MTAP gene is associated with ischemic stroke in Chinese Hans
Shu-juan Lia,1, Wen-li Hua,1, Dong-tao Liua, Feng-hui Suna, Qi Zhangb, Xin-chun Yangb, Shun-Zhang Yuc,
Li Jind,e, Xiao-feng Wangd,e,⁎
aDepartment of Neurology, Beijing Chaoyang Hospital, Capital University of Medical Sciences, Beijing 100020, China
bHeart Center, Beijing Chaoyang Hospital, Institute of Cardiovascular Disease, Capital University of Medical Sciences, Beijing 100020, China
cInstitute of epidemiology, School of public health, Fudan University, Shanghai, China
dState Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences and Institutes of Biomedical Sciences, Fudan University,
Shanghai 200433, China
eCMC Institute of Health Sciences, Taizhou 225300, Jiangsu Province, China
a b s t r a c ta r t i c l ei n f o
Received 2 February 2009
Received in revised form 6 April 2009
Accepted 8 April 2009
Available online 8 May 2009
Common pathogenic mechanisms may be involved in the prevalence of ischemic stroke and coronary heart
disease. Recently, genome-wide association (GWA) studies identified a chromosome region (9p21) that
confers the risk of coronary heart disease. In a hospital-based case–control study conducted in Chinese Hans,
we tested the hypothesis that the methylthioadenosine phosphorylase (MTAP) gene on chromosome region
9p21 is involved in the aetiology of ischemic stroke using a tagging single nucleotide polymorphism (tSNP)
strategy. We observed significant association of rs10118757 in the MTAP gene with ischemic stroke. The G
allele of rs10118757 was associated with an increased risk of stroke, with a per-allele OR of 1.31(95% CI,1.04–
1.65, p=0.025). The association remains after controlling for confounding factors including age, gender,
body mass index, smoking, alcohol drinking, hypertension, diabetes, and hyperlipidaemia (OR=1.38, 95 CI,
1.02–1.88, p=0.039). In addition, the GA+GG genotype of rs10118757 was associated with the increased
risk of an undetermined subtype of ischemic stroke (OR=2.14, 95 CI, 1.35–3.38, p=0.001). Further, we also
observed the combined effects of rs10118757 with alcohol drinking and hypertension, which increased the
risk of ischemic stroke. Our observations support the hypothesis that the MTAP gene may be involved in the
prevalence of ischemic stroke in Chinese Hans.
© 2009 Elsevier B.V. All rights reserved.
Recently, a major locus on chromosome 9p21 was found to be
associated with coronary heart disease or myocardial infarction in
several independent genome-wide association studies conducted in
Caucasians [1–4]. The region 9p21 contains the coding sequences of the
exons and spans approximately 63.3 kb. MTAP is a tumour suppressor
gene abundant in all normal tissues but often deleted in various
tumours. MTAP catalyses the reversible phosphorolytic cleavage of
methylthioadenosine leading to the production of methylthioribose-1-
coronary heart disease and stroke is still unknown.
[5–7], and the presence of stroke is also strongly associated with the
presence of coronary heart disease [8,9]. Stroke location is even an
independent predictor of long-term cardiac death or myocardial
infarction . In this study we aim to investigate the association of
MTAP, an important candidate locus of coronary heart disease on
addition, we postulated that interactions, if any, between genetic
variants and environmental exposures, would provide clues as to the
nature of the mechanisms involved in the aetiology of ischemic stroke.
Therefore,we alsosoughtto explore thecombined effects of the genetic
polymorphisms and the conventional risk factors of ischemic stroke.
2. Materials and methods
Cases included 355 patients, aged 27–82 years, who sustained a first
hospitalised non-fatal ischemic stroke in the Neurology Department of
brain MRI, and electrocardiography were performed in all patients. B-
mode carotid ultrasonography and echocardiography was performed
when clinically indicated. Cases were classified into stroke subtypes
(large vessel (34.4%), small vessel (29.9%), cardioembolic (9.6%), and
undetermined (26.2%)) using a modified TOASTclassification .
Controls include 430 subjects, aged 37–90 years, without a history
of stroke disease. These control subjects were hospitalised in the
Journal of the Neurological Sciences 284 (2009) 103–107
⁎ Corresponding author. School of Life Sciences, Fudan University, 220 Handan Rd.,
Shanghai 200433, China. Tel.: +86 21 65643714; fax: +86 21 65642426.
E-mail address: email@example.com (X. Wang).
1These two authors contributed equally.
0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
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departments of traumatic surgery, urinary surgery, hepatobiliary
surgery, cerebral surgery, oral surgery, ophthalmology, or otorhinolar-
yngology, between March, 2007 and July, 2008. The control subjects
were hospitalised for the following reasons: 27% due to traumatic
injuries,18.4% for acute or chronic infections,14% due to calculi in the
urinary system or gallbladder, 10.7% for hyperplasia of the prostate
gland, 7.2% for lumbar disc protrusion or hyperosteogeny, 5.1% due to
cataract, and 17.7% due to other conditions including indirect hernia,
liparomphalus, and polypus. Informed consent was obtained from all
subjects enrolled and the study was approved by the human ethics
committee of Chaoyang hospital.
2.2. Exposure information
the 6 months preceding the ischemic stroke; former smokers were
characterised as individuals who had smoked regularly for at least
6 months, but not during the year preceding the stroke; never and
former smokers were grouped into a single nonsmoking category for
statistical analysis. Alcohol consumption was defined as a dichotomous
variable where individuals consuming more than 3 drinks per week
were considered drinkers. Body mass index (BMI) was calculated as
weight/(height2) (kg/m2). A patient was classified as diabetic if they
had any previous diagnosis, history of antidiabetic medication use or
fasting levels of plasma glucose ≥7 mmol/l. Hyperlipidaemia was
defined as a total plasma cholesterol ≥5.72 mmol/l, and/or plasma
triglycerides ≥1.7 mmol/l, or current use of lipid-lowering drugs with
an established diagnosis of hyperlipidaemia. Individuals were classified
as hypertensive when their systolic pressure was ≥140 mmHg and/or
addition, any individual using antihypertensive agents was classified as
2.3. Selection of tSNPs and genotyping
When there is Linkage Disequilibrium (LD) in the genome, the
number of SNPs required for an association study depends on the
pattern of LD. A small number of tagging SNPs (tSNPs) is sufficient to
tagging SNPs of MTAP were selected using the publicly available
HapMap CHB databank (public data release 21 a/phase II, Jan. 2007;
http://www.hapmap.org/cgi-perl/gbrowse/hapmap_B35/). To iden-
tify common haplotype-tagging SNPs, the eligible SNPs were processed
using the Tagger program (implemented in Haploview, version 3.32).
We definedthecommonvariantsas thosewith minorallelefrequencies
(MAF) N5%, and set the LD measure r2threshold at 0.8. In total, 9 tSNPs
(rs7023474, rs7027989, rs4399022, rs7850937, rs10118757, rs3802392,
rs7047648, rs4129861, rs10117507) in the MTAP gene capturing 25
genotyped alleles were selected. Genotyping was conducted by Orchid
BioSciences using the GenomeLab SNPstream genotyping platform
2.4. Statistical analysis
The deviation from Hardy–Weinberg expectation for the genetic
variants was tested by a chi-square statistic. Continuous variables were
variables were tested using a student's t-test. Unconditional logistic
Clinical characteristics of the studied groups.
Mean age (SD)
Mean BMI (SD)
Association of the studied tSNPs with ischemic stroke in Chinese Hans.
SNP Genotype frequency (percentage)Allele frequency (percentage)
Major homozygote Heterozygote Minor homozygote
Major alleleMinor allele
S. Li et al. / Journal of the Neurological Sciences 284 (2009) 103–107
which was conducted under the assumption of three different genetic
models (dominant, additive, and recessive). Stratified analyses were
conducted toobservethemodificationeffectbygenetic polymorphisms
stroke. A logistic regression model was used to evaluate potential
multiplicative interaction effects. The departures from multiplicative
were assessed by including main effect variables and their product
terms in the logistic regression model when adjusting for potential
confounding factors. We considered smoking (yes=1, no=0), alcohol
consumption (yes=1, no=0), BMI (continuous), hypertension
(yes=1, no=0), diabetes (yes=1, no=0), and hyperlipidaemia
(yes=1, no=0) as potential confounding factors. Owing to limited
sample size, we dichotomised the genetic polymorphism by grouping
subjects into carriers and non-carriers of the risk allele in a gene–
environment interaction estimation. SPSS software version 13.0 for
Windows was used in statistical evaluations of the above data.
To construct the related haplotype, genotype data were used to
estimate inter-marker LD, measuring pair-wise D′ and r2and defining
LD blocks. Haplotypes for stroke subjects and control subjects were
inferred using the statistical software SHEsis and PHASE [14,15]. Tests
for the associations of each set of SNPs and haplotype with stroke
were estimated by using the Haploview software .
3.1. The clinical characteristics of studied subjects
The clinical characteristics of cases and controls are summarised in
Table 1. As expected, when compared with the control group, the
ischemic stroke group had higher BMI levels, higher percentages of
smokers and alcohol drinkers, and a higher percentage of patients
with hypertension, diabetes, and hyperlipidaemia. In a multivariate
logistic model accounting for covariates, smoking, alcohol drinking,
hypertension, and diabetes significantly increased the risk of ischemic
stroke, with adjusted ORs of 3.03 (1.94–4.73), 1.96 (1.20–3.22), 10.50
(7.16–15.41), and 1.60 (1.07–2.42), respectively.
3.2. Single-locus association of MTAP polymorphisms with stroke and
The genotyping success rates of the nine genotyped tSNPs ranged
from 97.7% to 99.6%. All tSNPs were consistent with Hardy–Weinberg
expectations in control groups. The genotype and allele frequencies of
the nine SNPs for the case and control subjects are listed in Table 2. Of
the nine tSNPs, rs7850937, rs10118757, and rs3802392 exhibited
significant association with ischemic stroke. However, in multivariate
analysis accounting for covariates including age, gender, BMI,
smoking, alcohol drinking, hypertension, diabetes, and hyperlipidae-
mia, only the G allele of rs10118757 was associated with an increased
risk of stroke, with a per-allele OR of 1.31(95%CI,1.04–1.65, p=0.025)
(additive model). The association remains after controlling for the
confounding factors of age, gender, BMI, smoking, alcohol drinking,
hypertension, diabetes, and hyperlipidaemia (OR=1.38, 95 CI, 1.02–
1.88, p=0.039) (Table 3).
The sample size allowed the observation of large vessel disease,
small vessel disease, and stroke of undetermined aetiology as distinct
ischemic stroke subtypes. The GA+GG genotype of rs10118757 was
associated with an increased risk of undetermined subtype of
ischemic stroke (OR=2.14, 95 CI, 1.35–3.38, p=0.001) (dominant
model). The association remains after controlling for confounding
factors (OR=2.55, 95 CI, 1.41–4.59, p=0.002) (Table 3).
3.3. The combined effects of MTAP polymorphism and environment
As shown in Table 4, elevation of ischemic stroke risk was obvious
among individuals with the AG+GG genotype for rs10118757 who
consume alcohol with adjusted combined ORs of 2.88 (95% CI, 1.38–
6.00), compared with low-risk individuals (who did not consume
alcohol with the AA genotype for rs10118757). Elevation of ischemic
stroke risk was also observed among individuals with the AG+GG
genotype for rs10118757 who had hypertension with adjusted
combined ORs of 15.38 (95% CI, 8.85–26.71), compared with low-
risk individuals (who did not have hypertensionwith the AA genotype
3.4. Haplotype association of MTAP polymorphisms with stroke
The overall LD of nine tSNPs of the MTAP gene is weak in Chinese
Hans (Fig. 1). LD analysis revealed that SNP rs7023474 was in
moderate LD with rs7027989 (D′=0.91; r2=0.62). The two SNPs
are 1108 bp apart in the MTAP gene. Haplotype analysis of rs7023474
and rs7027989 did not exhibit significant differential distribution of
the haplotype profile between ischemic stroke subjects and control
subjects (p=0.85) (data not shown).
Stroke is a multifactorial polygenic disease controlled by multiple
genetic and/or environmental factors that are assumed, at least to
some extent, to be subject to specific gene–environment and gene–
gene interactions. Because smoking, alcohol drinking, hypertension,
Significant single polymorphism associations within MTAP for all stroke subjects and for ischemic stroke subtypes in Chinese Hans.
SubjectsSNP Model Risk alleleOR (95% CI)
Adjusted OR (95% CI)Adjusted p
⁎Adjusted for gender, age, BMI, smoking, drinking, hypertension, hyperlipidaemia, and diabetes.
Combined effects of genetic and environmental risk factors on ischemic stroke risk.
Risk factorSNP Cases, N
Crude OR Adjusted ORa
aAdjusted for gender, age, BMI, smoking, drinking, hypertension, hyperlipidaemia, and
S. Li et al. / Journal of the Neurological Sciences 284 (2009) 103–107
and diabetes were significantly and independently associated with
ischemic stroke, notwithstanding the relatively small sample size, we
also explored the combined effects of the MTAP polymorphisms and
these environmental exposures in relation to ischemic stroke. We not
only observed that rs10118757 significantly increased the risk of
ischemic stroke, but also made preliminary observations that
rs10118757-drinking and rs10118757-hypertension interaction con-
tribute to the increased risk of ischemic stroke in Chinese Hans. To
date, few studies addressing the gene–environment effects on the
aetiology of stroke, and this is to the best of our knowledge the first
study that has explored the association between MTAP polymorph-
isms and the risk of ischemic stroke.
The SNP rs7850937 is located 7054 bp upstream of the rs10118757
in the fifth intron of the MTAP gene and rs3802392 is located 1581 bp
downstream of rs10118757 in the sixthintron of the MTAPgene.These
three SNPs are located in a different haplotype block in our sample,
since the pair-wise LDs of rs7850937, rs10118757, and rs3802392 are
weak. Remarkably, SNP rs10118757 may be a notable genetic marker
for ischemic stroke since the risk of conferring effect of its AG/GG
genotype on ischemic stroke and the combined effect of AG/GG
genotype with drinking and hypertension still exists after adjusting
for multiple confounding factors such as age, gender, smoking, alcohol
drinking, BMI, hypertension, diabetes, and hyperlipidaemia.
The functional significance of rs10118757 is still unknown. This
SNP may be in strong LD with a true causal geneticvariant somewhere
in proxy. However, intronic variants are considered to have the
potential to directly affect gene-expression levels in some cases .
Therefore, searching for the functional causal SNP in LD with
rs10118757, as well as performing a functional experiment to test
the effect of this polymorphism on the expression levels of the MTAP
gene, is needed.In addition, althoughthe observed geneticassociation
linked the MTAP gene to the aetiology of ischemic disease, the
pathophysiological mechanism by which MTAP influences suscept-
ibility to cerebral disease is yet unknown. Thus, the biological
mechanism underlying this association must also be addressed.
The major strength of this study lies in the fact that all cases were
patients with newly diagnosed ischemic stroke. The controls were
selected from the same hospital fromwhich the cases were derived. In
addition, the moderate sample size allowed us to take multiple
conventional risk factors into consideration when studying associa-
tions, which gave us the ability to conduct stratified analyses and
perform adjustments in multivariate models. However, several
limitations should be addressed when interpreting our results. (1)
Our study is a hospital-based case–control study and the sample size
of our study is relatively small. Misinterpretations leading to an
artificial association may potentially result from case–control studies,
especially when the sample size was relatively small . Therefore,
our results can only be used to give the implication for further
investigation. (2) Subtype analysis and gene–environment interaction
analysis further reduce the statistical power, making all conclusions
preliminary and subjects to the risk of false-positive associations. (3)
The tSNPs selected were based on the Hapmap Han genotyping data.
Consequently, our design may have been insufficient to capture all
associations. We may also have missed the effects of rare alleles (e.g.,
In summary, this study not only suggests that the MTAP
rs10118757 G allele is an independent risk factor of ischemic stroke
in Chinese Hans, but also that this allele is a modifier, modulating the
risk of ischemic stroke caused by hypertension and drinking risk
This work was supported by a grant from the National Science
Fund for Distinguished Young Scholars (30625016), a grant from the
Major Program of National Natural Science Foundation (30890034),
and a grant from Shanghai Municipal Health Bureau Fund for
Distinguished Young Scholars (2006Y22).
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Fig.1. Haploview LD graph of nine SNPs of MTAP. Standard colour scheme of Haploview
was applied for LD colour display. Each diamond for each SNP combination indicates the
pair-wise LD between all tSNPs. The measure of LD (r2) among all possible pairs of SNPs
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