Polymorphisms of VEGF, TGFβ1, TGFβR2 and conotruncal heart defects in a Chinese population
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- Wang, E., Wang, Z., Liu, S. et al. Mol Biol Rep (2014) 41: 1763. doi:10.1007/s11033-014-3025-9
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Genetic variants may determine susceptibility of congenital heart disease (CHD). To evaluate the impact of transforming growth factor-β1 (TGFβ1), TGFβ receptor II (TGFβR2) and vascular endothelial growth factor (VEGF) polymorphisms on conotruncal heart defects susceptibility, we genotyped six functional polymorphisms TGFβ1 rs1800469 C>T, TGFβR2 rs3087465 G>A, VEGF −2578C>A, −1498T>C, −634G>C and +936C>T in a hospital based case–control study of 244 conotruncal heart defects cases and 136 non-CHD controls in a Chinese population. Logistic regression analyses revealed that if the TGFβ1 rs1800469 CC homozygote genotype was used as the reference group, subjects carrying the CT variant heterozygote had a significant 0.48-fold decreased risk of conotruncal heart defects [odds ratio (OR) = 0.52; 95 % confidence interval (CI) = 0.30–0.88], subjects carrying the TT variant homozygote had a significant 0.47-fold decreased risk of conotruncal heart defects (OR 0.53; 95 % CI 0.28–1.00). In stratification analyses, the TGFβ1 rs1800469 C>T genotype was associated with a decreased risk for tetralogy of fallot in homozygote comparisons (OR 0.47; 95 % CI 0.22–0.99), a decreased risk for transposition of great artery in the dominant genetic model (OR 0.49; 95 % CI 0.28–0.87) and heterozygote comparisons (OR 0.45; 95 % CI 0.24–0.83). Our findings suggest that TGFβ1 rs1800469 C>T polymorphism was significantly associated with decreased risk of conotruncal heart defects. TGFβR2 rs3087465 G>A, VEGF −2578C>A, −1498T>C, −634G>C and +936C>T polymorphisms may not play a role in the susceptibility of conotruncal heart defects.
KeywordsVEGFTGFβ1TGFβR2PolymorphismsCongenital heart diseaseSusceptibility
Congenital heart disease
Endocardium transform to mesenchyme
Single nucleotide polymorphisms
Vascular endothelial growth factor
Congenital heart disease (CHD) is the leading non-infectious cause of death in children . In China, the prevalence of CHD was 73.2 per 10,000 births in high-prevalence areas . Chromosomal defects and environment teratogens only cause a small portion of the disease, most CHD has a multifactorial etiology and genetic variants may determine susceptibility of CHD . In all types of CHD, conotruncal heart defects count 15–20 % .
The heart is the first organ to form and function during development. In the process, endocardial cushion (EC) formation is a critical step and EC malformation may result in CHD [5, 6]. EC divides the heart tube into atria and ventricle, and the common outflow tract (OFT) into dorsal aorta and pulmonary artery , which calls for the process of endocardium transform to mesenchyme (EMT), occurring on the inner walls of the atrioventricular canal and OFT . Numerous signaling pathways contributed to the formation of EC, such as bone morphogenetic protein (BMP)/TGFβ, vascular endothelial growth factor (VEGF), nuclear factor of activated T lymphocytes (NFATc1), Notch, wingless and int (Wnt)/β-catenin, ErbB, and neurofibromatosis type 1 (NF1)/Ras .
The transforming growth factor-β1 (TGFβ1) gene is located on chromosome 19q13 . In the promoter region, one common single nucleotide polymorphism (SNP) C-509T (rs1800469) was identified in TGFβ1 in an eastern Asian population. TGFβ1 rs1800469 is located within an Yin Yang 1 (YY1) consensus binding site and increased promoter activity compared with the C allele [9–11]. Studies have shown that the -509T allele was significantly associated with higher levels of TGFβ1 in plasma and reduced T cell proliferation [12, 13]. TGFβ1 initiates downstream signaling events by binding to the TGFβ receptor II (TGFβR2), a constitutively active kinase, which activates type I receptors (TGFβR1) by the phosphorylation of serine and threonine residues the GS box and downstream signaling [14–16].
Mutations in the TGFβR2 have recently been described in patients with Marfan syndrome (MFS) . Heterozygous mutations in TGFβR1 and TGFβR2, have been reported in familial thoracic aortic aneurysms and dissections (TAAD), MFS and Loeys-Dietz syndrome (LDS) [17–20]. For the TGFβR2, Seijo et al.  reported a common G-875A variant (rs3087465) in the promoter region of the gene, and this base-pair change was demonstrated to enhance the activity of TGFβR2 transcription in normal epithelial cells and was shown to affect the specific binding with oligonucleotide probes in this 2875 position.
Spatiotemporal expression pattern of VEGF was the first indication of a specific role for VEGF in EC formation. It is reported that VEGF was required for proper heart morphogenesis at stages where the heart is still avascular , and VEGF-expressing endothelial cells in the cushion-forming region may be a unique subpopulation of endothelial cells predetermined to undergo EMT . The VEGF gene is located on the chromosome 6p12 and consists of eight exons, exhibiting alternate splicing to form a family of proteins . SNPs have been reported to be associated with differential VEGF expression in vitro . Two of these SNPs (−2578C>A, rs699947 and −1498T>C, also assigned as −460T>C, rs833061) are located in VEGF promoter region, one (−634G>C, rs2010963, also denoted G+405C) in exon 1 and one (+936C>T, rs3025039) in exon 8, corresponding to the 3′ untranslated (UTR) region .
To evaluate the impact of the six functioal SNPs, TGFβ1 rs1800469 C>T, TGFβR2 rs3087465 G>A, VEGF −2578C>A, −1498T>C, −634G>C and +936C>T, on conotruncal heart defects susceptibility, we performed genotyping analyses for the six SNPs in a hospital based case–control study of 244 conotruncal heart defects cases and 136 non-CHD controls in a Chinese population.
Patients and methods
The study protocol was approved by the Review Board of Peking Union Medical College (Beijing, China). All patients’ parents provided written informed consent to be involved in the study.
This study involved 244 patients with conotruncal heart defects and 136 non-CHD controls. Patients were consecutively recruited from the Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China) between December 2009 and July 2011. All patients were non-syndromic cases whose condition was diagnosed using ultrasonography and confirmed by surgery. Controls were non-CHD outpatients matched to cases by age (±3 years) and sex and recruited from the Affiliated People’s Hospital of Jiangsu University (Zhenjiang, China) and Changzhou Second People’s Hospital, the Affiliated Hospital of Nanjing Medical University (Changzhou, China) during the same time period, most of them with trauma or infectious diseases.
All subjects were genetically unrelated ethnic Han Chinese. Each subject and her/his parents were personally questioned by trained interviewers using a structured questionnaire to obtain information about maternal diabetes mellitus (DM), teratogenic contact during pregnancy, and family history of CHD in first-degree relatives (parents, siblings, children). Information about rubella, influenza, and febrile illnesses during pregnancy was also collected.
Cases with structural malformations involving another organ system or known chromosomal abnormalities were excluded. Exclusion criteria also included a positive family history of CHD in a first-degree relative (parents, siblings, children), maternal DM, phenyl ketonuria, maternal exposure to teratogens (e.g., pesticides, organic solvents), as well as rubella, influenza, and febrile illnesses during pregnancy. Controls with congenital anomalies were excluded.
Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF–MS)
Samples of venous blood (2 mL) were collected from patients using Vacutainer tubes. These were transferred to tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood with the QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany). Genotyping was undertaken by MALDI-ToF–MS as described previously . The steps of MALDI-ToF–MS including multiplex polymerase chain reaction (PCR), amplification, shrimp alkaline phosphates digest, IPLEX primer extension, clean resin, MALDI-ToF–MS analysis and data analysis.
Differences in the distributions of demographic characteristics, selected variables, and genotypes of variants between cases and controls were evaluated using the χ2 test. Associations between genotypes and the risk of conotruncal heart defects were estimated by computing the odds ratios (ORs) and their 95 % confidence intervals (CIs) using logistic regression analyses for crude ORs. The Hardy–Weinberg equilibrium was tested by a goodness-of-fit χ2 test to compare the observed genotype frequencies to the expected ones among control subjects. All statistical analyses were conducted with SAS 9.1.3 software (SAS Institute, Cary, NC, USA).
Comparison of TOF patients, TGA cases and controls by selective characteristics
Controls (n = 136)
TOF (n = 120)
TGA (n = 124)
Age, years (mean ± SD)
1.58 ± 1.61
1.55 ± 1.70
2.27 ± 4.49
SpO2 (%, range)
Isolated TOF (no, %)
72 (60.0 %)
TOF with ASD (no, %)
34 (28.3 %)
TOF with PDA (no, %)
4 (3.3 %)
TOF with ASD and PDA (no, %)
10 (8.3 %)
TGA with VSD (no, %)
52 (41.9 %)
TGA with ASD (no, %)
23 (18.5 %)
TGA with VSD and ASD (no, %)
30 (24.2 %)
TGA with VSD and PDA (no, %)
5 (4.0 %)
TGA with ASD and PDA (no, %)
9 (7.3 %)
TGA with VSD and ASD and PDA (no, %)
5 (4.0 %)
Arterial switch operation (no, %)
95 (76.6 %)
Transannular patching (no, %)
99 (82.5 %)
CPB time (min, mean ± SD)
123.21 ± 27.82
190.84 ± 53.01
Median ventilation time (h, range)
Median length of ICU stay (h, range)
Primary information for six genotyped SNPs
Regulome DB scorea
MAF in our controls
p value for HWE test
% Genotyping valuec
TGFβ1: rs1800469 C>T
TGFβR2: rs3087465 G>A
VEGF: rs699947 C>A (−2578C>A)
VEGF: rs833061 T>C (−1498T>C)
VEGF: rs2010963 G>C (−634G>C)
VEGF: rs3025039 C>T (+936C>T)
Main effects of SNPs on CHD risk
Homozygotes (for common alleles)
Homozygotes (for rarer alleles)
OR (95 % CI)
OR (95 % CI)
TGFβ1: rs1800469 C>T
TGFβR2: rs3087465 G>A
VEGF: rs699947 C>A
VEGF: rs833061 T>C
VEGF: rs2010963 G>C
VEGF: rs3025039 C>T
TGFβ1: rs1800469 C>T genotype frequencies among controls, TOF patients and TGA cases
Controls (n = 136)
TOF (n = 120)
TGA (n = 124)
No. (%) OR (95 % CI)
No. (%) OR (95 % CI)
TGFβ1: rs1800469 C>T
CT + TT
CC + CT
We prevously conducted a case–control study of NFATc1 genotype and perimembranous ventricular septal defect . In this case–control study, we invested the association of TGFβ1 rs1800469 C>T, TGFβR2 rs3087465 G>A and four VEGF SNPs, −2578C>A, −1498T>C, −634G>C, +936C>T, and risk of conotruncal heart defects in a Chinese population. We found that TGFβ1 rs1800469 C>T polymorphism was significantly associated with decreased risk of conotruncal heart defects.
TGFβ superfamily is one of the signaling pathways important for EC formation. Using in vitro explant assays of chicken and genetically modified mice, the involvement of TGFβ and BMP signaling pathways in cardiac cushion formation has been intensively studied . TGFβ inhibits the growth of many cell types . The TGFβ pathway has important roles in EMT via interactions with SMAD and via cross talk with other signaling pathways such as extracellular signal-regulated kinase, c-jun N-terminal kinase, PI3K/AKT and Rho-like guanosine triphosphatases . The TGFβ1 C-509T (rs1800469) polymorphism was well characterized in both functional evaluations and association studies. Grainger et al.  showed that TGFβ1 levels are under genetic control (heritability estimate, 0.54) with the C-509T (rs1800469) SNP responsible for 8.2 % of the additive genetic varian. The -509T (rs1800469) allele was significantly associated with higher levels of TGFβ1 than was the C allele and altered TGFβ1 transcription activity and the binding of transcription factor YY1 [9, 12].
TGFβ superfamily members signal through heteromeric complexes of type II and type I transmembrane serinethreonine kinase receptors . TGFβ receptors (TGFβR1, TGFβR2) polymorphisms have been associated with rare syndromes including aortic dilatation and familial thoracic aortic aneurysm [18, 19]. Magdoud et al.  reported TGFβ1 haplotypes were associated with recurrent pregnancy loss (RPL). Eerde et al.  found TGFβ1 rs1800469 C>T SNP was associated with vesico-ureteral reflux, which was also part of the spectrum of congenital anomalies. Yang et al.  found the CT genotype of TGFβ1 rs1800469 decreased the risk of asthma in a Chinese population. To the best of our knowledge, these results provide the first positive evidence for an association between TGFβ1 rs1800469 C>T SNP and conotruncal heart defects, however, its underlying molecular etiology remains to be elucidated.
VEGF play a critical role during EMT processes and mediate EC formation. Spatiotemporal expression pattern of VEGF should be tightly regulated and even a tiny disturbing could cause CHD. Evidence also showed that increased VEGF levels during right ventricular OFT development can lead to abnormal development of both cushion and myocardial structures. Furthermore, the importance of appropriate timing and dosage of VEGF during heart development was highlighted by shared cardiovascular developmental defects in animal models, including mice heterozygous for VEGF allele and transgenic mice, which have a two to three folds increased VEGF [35, 36]. It is reported that VEGF gene polymorphisms may also be related with CHD, such as TOF and pulmonary stenosis .
VEGF polymorphisms have been extensively investigated both in their function and in their associations with different diseases, including CHDs. A few studies have been conducted to evaluate the role of VEGF polymorphisms in CHD susceptibility and the focus was mainly on VEGF −634G>C. A study by Xie et al.  reported that VEGF -634C allele was significantly protective against VSD in a case–control study of 222 CHD patients and 352 controls. However, in a small case–control study in Hungary (102 CHD cases and 112 controls), Vannay et al.  found that VEGF -634C presented an increased risk for CHD. In a recent meta-analysis of the VEGF polymorphisms and risk of CHD, genotypes for the VEGF polymorphism might not be associated with the risk of CHD . In the present study, we genotyped four potentially functional VEGF SNPs in a Chinese population in 244 conotruncal heart defects cases and 136 controls and failed to find evidence for the significant associations of VEGF SNPs with CHD risk. The discrepancy could be resulted from the chance finding of small studies and/or ethnic difference in terms of genetic associations.
Considering TGFβ1 rs1800469 C>T mutant alleles in the control group, OR, case samples and control samples, the power of our analysis (α = 0.05) was 0.854 and 0.834 in 240 conotruncal heart defects cases and 135 controls with OR 0.52 and 0.53 respectively.
Several limitations of the study need to be addressed. First, we did not obtain DNA samples from the parents to evaluate the etiological role of TGFβ1, TGFβR2 and VEGF in conotruncal heart defects. Second, because our study was a hospital-based case–control study and selection bias could not be fully excluded, large population-based studies are warranted to further elucidate the role of TGFβ1, TGFβR2 and VEGF polymorphisms in susceptibility of conotruncal heart defects. Third, the sample size of this study was not large enough to evaluate the low penetrance effect of the SNPs.
In conclusion, our findings suggest that TGFβ1 rs1800469 C>T polymorphism was significantly associated with decreased risk of conotruncal heart defects. TGFβR2 rs3087465 G>A, VEGF −2578C>A, −1498T>C, −634G>C and +936C>T polymorphisms may not play a role in the susceptibility of conotruncal heart defects.
This study was supported by the Major National Basic Research Program in the People’s Republic of China (Program 973, 2010CB529508), the National Natural Science Foundation of China (30900630), China Postdoctoral Science Special Foundation (2012T50066, 2012M510353), Peking Union Medical College Postdoctoral Foundation (2011-XH6), the Jiangsu Province Health Department Program Grant (H201046) and the Jiangsu Province Natural Science Foundation (BK2009207, BK2009209).
Conflict of interest