

0.178

1.34

OR

0.0001

0.42

1.0850.373

0.652.76

95 %CI

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were 26.3 % (CC), 50 % (CT), and 23.8 % (TT) in cases and 20.9 % (CC), 64 %
(CT), and 15.1 % (TT) in controls. When the MRAS/rs9818870 CC homozygote
genotype was used as the reference group, the CT genotype did not show any
association with the risk of CAD (CT vs CC, OR 1.34, CI 0.652.76, p = 0.42). The
TT genotype was also not associated with an increased risk of CAD (TT vs CC, OR
0.497, CI 0.221.12, p = 0.093). In the recessive genetic model, when the MRAS/
rs9818870 TT genotype was used as the reference group, the combined CT/TT
genotypes did not show any association with the risk of CAD (CT/TT vs CC, OR
0.623, CI 0.291.32, p = 0.217). The polymorphism was not associated with the
risk of CAD in a dominant genetic model (CC vs TT/CT, OR 1.75, CI 0.793.83,
p = 0.16).
The genotype frequencies of the C12orf43/rs2258287 G/T polymorphism were
5.75 % (GG), 51.72 % (GT), and 42.53 % (TT) in cases and 2.99 % (GG), 16.42 %
(GT), and 89.59 % (TT) in controls. When the C12orf43/rs2258287 GG homozygote genotype was used as the reference group, the GT genotype was associated
with an increased risk of CAD (GT vs GG, OR 1.98, CI 0.3710.55, p = 0.42). The
TT genotype was also associated with an increased risk of CAD (TT vs GG, OR
5.97, CI 2.7313.03, p \ 0.0001). In the recessive genetic model, when the
C12orf43/rs2258287 TT genotype was used as the reference group, the combined
GT/TT genotypes were associated with an increased risk of CAD (GT/TT vs GG,
OR 0.178, CI 0.0850.37, p \ 0.0001). The polymorphism was not associated with
the risk of CAD in a dominant genetic model (GG vs TT/GT, OR 1.64, CI
0.289.85, p = 0.58). However, for another SNP, there was no difference in
genotype and allele frequencies between two groups.

Discussion
We checked the associations between the SNPs C12orf43 rs2258287 G/T and MRAS
rs9818870 C/T and the risk of CAD in a Pakistani population and found that the
C12orf43 rs2258287 G/T polymorphism may increase the risk of CAD in the
Pakistani subjects. C12orf43/rs2258287 is one of the members of a group of SNPs
in the 11q22.3 locus, which have been found to be strongly associated with CAD in
various studies (Marian 2015). The chromosomal region having this SNP includes
intron7 of the Hepatocyte nuclear factor-1a (HNF1A). HNF1A encodes a
transcription factor and is expressed specifically in the liver. Variants in HNF1A
can possibly lead to maturity-onset diabetes of the young, and may influence plasma
concentration of C-reactive protein, an influential threat pointer in support of
cardiovascular disease. Besides, a peril allele by the side of the HNF1A locus
(rs2258287) have been reported to increase plasma levels of LDL-C (Erdmann et al.
2009). M-Ras is a member of the Ras super family of small GTPases, which act as
molecular switches in diverse cellular functions and thereby regulate a variety of
biological processes. M-Ras has been implicated in the regulation of TNFastimulated LFA-1 activation and integrin-mediated leukocyte adhesion downstream
of various inflammatory cytokines (Stachon et al. 2013). The direct SNP rs9818870
contained by 3q22.3 is positioned into the 3untranslated region of MRAS next to a

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bunch of miRNA-binding sites (Erdmann et al. 2009). MRAS gene is 33 kb in size

and contains 5 exons (Erdmann et al. 2009). Studies in mice have shown that M-ras
is involved in tumor necrosis factor-a-stimulated Lymphocyte Function Associated
molecule 1 (LFA-1) synthesis into splenocytes; symptomatic of that M-ras might
take part in a function of the atherosclerosis development during adhesion
signaling. The M-ras protein is generally articulated in tissues and is present at
elevated levels in the cardiovascular system, principally in the heart (Ellis et al.
2011). The mortality rate is high due to CAD among men and women all over the
world. Women develop disease on average, after 10 years of men. However, the
reason for this is not clearly understood. Some CAD risk factors have been found to
affect the onset of disease in men and women separately. The high intensity of risk
factors (like hypertension, diabetes, and physical activity) at a younger age in males
increases the chances of CAD (Cunnington et al. 2010). The CAD cases included in
the present study were from both sexes. Our most rural areas are tied in various
customs and traditions based on gender discrimination. Women in these areas are
overburdened and face serious health issues. Poor living standards, interfamily
marriages, and the absence of health care make the women more susceptible to
CAD and other heritable and non-heritable diseases. These factors may be the
reason of the higher percentage of women CAD patients as compared to men.
Smoking is considered a major cardiovascular risk factor (Nakanishi et al. 2015).
Sometimes, it is taken as a habit, but in fact, nicotine changes the neurophysiology,
and the smoker feels comfort with inhaled nicotine. A smoker with nicotine also
inhales chemicals that influence vascular dysfunction, oxidation of lipids, and
thrombosis. Smoking results in vascular damage in the case of both active and
passive smoking. Cessation of smoking significantly reduces the chances of
readmission to hospitals, progression of disease, and mortality in CAD cases
(Armstrong et al. 2015). Smoking in association with CAD is considered a risk
factor for the onset of disease, because oxidative stress is involved in the
progression of CAD as cigarette smoke causes oxidative stress. In young adult (age
3235) patients with CAD with heavy smoking, oxidative stress indices are
significantly associated (0.01) with CAD severity (Anand et al. 2008). Our results
also show the information about the causative role of smoking for CAD. Wilson and
his colleagues have also reported that treatment of cardiovascular risk factors, like
smoking and hypertension, plays a critical role in the delayed onset and progression
of CAD in diabetic patients (Sunman et al. 2013). CAD and diabetes are
pathologically interrelated diseases. The severity of CAD is higher in diabetic
patients (Pipe et al. 2010). The onset of disease at an early age indicates an
underlying genetic cause. With increasing age, other risk factors also start
contributing to the development of CAD. The relationship between genetic factors
and CAD development has been described by many investigators. In the Korean
population, genes of endothelial function showed an association with CAD, but an
analysis for age gave significant results only in cases aged less than 51 years
(Haffner 2006). The disease diagnosis at early age (\54) depicts CAD family
history as the sole and unique risk factor, which is masked by age more than
62 years. Smoking is also found to be associated with the diagnosis of CAD at an
early age (Yu et al. 2008). The SNPs included in the current study have been

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reported in many GWASs to be associated with CAD and MI, but observed lack of
association for one SNP may be due to the possibility that different genes may be
involved in the onset of disease in young and old people. The MRAS gene
polymorphism shows association with CAD in white Europeans (ODonnell et al.
2011). This polymorphism shows no significant but minor association in Chinese
population (Liu et al. 2015). However, a significant association of C12orf/
rs2258287 with CAD was observed in our study sample. To our knowledge, the
present study was the first attempt to study such an association in the local
population. The frequency of the T allele was very high in our studied population. In
CAD subjects, the T allele frequency was 68 %. In the control group, the frequency
of T was also high (89 %) as compared to that of the G allele. With this high
frequency, the contribution of risk factors in the onset of CAD within a population
has become more critical.
The study had some limitations. The sample size in the current study is small, and
therefore the results need to be replicated in larger cohorts of the same ethnicity.
Secondly, the mean age of cases and controls is significantly different which may
have introduced bias in the study. Although the mean age of controls is
48.35 5.60 which indicates that the controls have been disease free and are
healthy for a long time, yet more reliable results may have been obtained with the
age-matched controls. Thirdly, the presence of a higher proportion of comorbidities
is also a limiting factor of the study. Despite these limitations, however, the current
study could successfully detect some positive association between a variant and the
risk of CAD which can be used to look into the genetic causes of CAD in more
detail in the unique Pakistani ethnic group.

Conclusion
In conclusion, we have shown that single-nucleotide polymorphisms may play an
important role in the progression of diseases like coronary artery disease. However,
as these diseases are complex and involve an interplay of various lifestyle,
environmental, and genetic factors, the results must be replicated in a larger sample
size to validate the results of the current study.

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