ECE1 Polymorphisms May Contribute to the Susceptibility of Sporadic Congenital Heart Disease in a Chinese Population

Abstract

Endothelin-converting enzyme-1 (ECE1) plays a key role in the development of a subset of neural crest lineages such as cardiogenesis. Genetic variants of ECE1 C338A (rs213045) and T839G (rs213046) have been shown to alter ECE1 expression. This observation led us to hypothesize that two polymorphisms might influence the susceptibility of sporadic congenital heart disease (CHD). We conducted a case–control study comprised of 945 CHD cases and 972 non-CHD controls in a Chinese population. We tested our hypothesis by genotyping ECE1 C338A and T839G and assessed their association with the risk of CHD. Compared with the 338 CC and the 839 TT genotypes, the ECE1 338 AA/AC and 839 TG/GG genotypes significantly increased the risk of CHD (adjusted odds ratio [OR]=1.38, 95% confidence interval [CI]=1.14–1.68; and adjusted OR=1.30, 95% CI=1.07–1.58, respectively). A combined analysis was performed that showed that the presence of 2–4 risk alleles (the ECE1 338A and 839G allele) increased the risk of CHD by 2.07-fold compared with 0–1 risk alleles. Furthermore, we found that the association between 2–4 risk alleles and CHD risk was stronger in females (adjusted OR=1.77, 95% CI=1.31–2.40) than males (adjusted OR=1.33, 95% CI=1.03–1.71), and in the phenotypes of Tetralogy of Fallot (adjusted OR=1.84, 95% CI=1.10–3.06) and perimembranous ventricular septal defect (pmVSD) (adjusted OR=1.74, 95% CI=1.35–2.24). Our results suggest that ECE1 polymorphisms may contribute to the susceptibility of sporadic CHD in a Chinese population.

Introduction

C ongenital heart disease (CHD) is a leading cause of perinatal mortality, with an incidence of ∼0.6–0.8% (Hoffman and Kaplan, 2002). Some syndromic forms of CHD have been shown to be caused by monogenic inheritance or an abnormal chromosome structure. However, most nonsyndromic CHD occurs sporadically (Bruneau, 2008). Despite the fact that sporadic CHD is traditionally explained by the multifactorial inheritance model, which includes many susceptibility genes, little progress has been made in the search for common variants of candidate genes that may account for the majority of sporadic CHD cases.

Endothelin-converting enzyme-1 (ECE1) plays a central role in the endothelin (ET)-mediated signaling pathways, such as the ET-1/ECE1/ET-A receptor and ET-3/ECE1/ET-B receptor pathways. These two pathways act independently in the development of a subset of neural crest lineages, such as cardiogenesis (Yanagisawa et al., 1998). Neural crest cells, which are the components of the craniofacial regions, migrate to the heart where they are essential for septation of the left ventricular outflow tract (Kirby et al., 1983). In ECE1 gene knockout mice, Yanagisawa et al. (1998) observed various malformations in both the cardiac conotruncal region and great vessels that were similar to those in ET-1 knockout mice and ET-A receptor-deficient mice (Kurihara et al., 1995; Clouthier et al., 1998; Yanagisawa et al., 1998).

Furthermore, ECE1 is an essential component in ET biosynthesis. ET-1 is known to be involved in the regulation of proliferation and differentiation of myocardium and smooth muscle cells (Irons et al., 1996). However, it is noteworthy that both ECE1^−/− and ET-A^−/− embryos showed complete penetrance of cardiovascular malformations in contrast to ET-1^−/− embryos, which showed a lower incidence of the same defects (Kurihara et al., 1995; Clouthier et al., 1998; Yanagisawa et al., 1998). These phenomena suggest that pro-ET-1 derived from the mother or the placenta is delivered to the developing heart and locally converted to ET-1 by ECE1, thus partially rescuing the cardiovascular phenotypes of ET-1^−/− embryos. In ECE1 knockout mice, ECE1 cannot come from the mother or placenta (Yanagisawa et al., 1998). Therefore, we hypothesized that different ECE1 genotypes may affect cardiac development and contribute to the susceptibility of sporadic CHD.

ECE1 C338A (rs213045) and T839G (rs213046) are the two common polymorphisms in the promoter region of the ECE1 gene. Studies have revealed that ECE1 C339A is associated with increased transcription activity (Funke-Kaiser et al., 2003). Therefore, we determined the genotypes at the C338A and T839G polymorphic sites in CHD patients and control subjects to evaluate the relationship between ECE1 polymorphism and CHD.

Materials and Methods

Study population

This study was done under full compliance with all government policies and the Helsinki Declaration. All blood samples were obtained legally from the affiliated hospitals of the Nanjing Medical University (NJMU) and used only for research purposes. Since the participants were children, written informed consent was obtained from their next of kin or guardians. The protocol and consent form were approved by the Institutional Review Board of NJMU, Nanjing, China. A total of 945 affected children with sporadic CHD, confirmed by cardiac surgery, and 972 unrelated healthy individuals were included in this case–control study. Subjects for the study were recruited from the Affiliated Nanjing Children's Hospital of Nanjing Medical University, Nanjing, China, between March 2009 and December 2011. Potential study subjects were first surveyed at the clinics using a short questionnaire to determine their willingness to participate in a research study, and then demographic information was obtained through a face-to-face interview. After the interview, 4 mL of venous blood was collected from each case or control. Cases who had other congenital disorders or known chromosomal abnormalities were excluded. Exclusion criteria for mothers included maternal diabetes mellitus, phenyl ketonuria, maternal teratogen exposure, or maternal therapeutic drug exposure during the intrauterine period (Jenkins et al., 2007). These exclusion criteria were necessary, since all of these factors can increase the risk of congenital anomalies. Controls were non-CHD outpatients from the same geographic area, who were age- and sex-matched to the CHD cases. They were recruited from the same hospital during the same time period, and most had a diagnosis of trauma or infection. Controls with any congenital anomalies were excluded. All subjects were genetically unrelated ethnic Han Chinese.

Genotyping

Genomic DNA was extracted from peripheral blood leukocytes by using the DNA Mini Kit (QIAGEN, NED). Genotyping was performed using the TaqMan method with the ABI 7900HT Real-Time PCR System, according to the manufacturer's instructions (Applied Biosystem). Our TaqMan Minor Groove Binder (MGB) probes were designed and synthesized by Applied Biosystem. The sequence of probes were 6FAMCCTCGAT GTGGCCCAMGBNFQ (rs213045-G), 7VICCCTCGATTTGG CCMGBNFQ (rs213045-T), 6FAMAGAACCCCAGAGAGG TMGBNFQ (rs213046-A), and 7VICAACCAAGAACCCCC GAGAMGBNFQ (rs213046-C). The polymerase chain reaction (PCR) primers for C338A (rs213045) and T839G (rs213046) were 5′-GTGGCAGATAACAAAAGTATCAG GAA-3′ (forward) and 5′-TTTGTCTGGTCTTTCTAGCATT AACC-3′ (reverse); 5′-TGTTCAGTTAGGGACTCAGGCC-3′ (forward) and 5′-GAAGCGGAGAGTCCTTTGGAA-3′ (reverse), respectively. About 10% of the samples were randomly selected for confirmation. The multiplex SNaPshot technique was used for genotyping. For rs213045, the PCR primers were 5′-TCCCCAGTGGCAGATAACAA-3′ (forward), 5′-AGACACACCTAAGGGCTGATG-3′ (reverse), and the extended primer was 5′-TTTTTTTTATCAGGAAG GTGCCCTCGAT-3′. For rs213046, the PCR primers were 5′-TATGAGGTGTTCAGTTAGGGACT-3′ (forward), 5′-CCT TGCTAGAAGCGGAGAGTC-3′, and the extended primer was 5′-TTTTTTTTTTTTTATCTGCTGGGTTAGACCTCTC-3′. The purified extension products were run on ABI3130XL (Applied Biosystems) and analyzed using GeneMapper 4.0 (Applied Biosystems). The results were 100% concordant.

Statistical analysis

Differences in the distributions of demographic characteristics, selected variables and frequencies of genotypes of the ECE1 C338A and T839G polymorphisms between the cases and controls were evaluated using a Student's t-test (for continuous variables) or a χ ² test (for categorical variables). The associations between the genotypes and risk of CHD were estimated by computing odds ratios (ORs) and their 95% confidence intervals (95% CIs) from an unconditional logistic regression analysis, with adjustment for possible confounders. The genotype frequencies of these two polymorphisms among controls were all in agreement with the Hardy–Weinberg equilibrium (p=0.146 for C338A; p=0.633 for T839G). We controlled the potential influence of multiple comparisons using the false discovery rate method. p<0.05 was considered statistically significant, and all statistical tests were two-sided. All statistical analyses were performed using SAS 9.1.3 software (SAS Institute).

Results

Characteristics of the study group

The characteristics of the CHD cases and controls included in the analysis are summarized in Table 1. There were no statistically significant differences between the cases and controls in age or gender (p=0.606 for age and p=0.558 for gender). Of the 945 CHD patients, 195 were diagnosed with cyanotic heart disease, 645 had a septation defect, 57 had patent ductus arteriosus (PDA), and 25 had a left-sided outflow obstruction defect.

Table 1.

Distribution of Selected Variables Between the Case and Control Subjects

	Cases		Controls
Variables	n	%	n	%	p^a
Age, years (mean±standard deviation)	2.87±0.18		2.94±0.17		0.606
Sex
Male	529	56.0	557	57.3	0.558
Female	416	44.0	415	42.7
CHD classification I
Cyanotic heart disease	195	20.6
Left-side obstruction defects	25	2.7
Septation defects	645	68.3
Patent ductus arteriosus	57	5.5
Other complex abnormality	28	3.0
CHD classification II
Septa and valve abnormalities only	701	74.2
Other CHD abnormalities	244	25.8
CHD classification III
Isolate CHD	559	59.2
Nonisolate CHD	386	40.8

The two-sided t test for age and the χ ² test for the distributions of gender.

CHD, congenital heart disease.

Associations between ECE1 polymorphisms and risk of CHD

The observed genotypes and allele frequencies for CHD cases and controls are shown in Table 2. The distributions of ECE1 C338A genotypes were different between cases and controls (p=0.003, p _trend=0.019). The logistic regression analysis revealed that the ECE1 C338A AC genotype, but not the AA genotype, was associated with a significantly increased risk of CHD compared with the CC genotype (adjusted OR=1.43, 95% CI=1.16–1.76). Furthermore, the ECE1 338AA/AC genotypes were significantly associated with an increased risk of CHD compared with the 338CC genotype (adjusted OR=1.38, 95% CI=1.14–1.68). For T839G polymorphism, the respective frequencies of TT and TG/GG were 28.0% and 72.0% among the cases compared with 33.7% and 66.3% among the controls (p=0.0074, p _trend=0.036). The TG/GG genotypes were associated with a 1.30-fold increased risk of CHD (adjusted OR=1.30, 95% CI 1.07–1.58) compared with the TT genotype.

Table 2.

Association of the ECE1 Polymorphisms with Risk of Congenital Heart Disease in a Chinese Population

	Cases (n=945)		Controls (n=972)
Genotypes	n	%	n	%	p^a	Adjusted OR (95% CI) ^b
C338A
CC	257	27.2	332	34.2	0.003	1.00 (reference)
AC	501	53.0	453	46.6	0.001	1.43 (1.16–1.76)
AA	187	19.8	187	19.2	0.053	1.29 (0.99–1.67)
AA+AC	688	72.8	640	65.8	0.001	1.38 (1.14–1.68)
A allele	875	46.3	827	42.5	0.019
C allele	1015	53.7	1117	57.5
Trend test					0.019
T839G
TT	265	28.0	328	33.7	0.025	1.00 (reference)
TG	499	52.8	467	48.1	0.008	1.32 (1.08–1.62)
GG	181	19.2	177	18.2	0.079	1.25 (0.96–1.63)
GG+TG	680	72.0	644	66.3	0.007	1.30 (1.07–1.58)
G allele	861	45.6	821	42.2	0.038
T allele	1029	54.4	1123	57.8
Trend test					0.036

The two-sided χ ² test for the distributions of genotype and allele frequencies.

Adjusted for age, sex in logistic regression model.

ECE1, Endothelin-converting enzyme-1, OR, odds ratio; CI, confidence interval.

Combined analysis of ECE1 C338A and T839G polymorphisms and the risk of CHD

Because the two single nucleotide polymorphisms (SNPs) (ECE1 C338A and T839G) were each associated with an increased risk of CHD, we combined the two polymorphisms based on the number of the risk alleles (338A and 839G) to further investigate the association between ECE1 polymorphisms and CHD. Before we did the combined analysis, we performed a linkage disequilibrium (LD) test between these two SNPs (r ²=0.72). We found that the difference in the distribution of the combined genotypes between the cases and controls was statistically significant (p<0.001) (Table 3). Since the frequencies of some combined genotypes were too low in the study population, we combined the 5 possible genotypes into just two groups (0–1 variants and 2–4 variants) based on the frequencies among the controls. This analysis showed that the distribution of these two groups between the cases and controls was significantly different (p<0.001). When we used the combined genotype with 0–1 variants as the reference, we found that the combined genotype with 2–4 variant alleles was associated with a statistically significantly increased risk of CHD (adjusted OR=2.07, 95% CI=1.46–2.94).

Table 3.

Cumulative Effect of the Two Risk Loci on the Risk of Congenital Heart Disease

	Cases (n=945)		Controls (n=972)
No. of risk alleles of the combined genotypes ^a	n	%	n	%	p^b	Adjusted OR (95% CI) ^c
0	250	26.5	268	27.6	<0.0001
1	19	2.0	96	9.9
2	481	50.9	406	41.8
3	25	2.7	68	7.0
4	170	18.0	134	13.8
Recombined groups
0–1	269	28.5	364	37.4	<0.0001	1.00 (reference)
2–4	676	71.5	608	62.6		2.07 (1.46–2.94)

The number represents the number of variant alleles; the variant alleles used for the calculation were the ECE1 338A and 839G alleles.

The two-sided χ ² test for distribution between the cases and controls.

Adjusted for age and sex in the logistic regression model.

We also evaluated the effect of the combined genotypes on CHD risk after the patients were stratified by sex or some specific CHD phenotypes. Since several patients had two or more types of cardiac defects, we chose only subjects with a single phenotype. As shown in Table 4, the association between 2–4 variants group and CHD risk was stronger in females (adjusted OR=1.77, 95% CI=1.31–2.40) than males (adjusted OR=1.33, 95% CI=1.03–1.71). There was also an increased risk of Tetralogy of Fallot (TOF) in individuals with 2–4 variants (adjusted OR=1.84, 95% CI=1.10–3.06) and perimembranous ventricular septal defect (pmVSD) (adjusted OR=1.74, 95% CI=1.35–2.24). However, we found no association between 2–4 variants and the risk of atrial septal defect (ASD) or PDA.

Table 4.

Stratification Analyses Between the Combined Genotypes and the Risk of Congenital Heart Disease

	Combined genotypes (case/control)
	0–1 variants		2–4 variants
Variable	n	%	n	%	p^a	Crude OR (95% CI)	Adjusted OR (95% CI) ^b
Sex
Female	102/152	24.5/36.3	314/263	75.5/63.4.	0.0002	1.78 (1.32–2.40)	1.77 (1.31–2.40)
Male	167/212	31.6/38.1	362/345	68.4/61.9	0.025	1.33 (1.04–1.71)	1.33 (1.03–1.71)
The phenotypes of CHD
TOF	21/364	24.7/37.5	64/608	75.3/62.6	0.019	1.83 (1.10–3.04)	1.84 (1.10–3.06)
Perimembranous ventricular septal defect	108/364	25.5/37.5	315/608	74.5/62.6	<0.001	1.75 (1.35–2.25)	1.74 (1.35–2.24)
Atrial septal defect	29/364	44.6/37.5	36/608	55.4/62.6	0.249	0.74 (0.45–1.23)	0.74 (0.45–1.24)
Patent ductus arteriosus	16/364	28.1/37.5	41/608	71.9/62.6	0.154	1.53 (0.85–2.77)	1.54 (0.85–2.80)

The two-sided χ ² test for distribution between the cases and controls.

Adjusted for age and sex in the logistic regression model.

Discussion

In this study, we observed that a significantly increased risk of CHD was associated with ECE1 C338A and T839G polymorphisms. When we evaluated these two polymorphisms together, the risk was significantly increased in the individuals carrying 2–4 risk alleles (ECE1 338A and 839G allele) compared with those who had 0–1 variants. These findings suggested that the two polymorphisms of the ECE1 genes may contribute to the susceptibility of sporadic CHD. To our knowledge, this is the first report to evaluate the association of ECE1 polymorphisms with CHD risk.

ECE1 C338A and T839G polymorphisms are located in the 5′-regulatory region of the ECE1 gene. For polymorphisms in the ECE1 promoter, several previous studies have detected potential associations with Alzheimer's disease, hypertension, and coronary artery disease (CAD) (Funalot et al., 2004; Wang et al., 2007; Scacchi et al., 2008; Jin et al., 2009a, b). One study showed that patients with CAD had a lower frequency of the ECE1 338A allele than controls, but the difference was not significant (p=0.11). Furthermore, the AA/AC genotypes decreased the risk of CAD only in individuals without the apolipoprotein E e*4 allele (adjusted OR=0.51; 95% CI=0.29–0.89; p=0.018) (Scacchi et al., 2008). Another study reported that individuals with ECE1 338AA/AC had a 1.58-fold increased risk of CAD compared with the CC genotype (adjusted OR=1.58; 95% CI=1.07–2.32; p=0.020) (Wang et al., 2007). Funke-Kaiser et al. (2003) observed that the 338A allele may increase transcriptional activity in transient transfection assays compared with the 338C allele by increasing allele-dependent binding affinities of the transcription factor E2F2 to the ECE1 promoter. In vivo, ECE1 mRNA expression levels were 2-fold higher in carriers of the 338A allele than in non-carriers. Although a synergistic effect on promoter activity was reported when 839G was combined with 338C, the ECE1 promoter region containing 839G did not appear to affect transcriptional activity; thus, T839G polymorphism may be a LD marker.

In our study, we observed that the ECE1 338A and 839G alleles may be the risk alleles for sporadic CHD. It has been suggested that increased plasma ECE1 levels may contribute to CHD. This may be attributed to higher ET-1 and ET-3 levels generated by increased ECE1 locally, and higher ET levels may disturb the development of a subset of neural crest lineages. Furthermore, functional redundancy of ET-A and ET-B receptors in cardiac development can also be decreased by higher ET levels (Yanagisawa et al., 1998). In our combined analyses, we observed that carrying 2–4 variants significantly increased the risk of CHD in females, but not in males. A similar phenomenon was reported in many other studies (Funke-Kaiser et al., 2003; Funalot et al., 2004; Wang et al., 2007; Jin et al., 2009a, b). Funalot et al. (2004) speculated that the gender-specific association might be due to a past effect of female hormones on ECE1 gene regulation or alternatively to the effects of androgens in men, which would abolish the influence of the genetic variants. ECE1 is expressed is both endothelial cells and steroidogenic cells of the corpus luteum. Its expression level depends on the phase of the menstrual cycle (Levy et al., 2001). Male hormones have been shown to raise plasma ET levels, whereas female hormones have been shown to decrease them (Polderman et al., 1993). We speculate that the gender differences we observed may be caused by a possible gender-specific modulation of ET system activation. It is tempting to speculate that the ECE1 promoter variants and ECE1 expression might have a greater impact on the risk of CHD in females than in males (Webb et al., 2000; Dubey et al., 2001). Furthermore, we evaluated the effects of the combined genotypes on the risk of specific CHD phenotypes. For our analysis, we selected 4 subgroups: TOF, perimembranous VSD, ASD, and PDA. We found that the combined genotypes (2–4 variants) were strongly associated with TOF and perimembranous VSD, but not PDA and ASD. Neural crest cells are known to migrate and contribute to the formation of outflow tract septation and conotruncal regions, so we suggest that the two SNPs of ECE1 may contribute to the risk of TOF and perimembranous VSD by disturbing the normal developmental process. Since ECE1 mainly contributes to the development of a subset of neural crest lineages, the two SNPs may not affect risk of ASD. Furthermore, since closure of PDA occurs after birth, it is probably influenced by additional factors unrelated to the function of ECE1.

Since we evaluated a hospital-based population, we could not avoid the possibility of selection bias of subjects that may have been associated with a particular genotype. Due to the low incidence of some phenotypes of CHD, we only chose 4 major phenotypes of CHD for our analysis of subgroups. For some rare phenotypes, the sample size was too small for a genetic association study. Therefore our analysis of subgroups could not confirm the results for these anomalies. Since our results were based on significant associations between ECE1 polymorphism and the risk of CHD, the results do not establish a causal relationship for each phenotype of CHD and should therefore be interpreted with caution. To validate our findings, additional functional studies are needed. Since our study was only conducted in a Chinese population, the results should be extrapolated to other ethnic groups cautiously.

In conclusion, we observed that the ECE1 C338A and T839G polymorphisms may confer susceptibility to sporadic CHD. Since our sample size was moderate and the statistical power of the study was limited, large population-based prospective studies are warranted to further elucidate the impact of ECE1 polymorphisms on the prevalence of CHD.

Conclusions

We observed that a significantly increased risk of CHD was associated with ECE1 C338A and T839G polymorphisms. Furthermore, we found that the association between 2–4 variants and CHD risk was stronger in females and in the phenotypes of TOF and perimembranous VSD. Our results suggest that polymorphisms of ECE1 may contribute to the susceptibility of sporadic CHD. More detailed information about environmental exposure and a gene–gene interaction are required to validate our findings.

Footnotes

Acknowledgments

We want to give our special thanks to the patients and their parents for participating in this study, and Dr. Zhen Wu (The Affiliated Children's Hospital of Nanjing Medical University) for blood sample collection. The study was supported by the National Nature Science Foundation of China (81070137) and 333 Foundation for the Academic Leader of Jiangsu Province (Xuming Mo).

Disclosure Statement

No competing financial interests exist.

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