Congenital heart disease (CHD) is the most common congenital defect (1). It is also the most common cause of mortality associated with congenital defects in newborns and is often associated with fetal loss. The prevalence of CHD has increased from 0.6% in the 1930s to 0.91% post-1995; however, this may, in part, be a result of improved diagnosis and increased frequency of monitoring. The highest rate of CHD prevalence was reported to be 9.3 cases per 1,000 live births in Asia (2). Although certain minor abnormalities, such as a minor ventricle septal defect (VSD) or atrial septal defect (ASD), do not have any functional impact, rarely affect the quality of patients’ lives and do not require intervention, the most complicated conditions require surgery or catheter intervention treatment. Certain complex lesions require multiple surgical procedures and close monitoring during the first few years of life. However, the etiology of CHD and its root cause remain to be elucidated. Therefore, exploration of genetic and environmental factors that may lead to an increased risk of CHD is of interest to patients with CHD and their families in terms of healthcare management and family planning. With the increasing number of patients with CHD, the assessment of the risk of recurrence in the children of patients with CHD has also become increasingly important (3). Furthermore, an enhanced understanding of CHD etiology may facilitate CHD prevention and clinical management of patients with CHD.

Approximately 20% of the cases of CHD result from known causes, such as genetic syndromes and teratogens. By contrast, little is known about the causes of the CHD in the remaining 80% of cases. It was hypothesized that these CHD lesions may be the result of interactions between genetic and environmental factors (4). Studies on CHD in the past decade have demonstrated that gene mutations of transcription factors, including T-box transcription factor 5 (TBX5), NK2 transcription factor related locus 5 protein (NKX2.5), and GATA binding protein 4 (GATA4), are able to cause CHD (5–8). A limited number of studies have examined the association between maternal lifestyle and risk of CHD. Maternal alcohol consumption during pregnancy is associated with birth defects in children (9–11). In the Baltimore-Washington Infant Study (12), maternal cigarette smoking of more than one pack per day was associated with two cardiac defects: Transposition with VSD and pulmonic stenosis (PS) (in females >34 years old) (12). Environmental factors affecting the rate of CHD have frequently been observed to act within the maternal preconceptional or fetal-placental-maternal context. The most common maternal risk factor is diabetes (13). Botto et al (14) reported that folate and folic acid-containing vitamin supplementation at the preconceptional stage has a protective effect against CHD.

The progress of novel genetic testing technologies, such as whole exome and genome sequencing, has greatly accelerated gene identification and led to the determination of novel candidate genes that may be involved in cases of CHD with unknown etiology (15). Traditional disease-gene linkage determination is predominantly based on analyzing a large number of families with multiple affected members, which is not practical for the analysis of CHD due to its rarity. A previous study conducted by our group analyzed 25 out of 61 familial pedigrees and found that the morbidity of CHD among the family members correlated with their genetic relationship, i.e. the risk of CHD was increased with the closeness of their genetic relationship. In addition, there was an increased incidence of threatened abortion in early pregnancy in patients with familial CHD as compared with those with sporadic CHD (16). Therefore, the present study design extended the familial pedigree collection and included additional families, as well as families with twins affected by CHD, to further investigate the genetic relationships among affected patients, their family members with CHD, familial CHD history and six key environmental factors. Results from the present study provide insights into the pathogenesis, intervention and prevention of CHD in China.

It is generally accepted that CHD is a multifactorial disease and that genetic and environmental factors are involved in the development of the disease (4). Genetic variations interact with each other, and, together with environmental factors, increase the susceptibility to CHD. A number of studies have focused on the etiology of CHD during the past decades (17,18). However, the exact pathogenesis of CHD remains to be elucidated. Previous studies have revealed that CHD has the potential to aggregate in families with its recurrent risk in relatives being higher than in the general population and positively correlated with genetic relationship (17,19). The results of the present study are in accordance with previous studies in terms of CHD being more likely to occur when the first-degree relatives of patients with CHD were affected with CHD. The CHD recurrence is significantly higher in the first-degree relatives compared with that of the second-degree relatives, implying that genetic factors have a key role in the development of CHD.

A previous study reported that the risk of recurrence in children of families in which one or both of the parents are affected by CHD is significantly higher than in children whose siblings are affected and their parents unaffected by CHD. Furthermore, the risk is higher when the mother is affected by CHD, compared with when the father is affected (20). When both parents are unaffected by CHD, the recurrence risk in the siblings of an affected individual is 1–6% for the majority of CHD lesions (21,22). The present study revealed that the recurrence risk of CHD was significantly higher in patients whose mother rather than father was affected, which suggested that maternal genetic factors may contribute to the development of CHD. By contrast, no difference in the risk of recurrence between individuals whose parents were affected and those whose siblings were affected was identified. These findings are rather inconclusive due to the limited number of patients and family members in the present study. However, to the best of our knowledge, the present study was the first to show that the recurrence risk was higher in patients whose sister was affected than in those whose brother was affected (P<0.01). This is another indication of the influence of maternal genetic factors in the development of CHD.

The twin studies conducted in the present study, although limited by complicated factors, provided the most reliable source for separating genetic contributions to CHD from environmental influences. Monozygous (identical) twins have the same genetic background; however, they are raised separately under the influence of different environmental factors. The concordance rate in monozygotic twins was compared with that of dizygotic (fraternal) twins to evaluate the genetic component (heritability) of the development of CHD. In the 21 twins in the present study, the concordance rate in monozygotic twins was 94.4%, while the concordance rate in dizygotic twins was only 33.3%. These results emphasize the involvement of multiple factors in the development of CHD, despite the low group size of dizygotic twins (n=3). It is of note that TOF and VSD occurred concurrently in three sets of twins, indicating that TOF and VSD may have similar mechanisms (20,22).

Environmental factors associated with CHDs have been observed to act in a maternal preconceptional or fetal-placental-maternal context. As aforementioned, mothers with pre-gestational diabetes have a five-fold increased risk in giving birth to children with CHD (5). Other environmental factors, although not conclusive, have also been reported (9–11). Shillingford and Weiner (23) reported that ASD was the most frequent cardiac anomaly in neonates with fetal alcohol syndrome caused by maternal alcohol consumption. A case-control study in Spain showed that a higher risk of developing CHDs was present in a group with the highest level of prenatal exposure to alcohol (24). Furthermore, Torfs and Christianson (25) reported that maternal cigarette smoking was associated with specific defects (atrioventricular canal, ASD and TOF). However, another study found no association between any heart defect and maternal smoking (26). Therefore, the present study considered six major environmental factors relevant for investigating their association with the development of CHD. It was revealed that threatened abortion in early pregnancy was significantly higher in patients with familial CHD than in patients with sporadic CHD, while for smoking, we observed that was it marginally sigificantly assocaited with familial CHD (P=0.05), which we consider to be due to the small sample size in the present study and therefore requires further study to investigate this association.

The present study confirmed previous findings and revealed additional mechanisms of CHD and potential environmental risk factors. Larger patient groups and a larger database of environmental factors are required to further verify the results of the present study. We aim to continue and extend the collection of pedigrees with detailed family histories, including the medical history of the mother and vitamin supplement intake during pregnancy. A total of 61 families have been enrolled in this continuing study and their blood samples have been collected for further analysis. Genetic analysis via next-generation sequencing and characterization of patients with CHD is ongoing.

In conclusion, the present study provided supportive information towards an enhanced understanding of the mechanism of the development of CHD. The results have laid a foundation to further investigate CHD-associated gene identification and provide a fundamental approach to establish individual genetic counseling for patients with CHD and their families. The presented analysis of pedigrees may facilitate research and clinical investigation to further explore and identify the pathogenic linkage of CHDs as well as the molecular mechanisms and genes involved in the development of CHD.