Associations between homocysteine metabolism related SNPs and carotid intima-media thickness: a Chinese sib pair study
Associations between homocysteine metabolism related SNPs and carotid intima-media thickness: a Chinese sib pair study
Kexin Sun 0 1 2 3
Jing Song 0 1 2 3
Kuo Liu 0 1 2 3
Kai Fang 0 1 2 3
Ling Wang 0 1 2 3
Xueyin Wang 0 1 2 3
Jing Li 0 1 2 3
Xun Tang 0 1 2 3
Yiqun Wu 0 1 2 3
Xueying Qin 0 1 2 3
Tao Wu 0 1 2 3
Pei Gao 0 1 2 3
Dafang Chen 0 1 2 3
Yonghua Hu 0 1 2 3
0 Beijing Center for Disease Prevention and Control , No.16 He Pingli Middle Street, Dongcheng District, Beijing 100013 , China
1 Department of Epidemiology and Biostatistics, Capital Medical University , 10 You'anmenwai Xitoutiao, Beijing 100069 , China
2 Department of Epidemiology and Biostatistics, Peking University Health Science Center , 38 Xueyuan Road, Beijing 100191 , China
3 Pingshan New District Center for Disease Control and Prevention , Shenzhen 518118, Guangdong , China
Carotid intima-media thickness (CIMT) is a good surrogate for atherosclerosis. Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. We aim to investigate the relationships between homocysteine (Hcy) related biochemical indexes and CIMT, the associations between Hcy related SNPs and CIMT, as well as the potential gene-gene interactions. The present study recruited full siblings (186 eligible families with 424 individuals) with no history of cardiovascular events from a rural area of Beijing. We examined CIMT, intima-media thickness for common carotid artery (CCAIMT) and carotid bifurcation, tested plasma levels for Hcy, vitamin B6 (VB6), vitamin B12 (VB12) and folic acid (FA), and genotyped 9 SNPs on MTHFR, MTR, MTRR, BHMT, SHMT1, CBS genes. Associations between SNPs and biochemical indexes and CIMT indexes were analyzed using family-based association test analysis. We used multi-level mixed-efects regression model to verify SNP-CIMT associations and to explore the potential genegene interactions. VB6, VB12 and FA were negatively correlated with CIMT indexes (p < 0.05). rs2851391 T allele was associated with decreased plasma VB12 levels (p = 0.036). In FABT, CBS rs2851391 was signii - cantly associated with CCA-IMT (p = 0.021) and CIMT (p = 0.019). In multi-level mixed-efects regression model, CBS rs2851391 was positively signiicantly associated with CCA-IMT (Coef = 0.032, se = 0.009, raw p < 0.001) after Bonferoni correction (corrected α = 0.0056). Genegene interactions were found between CBS rs2851391 and BHMT rs10037045 for CCA-IMT (p = 0.011), as well as between CBS rs2851391 and MTR rs1805087 for CCAIMT (p = 0.007) and CIMT (p = 0.022). Signiicant asso - ciations are found between Hcy metabolism related genetic polymorphisms, biochemical indexes and CIMT indexes. There are complex interactions between genetic polymorphisms for CCA-IMT and CIMT.
Homocysteine; Single nucleotide polymorphism; Carotid intima-media thickness; Atherosclerosis; Sib pair; Gene-gene interaction
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* Yonghua Hu
Carotid intima-media thickness (CIMT), a technique to
monitor carotid artery wall alterations using
ultrasonography, is a noninvasive measurement of preclinical
atherosclerosis [1]. It has often been considered as a surrogate of
early atherosclerosis and is widely used for cardiovascular
risk stratiication [ 2].
Hyperhomocysteinemia is an independent risk factor for
arteriosclerotic vascular diseases [3, 4]. However, the
association between elevated plasma homocysteine (Hcy) and
CIMT remains controversial [5]. This prompts the need for
genetic association studies to investigate the involvement of
Hcy in early stage of atherogenesis.
Hcy can be inluenced by genetic factors. Altered func
tioning of catalytic enzymes in Hcy metabolic pathways
caused by gene mutations may lead to inhibition of certain
pathways and elevation of plasma Hcy level [6, 7].
Methylenetetrahydrofolate reductase (MTHFR), methionine
synthase (MTR), methionines synthase reductase (MTRR),
betaine-homocysteine methyltransferase (BHMT) are
enzymes in Hcy remethylation pathway.
Cystathionineβ-synthase (CBS) is the key enzyme in Hcy
transsulfuration pathway. Serine hydroxymethyltransferase 1 (SHMT1)
sequesters 5-methyltetrahydrofolate, a co-substrate for
homocysteine remethylation, thus to inhibit Hcy
metabolism through methionine synthesis pathway. Genetic
association studies between encoding genes of these enzymes
and cardiovascular studies are previously studied [8, 9].
Diferent enzymes in Hcy metabolic pathways interact
with each other. Complex interactions between variations
of corresponding genes, especially interactions between
functional polymorphisms, can contribute to Hcy metabolic
disorder and subsequent diseases. Interactions between
MTHFR 677 C>T (rs1801133) and CBS 844ins68 [10–
12], MTHFR 677 C>T and MTRR 66 A>G (rs1801394)
[13] were found with regard to Hcy. Same interactions were
shown to elevate neural tube defects risk [14]. However, the
interactive efects of Hcy related SNPs on CIMT were not
fully explored.
To investigate the associations between Hcy
metabolism related SNPs, Hcy related biochemical indexes and
CIMT, we conducted a sib pair study in Chinese
population and genotyped 9 cardiovascular diseases associated
SNPs on 6 Hcy metabolism related genes using candidate
gene approach. The aims of the present study are: (1) to
explore associations between SNPs and Hcy, vitamin B6
(VB6), vitamin B12 (VB12), folic acid (FA) levels; (2) to
test the associations between SNPs and CIMT indexes; (3)
to explore the potential gene–gene interactions for CIMT
indexes.
The current study nested in Fangshan Family-based
Ischemic Stroke Study In China (FISSIC) program.
FISSIC has been described in details elsewhere [15]. In brief,
it is an ongoing family-based genetic epidemiological study
starting from June 2005. We recruited Northern Chinese
Han pedigrees from communities in Fangshan District,
Beijing, China. The inclusion criteria for this study were: (1)
>40 years old; (2) full siblings; (3) with no medical history
of cardiovascular events (including angina pectoris,
myocardial infarction, sudden cardiac death, ischemic stroke,
hemorrhagic stroke, transient ischemic attack, or other
cardiovascular diseases) or antihyperlipidemia treatment at
base line. The exclusion criteria were: (1) receiving extra
vitamin B or folic acid supplements regularly; (2)
receiving drugs which can inluence Hcy levels (methotrexated,
isoniazide, azathioprine, thiazide diuretics); (3) incomplete
genotyping data or other important parameters.
The present study included 186 eligible families with
424 individuals. Written informed consent was obtained
from every participant. This project was approved by the
Ethics Committee of Peking University Health Science
Center, Beijing, China.
Carotid intima-media thickness measurement
Every participant was scanned by one of the two trained
ultrasonographers with high-resolution B-mode ultrasound
system (Acuson Inc., Mountain View, CA, USA), using
7.5- to 10.0-MHz linear transducers according to study
protocol.
Bilateral carotid arteries were scanned from its origin
to bifurcation in transverse plane through an anterolateral
approach. Longitudinal views were obtained in three
segments: (1) distal common carotid artery (CCA) (2 cm
proximal to the dilatation of the carotid bulb); (2)
proximal CCA (1 cm proximal to the dilatation of the carotid
bulb); (3) bifurcation (1 cm proximal to the low divider).
The B-mode ultrasound images were recorded for at least 6
cardiac cycles when the intimal–lumen interface of far wall
was displayed as a sharp-edged continuous straight line.
IMT is a double-line pattern represented as the area
between the artery luminal edge and media-adventitia
interface [1]. IMT was analyzed by 3 qualiied readers
blinded to participants’ information using semi-automatic
edge-detection software (Vasular Research Tool Carotid
Analyzer, Medical Imaging Applications, Coralville, IA)
in regions free of plaques. The mean IMT of far wall was
recorded and was involved in further calculation. In each
segment, we measured IMT both in diastole and systole
frames and averaged the two values. The common carotid
artery IMT (CCA-IMT) was the mean of 4 values obtained
in bilateral distal and proximal CCA segments. The
bifurcation IMT (Bif-IMT) was the mean of values in bilateral
bifurcation segments. Carotid IMT (CIMT) was the mean
of IMT in all segments.
To control the quality of IMT measurement, the
reliability of scanning and reading procedures was evaluated. The
intra-class correlation coeicients for two sonographers
were both over 0.93, while the inter-class correlation
coeficient was 0.90. The intra-reader correlation coeicients
were 0.95 and 0.90 for CCA-IMT and Bif-IMT. The
interreader correlation coeicients were 0.88 and 0.86 for
CCAIMT and Bif-IMT.
Anthropometric measurements and biochemical
analyses
The details of anthropometric measurements [including
height (m), weight (kg), body mass index (BMI, kg/m2),
systolic blood pressures (SBP, mmHg), diastolic blood
pressures (DBP, mmHg), smoking status, drinking status]
and biochemical analyses [fasting blood glucose (FBG,
mmol/L), total cholesterol (TC, mmol/L), total
triglycerides (TG, mmol/L), high density lipoprotein cholesterol
(HDL-C, mmol/L), low density lipoprotein cholesterol
(LDL-C, mmol/L)] were described elsewhere [15].
Medical history of cardiovascular events was collected through
local surveillance system consisting of community
medical centers, township hospitals and district hospitals.
Current and ex-smokers were both treated as smokers during
the analysis, as well as drinkers. Hypertension was deined
as a diagnosis of hypertension, antihypertensive therapy,
SBP ≥ 140 mmHg or DBP ≥ 90 mmHg during examination.
Diabetes was deined as a diagnosis of diabetes, antidia
betic therapy, or FBG ≥ 7.0 mmol/L.
Assessment of homocysteine, vitamin B6, vitamin B12
and folic acid levels
We collected fasting venous blood sample from every
participant. Hcy (μmol/L) (Mindray, Shenzhen, China), VB6
(pg/ml), VB12 (pg/ml), FA (pg/ml) (Cusabio, Wuhan,
China) and plasminogen activator inhibitor-1 (PAI-1, pg/
ml) (Boster, Wuhan, China) concentrations were assessed
by commercially available ELISA kits using TECAN
GENios Plus enzyme-linked immunosorbent assay reader
(TECAN, Grödig, Austria).
SNPs selection and genetic analysis
We investigated 9 single nucleotide polymorphisms (SNPs)
in 6 well-studied candidate genes (MTHFR, MTR, MTRR,
BHMT, SHMT1, CBS) involved in homocysteine
metabolism. SNPs previously reported to be associated with
homocysteine levels or cardiac-cerebral vascular diseases
were selected.
DNA was extracted from blood samples. We performed
DNA genotyping using MassARRAY iPLEX platform
(Sequenom Inc, San Diego, California, USA) following the
manufacturer’s protocol. We assessed SNP genotypes by
MassARRAY Typer Analyzer version 4.0. The call rates
for 9 SNPs were all above 99.0%. To verify reproducibility,
a randomly chosen subgroup of 5% samples went through
repeat analysis. The results of these duplicated samples
were 100% consistent.
Statistical analysis
We analyzed TG, Hcy, VB6, VB12, FA, PAI-1 data on
natural logarithmic scale owing to their skewed distribution.
Continuous variables were described as the mean ±
standard deviation and Student’s t test was adopted to compare
means across gender groups. Categorical variables were
described as frequency and proportion, and Pearson’s χ2
test was used for comparisons between gender groups.
We investigated correlations between Hcy related
biochemical indexes and CIMT indexes, and the mean
levels of these biochemical indexes in every SNP genotype
groups.
We estimated Hardy–Weinberg equilibrium (HWE) for
SNPs in all participants and found no violation (Table 4).
When investigated under additive genetic models,
genotypes with 0, 1 or 2 risk allele copies were coded as 0, 1
and 2 respectively. When investigated under dominant
genetic models, genotypes with 0 risk allele copies were
coded as 0, while genotypes with 1 or 2 risk allele copies
were coded as 1.
Family-based association test in genetic analyses
(FBAT) is a method testing for linkage as well as
association using familial data. It is built on the original
transmission disequilibrium test (TDT) method and is suitable for
extended pedigrees such as sib pairs [16]. We used FBAT
to estimate associations between SNPs and Hcy related
biochemical indexes, as well as CIMT indexes under additive
models.
To replicate the signiicant indings in FBAT, we
employed multi-level mixed-efects regression model,
which can accommodate correlation between full siblings.
Bonferoni-corrected α value was used as the level of
signiicance. SNPs with p < 0.05/9 = 0.0056 were considered
signiicant.
We estimated gene–gene interactions between SNPs
signiicantly associated with CIMT indexes and other SNPs
under dominant model by adding multiplicative terms in
multi-level mixed-efects regression models.
FBAT analysis was conducted using PBAT software
(v3.6). Other statistical analyses were performed by STATA
(version 13, Stata Corporation, Texas, USA).
Basic characteristics of participants and sib pairs
The correlations between CCA-IMT and Bif-IMT were
0.754 and 0.786 in men and women respectively. Compared
with males, females had lower rates of smoking and
drinking. Females also had lower SBP and DBP levels, as well
as higher HDL-C levels. The CIMT indexes for females
Table 1 Basic anthropometric characteristics of participants
424 237 (50.2) 187 (49.8) –
53.9 ± 9.3 54.4 ± 9.5 53.2 ± 9.0 0.231
185 (43.6) 164 (69.2) 21 (11.2) <0.001^
131 (30.9) 117 (49.4) 14 (7.5) <0.001^
26.0 ± 3.5 25.9 ± 3.4 26.0 ± 3.7 0.733
136.4 ± 20.1 139.2 ± 19.0 133.6 ± 20.8 <0.001^
83.3 ± 11.8 85.5 ± 12.0 81.2 ± 11.2 <0.001^
5.69 ± 2.21 5.81 ± 2.24 5.57 ± 2.18 0.097
3.76 ± 1.04 3.75 ± 1.01 3.77 ± 1.06 0.770
0.37 ± 0.63 0.41 ± 0.64 0.31 ± 0.61 0.102
1.20 ± 0.43 1.17 ± 0.40 1.23 ± 0.45 0.046^
2.64 ± 0.67 2.65 ± 0.71 2.62 ± 0.63 0.661
2.63 ± 0.46 2.67 ± 0.42 2.59 ± 0.50 0.182
9.09 ± 0.58 9.05 ± 0.58 9.13 ± 0.59 0.314
6.38 ± 0.60 6.38 ± 0.59 6.39 ± 0.60 0.867
8.85 ± 0.51 8.85 ± 0.50 8.85 ± 0.52 0.966
11.58 ± 0.76 11.63 ± 0.67 11.52 ± 0.86 0.278
0.75 ± 0.01 0.77 ± 0.17 0.72 ± 0.01 0.002^
0.80 ± 0.18 0.82 ± 0.19 0.77 ± 0.15 0.004^
0.76 ± 0.16 0.78 ± 0.17 0.73 ± 0.14 0.001^
73 (17.2) 42 (17.7) 31 (16.6) 0.757
Continuous variables were described as mean ± standard deviation.
Categorical variables were described as frequency (proportion)
BMI body mass index, kg/m2; SBP systolic blood pressure, mmHg;
DBP diastolic blood pressure, mmHg; FBG fast blood glucose,
mmol/L; TC total cholesterol, mmol/L; TG total triglycerides,
mmol/L; HDL-C high-density lipoprotein cholesterol, mmol/L;
LDLC low-density lipoprotein cholesterol, mmol/L; Hcy homocysteine,
μmol/L; VB6 vitamin B6, pg/ml; VB12 vitamin B12, pg/ml; FA folic
acid, pg/ml; PAI-1 plasminogen activator inhibitor-1, pg/ml;
CCAIMT common carotid artery intima-media thickness, mm; Bif-IMT
bifurcation artery intima-media thickness, mm; CIMT carotid artery
intima-media thickness, mm. TG, Hcy, VB6, VB12, FA, PAI-1 were
analyzed on natural logarithmic scale
*p values for comparisons across diferent gender groups, ^p < 0.05
were thinner. The prevalence of hypertension in females
was lower than in males (Table 1).
Correlations between biochemical indexes and carotid
intima-media thickness
VB6 (CCA-IMT: Corr = −0.283, p < 0.001; Bif-IMT:
Corr = −0.234, p < 0.001; CIMT: Corr = −0.277,
p < 0.001), VB12 (CCA-IMT: Corr = −0.194, p = 0.002;
Bif-IMT: Corr = −0.173, p = 0.007; CIMT: Corr = −0.198,
p = 0.002) and FA (CCA-IMT: Corr = −0.371, p < 0.001;
Bif-IMT: Corr = −0.280, p < 0.001; CIMT: Corr = −0.363,
p < 0.001); were negatively correlated with CIMT indexes.
No correlations were found between Hcy, PAI-1 and CIMT
indexes (Table 2).
Associations between homocysteine metabolism related
SNPs and biochemical indexes
rs1801133 T allele was associated with elevated plasma
Hcy levels (p = 0.012). rs1532268 A allele was associated
with elevated plasma VB6 levels (p = 0.010). rs2851391 T
allele was associated with decreased plasma VB12 levels
(p = 0.036) (Table 3).
Associations between homocysteine metabolism
related SNPs and carotid intima-media thickness
in family-based association test analysis
When analyzing associations between 9 SNPs and
CIMT indexes using family-based association test
analysis (FBAT), we found that CBS rs2851391 was signii
cantly associated with CCA-IMT (p = 0.021) and CIMT
(p = 0.019), MTRR rs1532268 was signiicantly associated
with CCA-IMT (p = 0.040). No signiicant association was
found between Bif-IMT and homocysteine metabolism
related SNPs (Table 4).
Table 2 Correlations between
biochemical indexes and carotid
intima-media thickness
Hcy homocysteine, VB6 vitamin B6, VB12 vitamin B12, FA folic acid, PAI-1 plasminogen activator
inhibitor-1, CCA-IMT common carotid artery intima-media thickness, Bif-IMT bifurcation artery intima-media
thickness, CIMT carotid artery intima-media thickness, Corr correlation coeicient
^p < 0.05
Table 3 Associations between homocysteine metabolism related SNPs and biochemical indexes
HWE p value
p for CCA-IMT
p for Bif-IMT
Family-based association test analysis was under additive model, and adjusted for age, sex, smoking, BMI, diabetes, SBP, TG, LDL-C
HWE Hardy–Weinberg equilibrium, MAF minor allele frequency, described as minor allele (minor allele frequency)
#Fam: Numbers of informative families, ^p < 0.05
Associations between homocysteine metabolism related
SNPs and carotid intima-media thickness in multi-level
mixed-efects regression model
millimeters (mm) thickening of CIMT. No associations
between SNPs and Bif-IMT or CIMT reached Bonferoni
corrected α threshold (Table 5).
To replicate FBAT result, we further analyzed
associations between 9 SNPs and CIMT indexes using multi-level
mixed-efects regression model. SHMT1 rs11868708 was
associated with CCA-IMT (Coef = 0.007, se = 0.008,
raw p = 0.013) and CIMT (Coef = 0.011, se = 0.005, raw
p = 0.024). CBS rs2851391was associated with
CCAIMT (Coef = 0.032, se = 0.009, raw p < 0.001), Bif-IMT
(Coef = 0.025, se = 0.011, raw p = 0.019) and CIMT
(Coef = 0.023, se = 0.010, raw p = 0.018).
After Bonferoni correction, CBS rs2851391 remained
signiicantly associated with CCA-IMT (α = 0.0056).
Compared with individuals with no T alleles on
rs2851391, every extra T allele can result in 0.032
Gene–gene interactions between CBS rs2851391
and other homocysteine related SNPs on carotid
intima-media thickness
Gene–gene interactive efects were analyzed under domi
nant genetic model in multi-level mixed-efects regression
model. An interaction was found between CBS rs2851391
and BHMT rs10037045 for CCA-IMT (p = 0.011). The
combination of CBS rs2851391 CT/TT and BHMT
rs10037045 AT/TT genotypes can increase CCA-IMT
by 0.080 mm. Interactions were found between CBS
rs2851391 and MTR rs1805087 for CCA-IMT (p = 0.007)
and CIMT (p = 0.022). The combination of CBS rs2851391
Index changes
per risk allele#
Index changes
per risk allele#
Coef coeicients, SE standard error
*Adjusting for age, sex, smoking, BMI, diabetes, SBP, TG, LDL-C
#Coeicients for SNPs in mixed models, ^p < 0.05, ^^p < 0.01
Table 6 Gene–gene interactive efects of homocysteine related SNPs on CIMT indexes
SNP1: CBS rs2851391 × SNP2:
BHMT rs10037045
Index changes if risk allele of SNP1 existsa
Index changes if risk allele of SNP2 existsb
Index changes if risk alleles of both SNPs coexistc
p for interaction
Index changes if risk allele of SNP1 existsa
Index changes if risk allele of SNP2 existsb
Index changes if risk alleles of both SNPs coexistc
p for interaction
Adjusting for age, sex, smoking, BMI, diabetes, SBP, TG, LDL-C
aCoeicient for SNP1 in full mixed model
bCoeicient for SNP2 in full mixed model
cSum of coeicients for SNP1, SNP2 and interaction term in full mixed model
^p < 0.05, ^^p < 0.01
Index changes
per risk allele#
CT/TT and MTR rs1805087 AG/GG genotypes can
increase CCA-IMT by 0.101 mm and CIMT by 0.089 mm
(Table 6).
The accessibility and reproducibility of CIMT
measurement varies signiicantly at diferent carotid artery segments
[over 90% for CCA, 60–80% for bifurcation and 30–50%
for internal carotid artery (ICA)] [17]. In the present study,
we recorded images of bilateral ICA. However, ICA IMT
can only be clearly measured in 38.6% participants. Thus
it was not involved in further statistical analysis. The
associations between CIMT at various segments and
cardiovascular diseases are also diferent. Thickening of CCA-IMT
may relect the pathological changes of medial hypertrophy
as a result of smooth muscle cell hyperplasia or ibrocel
lular hypertrophy rather than the formation of
atherosclerotic plaque [18]. Epidemiological studies suggested
CCAIMT as a strong predictor for cardiovascular events. It was
associated with myocardial infarction and stroke [2, 19],
which was later proved by a meta-analysis [20]. Carotid
bifurcation is a region where blood in carotid artery lows
into two vessels of unequal sizes. The consequent sudden
changes of hemodynamic factors, such as variations in low
velocity, shear stress and turbulence, make carotid
bifurcation an atherosclerosis-prone location [21, 22]. Ebrahim
et al. reported that the associations between CCA-IMT
and stroke were independent of carotid plaques, whereas
associations between Bif-IMT and ischemic heart disease
can be largely explained by the presence of carotid plaques
[23].
B-group vitamins are essential coenzymes in Hcy
metabolism. Evidences indicated that increased CIMT
was associated with higher Hcy and lower folic acid levels
[24]. Although we observed no associations between Hcy
levels and CIMT indexes, signiicant negative associations
between FA, VB6, VB12 and CIMT indexes were found in
the present study. B vitamins (FA, VB6, VB12) can prevent
atherogenesis through their antioxidant properties and their
ability to lower plasma homocysteine levels [25, 26].
However, the efect of folic acid and vitamin B supplementation
on CIMT progression was still controversial [27].
Plasminogen activator inhibitor-1, a component of the
plasminogen/plasmin system, was considered as a link
between inlammation, insulin resistance and vascular
risks, such as atherogenesis and atherothrombosis [28, 29].
PAI-1 was suggested to be a predictor for cardiovascular
disease [30]. However, associations between PAI-1 and
CIMT were not consistent in diferent studies [ 31, 32]. We
tested PAI-1 in the present study but found no associations
between PAI-1 and CIMT indexes.
MTHFR 677 C>T (rs1801133) is the most frequently
studied functional genetic polymorphism proved to be
independently associated with folate, homocysteine, and
vascular endothelial dysfunction [33–35]. However, association
between MTHFR 677 C>T genotype and CIMT in difer
ent ethnic populations remains controversial [5, 36–38].
There is also no evidence that MTHFR 677 C>T has an
efect on coronary artery disease [ 39]. In the present study,
we observed an association between rs1801133 T allele
and elevated plasma Hcy levels, but found null
association between MTHFR 677 C>T and CIMT indexes. These
results indicated that the thickening of CIMT might be a
pathological process resulting from complex metabolic
disorders rather than isolated hyperhomocysteinemia.
SHMT1 rs11868708 risk allele conferred a 1.46-fold
risk of ischemic stroke in Singaporean Chinese. The total
number of risk alleles on MTRR rs16879248, SHMT1
rs11868708, TCN2 rs1173570 showed a cumulative efect
on ischemic stroke [40]. These three SNPs were genotyped
in the present study. The association between SHMT1
rs11868708 and CCA-IMT (raw p = 0.013) didn’t reach
Bonferoni corrected α threshold. More studies are needed
to investigate the role SHMT1 rs11868708 plays in
atherogenesis and subsequent cardiovascular diseases.
Cystathionine-β-synthase (CBS) transforms
homocysteine to cystathionine in the assistance of vitamin B6. CBS
68-bp insertion (844ins68) carriers have been reported to
lower plasma Hcy levels [41]. Rs2851391 located in the
intron of CBS. A GWAS meta-analysis showed that CBS
rs234709 and CBS rs2851391 were associated with Hcy
concentrations [9]. Zinck et al. discussed that rs2851391
variants might reduce the activity of CBS, and thus was
positively associated with homocysteine levels [42].
Rs2851391 genetic polymorphism is associated with neural
tube defects. Its risk genotype has a 2.0-fold risk of spina
biida [ 43]. Hsu et al. indicated that rs2851391
polymorphisms were associated with changes in postmethionine
load Hcy levels, but it was not associated with recurrent
stroke risk [44]. In the present study, we identiied CBS
rs2851391 as a risk factor for CCA-IMT but not Bif-IMT.
This might result from the diferent pathology for
CCAIMT and Bif-IMT thickening. CBS rs2851391 T allele
showed a marginal association with decreased plasma
VB12 level, which might be a possible explanation for its
association with CCA-IMT.
Methionine synthase (MTR) catalyzes the
remethylation of homocysteine to methionine [45]. MTR rs1805087
(A2756G) is a well-studied genetic polymorphism. Laraqui
et al. deined Hcy level ≥15 μmol/l as hyperhomocyteine
mia (HHcy), and found that MTR rs1805087 (A2756G)
G allele contributed a 2.0-fold risk for HHcy [46]. The G
allele can also increase DNA methylation level [47]. Masud
et al. reported signiicant associations of rs1805087 with
coronary artery disease under additive and dominant
models [48].
Gene–gene interactions between homocysteine related
SNPs are studied before. Many studies focus on
interactions between MTR rs1805087 and genetic polymorphisms
of other homocysteine metabolism related genes. The
combination of MTR A2756G AG/GG, MTHFR 677 CT/TT
and MTHFR 1298 AC/AA genotypes contributes signii
cantly to extremely high Hcy plasma levels [49].
Interaction between MTR A2756G and MTRR A66G may lead
to an increase of infants’ neural tube defects risks [50]. An
existence of gene–gene interaction between rs1801133,
rs662 and rs1805087 was reported for coronary artery
disease [48]. MTR rs1805087 is located in the
AdoMetbinding region, this polymorphism results in a
nonconservative substitution of aspartic acid residue by glycine
residue, which could inluence the secondary structure
and function of the protein [51]. In the present study, we
observed an association of distinct CBS rs2851391/MTR
rs1805087 genotype combination with elevated CCA-IMT
and CIMT, which might be an indication of interaction
between Hcy remethylation pathway and transsulfuration
pathway. BHMT rs10037045 is reported to be a risk
factor for ischemic stroke [52]. This SNP is not well studied
before. More researches are needed to understand the role
BHMT rs10037045 plays in atherogenesis and
cardiovascular diseases, and its interaction with CBS rs2851391.
Compared with parent-ofspring trios study, sib pair
study is more suitable for late-onset diseases, since the
parents’ phenotypic and genotypic traits may not be available
at the time ofspring develops the certain disease. What’s
more, sib pairs are more closely matched for age and
environmental factors than other relative pairs [53]. The shared
genetic background for siblings from same pedigree
prevented the problem of population stratiication, a cause for
false positive associations in population based case-control
studies. As a consequent, the power of sib pair study is
relatively low [54].
The present study has some limitations. Firstly, we only
selected 9 SNPs located on homocysteine related genes
using candidate gene approach. These 9 SNPs are
previously reported to be associated with Hcy levels or
cardiovascular diseases, or to have interactions with risk SNPs
for cardiovascular diseases. Thus, the aim of the present
family-based sib pair study is to verify positive results
found in population based studies, rather than to identify
new risk SNPs. Secondly, the present study is a
crosssectional study. The causality of the associations is thus
compromised. However, since human genetic proile is
unchangeable during life time, the genetic risk factors can
be considered as being prior to disease occurrence. On the
other hand, this can’t deny the need for longitudinal
studies to elucidate the relationships between homocysteine
related SNPs and CIMT or CIMT changes. Thirdly, CIMT,
especially CCA-IMT, is a good surrogate for
cardiovascular events. The associations between risk SNPs and CIMT
indexes suggest a possible inluence of these SNPs on
cardiovascular diseases. However, it is of greater value to
directly elaborate the associations between SNPs and
individual diseases. FISSIC study is an ongoing family-based
genetic epidemiological study. Our next goal is to
investigate the role genetic risk factors play in ischemic stroke,
coronary artery disease and type 2 diabetes.
Signiicant associations are found between Hcy related
genetic polymorphisms, biochemical indexes and carotid
intema-media thickness. There are complex interactions
between Hcy related genetic polymorphisms.
Acknowledgements The authors gratefully thank for staf of FIS
SIC study and all the study participants. This study is supported by
the Key Project of National Natural Science Foundation of China
(81230066) and the National Natural Science Foundation of China
(81102177, 81172744, 81473043).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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