In recent years, chromosomal imbalances observed under the microscope have been considered a significant contributor to CHD7. This study used fetal ultrasound imaging to establish isolated CHD, non-isolated CHD, non-CHD, and control groups and described the distribution and frequency of CNVs in each group to evaluate the feasibility of SNP array in identifying the etiology of CHD fetuses. The overall detection rate of abnormal CNVs in isolated CHD and non-isolated CHD groups was 8.2% (14/170) and 14.7% (10/68), respectively, while in the non-CHD and control groups, the rates were 5.6% (30/538) and 3.2% (42/1316). Previous studies have shown that the detection rate of chromosomal microarrays for congenital heart disease ranges from 4–20%15, while another study reported detection rates of 6% for ultrasound abnormalities and approximately 1.7% for non-ultrasound structural abnormalities in older women and those with positive results in chromosomal screening16. Compared to previous studies, this research exhibits a similar detection rate for fetal congenital heart defects and other ultrasound structural abnormalities. However, the detection rate for cases without ultrasound structural abnormalities is slightly higher. This difference may be attributed to variations in the array platform used, the resolution of the array, and the reporting practices of individual clinical laboratories. Additionally, as new literature and public data sharing contribute to an expanding understanding of genomic regions associated with specific diseases, annual reviews of the same dataset have led to an increase in disease-associated cases to 1.8% while reducing the number of cases classified as variants of uncertain significance to 0.9%17.
A recent meta-analysis of 45 studies revealed that the overall prevalence of chromosomal abnormalities in isolated congenital heart defects (CHD) and non-isolated CHD was 16% and 37%, respectively. Among non-isolated CHD cases, the prevalence of aneuploidy (19%), other CNVs (excluding 22q11) (4%), and trisomy 18 were higher than in isolated CHD cases18. Our study demonstrated that the overall prevalence of chromosomal abnormalities in the isolated CHD group and non-isolated CHD group is 11.8% and 42.6%, respectively. Comparative analysis showed that the non-isolated CHD group has the highest prevalence of aneuploidy and overall chromosomal abnormalities, with statistically significant differences compared to the isolated CHD group, non-CHD group, and control group. The non-isolated CHD group had the highest prevalence of T18, and there was no statistically significant difference in the prevalence of T21 compared to the isolated CHD group, consistent with the meta-analysis. However, there was no statistically significant difference between the non-isolated CHD group and isolated CHD group (14.7% vs. 8.2%, p = 0.134) in prevalence of pathogenic CNV, and even after excluding 22q11, the non-isolated CHD group still showed no statistically significant difference in pathogenic CNV prevalence compared to the isolated CHD group (10.3% vs. 5.3%, p = 0.269), which differs from the meta-analysis. Furthermore, our study found that the prevalence of aneuploidy cases and pathogenic CNVs was slightly higher than in the meta-analysis, possibly due to differences in the number of subjects included in each group.
The traditional chromosomal karyotype resolution is typically in the range of 5-10Mb. Our research indicates that there was no statistically significant difference in the distribution frequency of pathogenic CNVs and variants of uncertain significance CNVs between isolated and non-isolated CHD groups. The significance of CNV segment size typically depends on their location in the genome and the genes or non-coding regions they contain. Smaller CNV segments may have localized effects on gene expression, which could impact an individual's phenotype and disease risk. Larger CNV segments may affect multiple genes, thus influencing various phenotypes and disease risks19. Some studies suggest that the etiology of isolated CHD is multifactorial, with some cases being attributed to single genes. Non-isolated CHD is associated with various causes, including chromosomal and sub-chromosomal abnormalities, single-gene syndromes, epigenetic factors, and environmental influences15. The results of our study show that in the non-CHD group, pathogenic CNVs larger than 10Mb account for 50% of the cases. These larger segments may be considered partial aneuploidy, and non-CHD with multiple systemic developmental abnormalities are more likely to manifest as chromosomal abnormality syndromes. In contrast, isolated CHD identified by ultrasound is more likely to be caused by smaller chromosomal segments or gene variations.
In the four groups of pathogenic CNVs, we identified a total of 15 cases involving the 22q11.2 region, including 11 deletions and 4 duplications. The 22q11.2 microdeletion is currently recognized as a syndrome associated with congenital heart defects, with severity ranging from non-survivable to subclinical, or even without a CHD phenotype. The main known causative gene for this syndrome is TBX1. The 22q11.2 microduplication syndrome complements the 22q11.2 microdeletion syndrome, sharing common features but exhibiting considerable phenotypic variation, with a CHD occurrence rate of approximately 25%20, 21. Furthermore, we observed one case each of 22q11.1q11.21 microduplication in the isolated congenital heart defect group and the control group. This region encompasses a critical area within the 22q11.21 recurrent region (Cat Eye Syndrome, CES). However, it does not involve the 22q11.2 region associated with DiGeorge syndrome/Velocardiofacial syndrome (DGS/VCFS)22. Upon pairwise comparison, we found that the occurrence rate of 22q11.2 abnormalities in the isolated CHD group and the non-isolated CHD group did not exhibit statistically significant differences (2.9% vs. 4.4%, p = 0.865), consistent with some previous reports18, 23. However, both the isolated and non-isolated CHD groups had higher occurrence rates than the non-CHD group (p = 0.012, p = 0.008) and the control group (p = 0.001, p = 0.001). This study identified 3 cases of 22q11.2 microdeletion and 2 cases of microduplication in the control group without ultrasound structural abnormalities. This may be related to the lack of obvious neurological and psychiatric characteristics in the prenatal stage and the absence of typical facial features.
Additionally, among the four groups, a total of 9 cases involved the 15q11.2. The occurrence rates were as follows: isolated CHD group 0.6% (1/170), non-isolated CHD group 1.5% (1/68), non-CHD group 1.1% (6/538), and control group 0.1% (1/1316). After pairwise comparisons, there were no statistically significant differences between the isolated CHD group and the non-isolated CHD group (p = 0.522). The nature of the 15q11.2 BP1-BP2 microdeletion has been a subject of controversy. Its prevalence in CMA-tested populations is approximately 0.57%-1.27% 24. Clinical phenotypes are mainly associated with neurodevelopmental disorders, developmental and language delays, and autism spectrum disorders, with a relatively low penetrance of 10–12%25. The 15q11.2 BP1-BP2 microdeletion encompasses four highly conserved non-imprinted genes, NIPA1, NIPA2, CYFIP1, and TUBGCP5. Currently, there is no established independent connection between this microdeletion and heart morphology. Some studies suggested that the 15q11.2 BP1-BP2 microdeletion has a relative frequency of 3.4% in intellectual disability, 2% in schizophrenia, and 2.1% in epilepsy, with no increased risk of cardiac malformation or autism, making it of limited clinical significance, and it has been suggested to be classified as a "mildly pathogenic factor." 26. In 2015, the UK Genomic Medicine Committee even proposed not to report the 15q11.2 BP1-BP2 microdeletion in prenatal diagnosis27. However, a recent study by the Williams team indicated an increased risk of cardiovascular malformation associated with the 15q11.2 BP1-BP2 microdeletion, with cardiovascular malformation being more common but not necessarily severe25. In our study, both isolated and non-isolated CHD groups had 15q11.2 microdeletion, and both exhibited ventricular septal defects, which aligns with the findings by Williams et al. In the non-CHD group, four cases of 15q11.2 microdeletion only showed increased nuchal translucency on ultrasound.
In variants of uncertain significance CNVs, small segmental duplications were predominant across the four groups. It was observed that 2q13 was involved in all four groups, with eight cases of microdeletions and one case of microduplication in total. The pathogenic nature of 2q13 is not well understood. Several studies have indicated that duplications and deletions of 2q13 are risk factors for developmental delay and anomalies. Wolfe’s research found an increased prevalence of attention deficit hyperactivity disorder (ADHD) in individuals with defects associated with the 2q13 locus, with 30% of defect carriers having heart defects, whereas no defects were observed in carriers of duplications28. Other researchers have noted that with chromosome 2q13 phenotypes, deletions are more enriched in cardiovascular disease, while duplications are associated with craniofacial features29. In our study, two cases of 2q13 deletion were found in individuals with CHD, and both were isolated cases. Their phenotypes were complete transposition of the great arteries with pulmonary artery stenosis and anomalous origin of the right pulmonary artery with pulmonary artery stenosis, which aligns with the above results. Since 2q13 can also be present in the normal population and given the current lack of large-sample data, further research may be needed to explore the pathogenic genes and phenotypes associated with heart defects.
Additionally, we also observed that the isolated CHD group had a Dup(9p24.3p24.1), the control group had a Dup(9p24.3), the non-isolated CHD group had a Dup(16q11.2q12.1), and the non-CHD group had a Dup(16q12.1), all of which involved partially overlapping regions. The 9p24.3 duplication segment contains genes like DOCK8 and may be associated with autism spectrum disorders, intellectual disabilities/developmental delay, and other conditions. However, whether the 9p24.3p24.1 segment is related to congenital heart disease phenotypes has not been reported in the literature30. All four groups of CNVs involved chromosome 16, but the specific segments affected were different. Chromosome 16 is one of the most enriched chromosomes for segmental duplications, and 16p is one of the more unstable regions in the genome, with over 10% of the 16p euchromatic regions consisting of highly complex low-copy repeats31, 32. The shared regions affected by CNVs in the non-isolated CHD group and the non-CHD group have rare clinical phenotypes reported in the literature. This suggests that, apart from genetic factors, other factors such as environmental influences may also play a role.
Study limitation
The study has a large overall sample size, but it still had limitations. The number of samples involving CHD is relatively small, especially for non-CHD. Ultrasound has certain limitations, and some cases with mild neurodevelopmental or craniofacial abnormalities may be challenging to classify into specific groups. Fetal and other associated symptoms may become more apparent as gestational weeks progress. Additionally, the study lacked parental validation and follow-up data.