The NKX2-5 gene is located on chromosome 5q35.1 and consists of two coding exons that encode a protein consisting of 324 amino acids. Similar to other members of the NK2 family of transcription factors, it contains a highly conserved homeodomain (HD), which encompasses a helix-loop-helix domain with three alpha helices responsible for recognizing and binding specific DNA sequences (Chen and Fishman, 1996; Dixit et al., 2021). A transient upregulation of NKX2-5 expression occurs during conduction system development, indicating a crucial role of this gene in the maturation and establishment of the conduction system through modulation of gap junction and ion channel protein expression (Pashmforoush et al., 2004). Although the association between NKX2-5 gene variants and various congenital heart diseases is well-established, the genotype-phenotype correlation of these variants in heart diseases remains unclear (Zhu et al., 2000). Our study implemented the genetic test of a large non-syndromic congenital heart disease family in China, with the objective of identifying the underlying cause and providing more precise information for subsequent genetic counseling.

The heart is the first organ to form during embryonic development and is an essential prerequisite for embryo growth and survival, as it provides adequate oxygen and nutrients through the circulatory system. The formation of a fully functional four-chamber heart results from the precise coordination of cell differentiation processes that integrate multicellular morphogenesis in the early embryo. CHDs are the most common birth defects in humans, with an estimated prevalence of up to 1% for live births and 10% for stillbirths (Xu et al., 2017; Akazawa and Komuro, 2005; Fahed et al., 2013; Marelli et al., 2014; Mozaffaria et al., 2015).

Cardiac development is a complex biological process that necessitates the integration of cellular commitment, morphogenesis, and excitation-contraction coupling. Numerous empirical findings have demonstrated the indispensable role of NKX2-5 in regulating septal formation and facilitating the maturation and maintenance of atrioventricular node function during cardiac morphogenesis (Pashmforoush et al., 2004; Bruneau, 2008; Harvey, 2002). Consequently, genetic variations in NKX2-5 can lead to nonsyndromic congenital heart disease (Schott et al., 1998). The association between genetic variants of the NEX2-5 gene and cardiovascular abnormalities has been confirmed in various animal models, including Drosophila, frog, and mouse (Tanaka et al., 2002; Harvey, 2002). Knockout of the NKX2-5 gene can cause cardiovascular abnormalities and impaired heart development.

Variants in NKX2-5 gene can cause various congenital heart defects, indicating that this transcription factor is involved in multiple pathways of heart development, including atrial, ventricular, and septal development, as well as atrioventricular conduction and atrioventricular valve formation (Benson et al., 1999). The families in the present study exhibited diverse cardiac abnormalities, similar to those observed previously.

The expression of NKX2-5 occurs in the heart and cardiac progenitor cells during early stages of development when the two cardiac primordia are symmetrically positioned in the anterolateral mesoderm. Although different nonsense variants of the NKX2-5 gene lead to the same cardiac abnormal phenotype, there are differences in transcriptional activation ability and DNA binding ability among various variants. The apoptosis of myocardial cells may be one of the reasons why certain nonsense variants induce heart defects (Zhu et al., 2000).

The NKX2-5 gene variants have been identified in numerous cardiac disorders, including tetralogy of fallot, nonsyndromic CHDs, ASD, patent foramen ovale, and others. Although the association between NKX2-5 gene variants and various congenital heart diseases is well-established, the genotype-phenotype correlation of these variants in heart diseases remains unclear (Goldmuntz et al., 2001; Gioli-Pereira et al., 2010; Belvís et al., 2009; Watanabe et al., 2002). The Chinese family predominantly exhibit atrial septal defect in our study, while other cardiac abnormalities observed include tetralogy of fallot, and patent foramen ovale, etc. The same nonsense variant (NM_004387.4: c.342C>A, p.(Cys114*)) in NKX2-5 gene is associated with variable disease severity and cardiac phenotype even within the family. Growing evidence supports oligogenic and multifactorial models of CHDs (Gifford et al., 2019). However, since our study only performed Trio-WES on the proband and parents, there are limitations in analyzing other genetic factors beyond the rare likely pathogenic variant in this family.

This variant has been submitted to the ClinVar database and is associated with atrial septal defect 7 (ASD7), where it has been classified as pathogenic (Accession:RCV002889896.2). However, this variant has not been reported in the literature in individuals affected by NKX2-5-related disorders. In addition, the ClinVar database also includes other variants in the NKX2-5 gene that affect the same amino acid(NM_004387.4:c.342C>G(p.Cys114Trp); c.340T>G(p.Cys114Gly)), but these are all classified as uncertain significance (VUS) and lack supporting literature reports. Additionally, the gnomAD database contains LoF variants for NKX2-5 gene. This observation may be attributed to variable expressivity of the gene-related phenotypes, suggesting that some asymptomatic individuals might have been included in the control population database. Notably, the two variants rs1761374435 and rs1761372394, annotated as loss-of-function (LoF) in gnomAD, show relatively high population carrier frequencies (0.0003431 and 0.0001467 in East Asian populations, respectively). Through UCSC genome browser analysis, we identified that both variants are located within intronic regions of the MANE-select transcript, suggesting their functional impact may be limited.

To date, the HGMD Professional database has documented loss-of-function variants in the NKX2-5 gene across dozens of cases involving various cardiovascular disorders, suggesting sufficient evidence of haploinsufficiency for this gene. The NKX2-5 protein contains an evolutionarily conserved HD domain located at amino acid positions 145 to 197, which specifically functions to recognize and bind consensus DNA motifs. In this present study, a heterozygous variant of NKX2-5(NM_004387.4: c.342C>A, p.(Cys114*)), was identified in a family with nonsyndromic congenital heart disease, which is expected to result in the production of a truncated protein. Therefore, it is plausible to consider this variant as the underlying cause of the disease within this particular family. However, the specific impact of this variant on the protein requires further functional experimental validation. For example, the expression of truncated transcripts can be confirmed through transcript data (such as RT-PCR), and the loss of transactivation activity can be assessed via functional experiments (such as luciferase reporter assays).

Small ASD may close spontaneously during infancy or childhood, while large ASD in adulthood may lead to massive blood shunts from left to right, leading to congestive heart failure, atrial arrhythmia, sudden cardiac death and other serious abnormalities. The ultimate therapeutic approach for certain patients involves heart transplantation, and a precise genetic diagnosis holds great significance in terms of treatment and subsequent guidance on fertility (Ellesøe et al., 2016; van der Meulen et al., 2022).

The findings suggest that the same variant may manifest diverse phenotypes and varying severity of cardiac abnormalities even within the family. An early and definitive genetic diagnosis can provide precise information for subsequent treatment and fertility counseling.