Generally, human cilia are classified into two types: non-motile cilia (also known as primary cilia) and motile cilia. The critical function of primary cilia involves the signaling pathway and the regulation of the cellular environment, while respiratory tract mucous clearing, transport, and movement of cerebrospinal fluid, as well as gametocytes, are performed by the motile cilia.51 Dextrorotatory of the cilia produces a one-way nodal flow toward the left; hence transmission of determinant molecules is then toward the lateral plate mesoderm on the left, which produces left–right patterning asymmetry of the organs.9 Cilia generate leftward fluid flows in an embryonic “left–right organizer,” which directs asymmetric activation of the Nodal signaling pathway.52 This pathway guides the asymmetric morphogenesis of developing organs, including the heart.53 Studies in mice have shown that cilia transduced Hedgehog signaling coordinates left–right patterning with heart looping and differentiation of the heart tube.54

Studies on PCD or cilia dysfunction in HTX suggested that the genes involved in ciliary function play essential roles in determining the left–right patterning.6 Examples of genes involved in ciliary structure and function are DNAH11, DNAH5, DNAH7, and DNAI1.55 At the embryonic node, abnormalities in either motile or non-motile cilia may alter the left nodal flow, leading to HTX.51 About 12.1% of patients diagnosed with PCD and about 8.9% of patients without PCD were associated with HTX.56 A recent study by Liu et al. (2019) mentioned that roughly half of the patients with PCD were reported to have HTX associated with CHD.6 HTX and CHD are caused by multiple genes while PCD is a monogenic recessive illness with genetic heterogeneity.6,47 The connection between HTX, CHD and PCD needs to be investigated further, considering the effect of ciliary function on HTX and CHD from past studies.

Heterotaxy syndrome arises from intricate dysregulations in multiple signaling pathways during embryonic development. The Nodal, Hedgehog, and canonical WNT pathways play pivotal roles in establishing left–right asymmetry.57–59

The nodal signaling pathway is a significant component in left–right patterning determination during embryo development.60 In the Nodal signaling pathway, information is transmitted by type I and II receptors and EGF-CFC co-receptors, and the consequences of the receptor activation are SMAD2 and SMAD3 phosphorylation (Figure 2(A)).60,61 This enables SMAD2/SMAD3 binding to SMAD4, causing interactions with other transcription factors and target genes regulations in the nucleus. The heterodimeric SMAD complex may interact with DNA binding proteins (e.g. FOXH1 and MIXER) or simply bind to DNA.62 This process involves both extracellular and intracellular components.61 Proteins from the Nodal signaling pathway i.e., NODAL, LEFTY and PITX2 are expressed asymmetrically on the left side while LEFTY1 is expressed on the midline of the lateral plate mesoderm (LPM).15 The Nodal signaling pathway is dose sensitive, where any abnormality in signal transduction will affect Nodal expression, risking abnormal patterning and lead to HTX.62 Damaged Activin/Nodal-Smad signaling and reduction in signal transduction were observed in functional analysis of NODAL variants, supporting the dose sensitive hypothesis of the pathway.60,62 In an animal study, NODAL was found to have an important function in primitive streak and embryonic mesoderm development.63 To maintain balanced expression of Nodal, antagonists which are the Lefty proteins, act as negative feedback toward the Nodal expression.15,60 In a targeted study on mice, all four Lefty1 exons were removed using targeting vector, which resulted in abnormality of left–right patterning as Nodal was expressed bilaterally in the LPM.64 This supports the postulated mechanism of either the absence or bilateral expression of NODAL in NODAL signaling that causes HTX.62,64

Figure 2.

Three signaling pathways suggested to be involved in causing HTX. (A) Nodal signaling pathway. NODAL binds to its receptors (type 1, ActRII and type 2, ALK4) and co-receptor (EGF-CFC). SMAD2/3 are phosphorylated and bind with SMAD4, forming a complex. The SMAD complex enters the nucleus to regulate target genes transcription either by binding to the DNA binding protein or directly binds to the DNA. To keep the balance of NODAL expression, LEFTY acts as the negative feedback protein where it either binds to NODAL of the co-receptor when NODAL is expressed in abundance. Repression of the target genes may lead to absence or bilateral expression of NODAL leading to laterality defects and HTX. (B) Hedgehog signaling pathway. SMO inhibition by PTCH is lifted when Hh ligands bind to PTCH. This allows downstream cascade as SuFu stops its binding to GLI and GLI is able to enter the nucleus for target gene transcription. Binding of SuFu to GLI is a negative regulation of the pathway as the binding produces GLI-repressor. Presence of ZIC3 will inhibit the production of GLI-repressor and affect the gene transcription process in the nucleus. Repression of the IFT genes will cause disruption of the ciliary structure and function which also will cause laterality defects and HTX. (C) Canonical WNT signaling pathway. WNT binds to its receptor, FZD and co-receptor, LRP5/6. The binding induces dissociation of a complex consisting of APC, AXIN/DVL/GSK3/CK1/β-catenin. The result of the dissociation is the release of β-catenin which is then able to enter the nucleus to regulate target genes transcription by interacting with TCF. ZIC3 binding to TCF has the ability to repress β-catenin, hence affecting the WNT signaling pathway. It is suggested that β-catenin repression will give effect of suppressing the WNT pathway. Another suggested effect is reduction or accumulation of β-catenin. Both lead to failure in forming the dorsal axis, disrupting organ arrangements and cause HTX.

The Hedgehog signaling pathway is another significant component in mammals’ organ development where the Sonic Hedgehog (Shh), Indian-Hedgehog (Ihh) and Desert-Hedgehog (Dhh) are the three main Hedgehog proteins in the pathway.65 The binding of Hedgehog ligands to the PTCH will stop its inhibition on the SMO, hence allowing the downstream pathway. The downstream pathway includes interactions with GLI family (Figure 2(B)), where repressed SMO prevents GLI movement into the nucleus and retains it in the cytoplasm, by binding GLI to SuFu.65,66 The binding of GLI to SuFu does not only prevent target gene transcription by GLI but also leads to production of the GLI protein repressor by proteasome.48,67 ZIC3 interactions with the GLI in the pathway were found to cause alterations to the ratio of activator to repressor of GLI.48 The alterations of the GLI activator and repressor lead to target gene repression such as IFT genes (important for cilia structure and maintenance).67,68 Repression of the IFT genes leads to problems in the cilia structure and function,67,68 hence, causing HTX.

Apart from the Hedgehog signaling pathway, the WNT signaling pathway disruption was also inferred to have caused alterations to the left–right patterning. Asymmetry left–right patterning can be initiated by multiple WNT ligands.48 In the pathway, WNT binds to its receptor and co-receptor (FZD and LRP 5/6, respectively),which then initiates the dissociation of a complex (APC, AXIN/DVL/GSK3/CK1/β-catenin) and releases β-catenin from the complex (Figure 2(C)).69 β-Catenin interacts with TCF to regulate target genes transcription.48 Loss of Wnt3a in the dorsal posterior node, for example, causes aberrant left–right patterning due to delayed expression of certain proteins such as Nodal, Lefty2 and Pitx2 bilaterally in the LPM.70ZIC3 is one of the genes that has been suggested to have an effect on the WNT pathway leading to HTX.48 Fujimi et al. (2012) performed an animal study and discussed WNT signaling pathway suppression by ZIC3 and the effect of ZIC3 interaction in the pathway.71 They hypothesized that WNT signaling suppression by ZIC3 is tissue-specific as the observation of some mammalian cell lines did not show suppression of the WNT signaling.71 It is also hypothesized that ZIC3 interaction with TCF could repress WNT signaling.48 Reduction or overaccumulation of the maternal β-catenin leads to failure in dorsal axis development which is important in determining the arrangement of organs during embryogenesis.72

Dysfunctions in one pathway can influence the others, disrupting the precise coordination needed for proper left–right axis formation.73,74 For instance, variants in genes involved in the Nodal pathway, such as NODAL, ACVR1, and ACVR1B, can lead to abnormal Nodal signaling. This, in turn, affects Hedgehog signaling as Nodal induces the expression of Sonic Hedgehog (SHH) and reciprocally Hedgehog regulates Nodal-related genes.58,73 Concurrently, dysregulation in the canonical WNT pathway can impact the expression of Lefty proteins, key inhibitors of Nodal signaling.58 This disturbs the cross-talk between Nodal and canonical WNT pathways and influences Hedgehog signaling through altered SHH expression.74 The cumulative effects of these dysregulations disrupt the finely tuned interaction among the pathways, leading to the misalignment of internal organs, including the heart, and the manifestation of HTX. This asymmetry is also influenced by cilia activity and their role in creating the left–right organizer flow dynamics.75 It is crucial to note that HTX is a genetically heterogeneous condition, and not all cases can be attributed to variants in these pathways alone, indicating the involvement of other genetic and non-genetic factors in the development of the disorder.

DNAH11 is one of the genes responsible for forming the protein required in cilia structure. Apart from HTX, variants in DNAH11 are also linked to PCD.40 When comparing patients with and without CD, it was discovered that the mutation rate of the gene varied, with the rate of mutation being greater in patients with CD.6DNAH11 variants were predicted to affect splicing and have damaging effects on the protein, potentially leading to the development of heterotaxy and congenital heart defects.41 A study by Liu et al. (2019) found that out of a total of 34 variants, 14 were variants in the DNAH11 gene, which is the highest number of variants found in one gene in the study.6 In a family, two children were found to have the same DNAH11 variants (c.G3426-1A and c.C4306T); however, only the proband was affected with HTX while the other sibling was not.76 According to Xia et al. (2021), this occurrence is attributed by incomplete penetrance or the presence of other variants that inhibit the DNAH11 variants to develop HTX phenotypes.76

The variants are inherited in an autosomal recessive manner where various types of variants such as stop-gain, frameshift, missense, and splice site were reported.6,76Table 2 lists previously reported DNAH11 variants.

Dynein axonemal heavy chain 5 is another gene that is associated with HTX due to its function in encoding proteins for the ciliary structure, specifically the outer dynein arm.78 It is mainly associated with PCD, and PCD caused by DNAH5 has been associated with laterality defects including situs ambiguous or HTX.78 The gene is inherited in an autosomal recessive manner and trans-heterozygosity of DNAH5 with DNAH6 and DNAH11 has been observed to cause HTX (Table 3).6,47

Zinc finger in cerebellum 3 is a gene from the GLI superfamily that acts as a transcription factor during neurogenesis, and previous studies using animal models suggested that reduced expression and/or activity of ZIC3 protein during early embryogenesis affects the laterality patterning.81ZIC3 is a gene with significant evidence of its pathogenicity in causing HTX, where at least 36 ZIC3 variants have been discovered to be pathogenic since the first ZIC3 variant was found in 1997.82 Variants in the ZIC3 gene have been shown to have a significant impact on heterotaxy, particularly in male patients with abnormal arrangement of thoracic and visceral organs.81 These variants, which are predominantly located in the zinc finger domains or result in truncation before these domains, have been found to cause abnormal trafficking of mutated ZIC3 proteins.83 Functional analysis in zebrafish models has further confirmed the pathogenic nature of these variants, with frameshift variants showing full non-functionality and a missense variant showing partial activity compared to the wild-type.84 Additionally, variants in the N-terminal and zinc finger-binding domains of ZIC3 have been found to result in the loss of reporter gene transactivation and aberrant cytoplasmic localization, respectively.82

A functional investigation of a novel hemizygous mutation (c.890G>T, p.C297F) in the ZIC3 gene revealed that this specific mutation reduces the transcriptional activity of ZIC3. It affects the binding affinity of the GLI-binding site and leads to abnormal cellular localization when introduced into transfected cells. In addition, the variant (p.C297F) was found to affect protein structure and DNA-binding ability.82 Other than that, ZIC3 is the earliest gene associated with HTX reported and the only gene to be inherited by X-linked inheritance of HTX.83 D’alessandro et al. (2011) reported that the women in the studied family were carriers with no phenotypic manifestations, while several male family members manifested congenital heart diseases.85 One of the male family members was also found to carry the variant; however, no morphological defects of the organs were observed, which was confirmed to be due to incomplete penetrance of the ZIC3 gene variant.85 Five out of 100 familial HTX was found to be caused by ZIC3 variants, while 1 out of 100 were sporadic cases.35,85 Previously reported ZIC3 variants are listed in Table 4.

NODAL protein is a secreted molecule from the Nodal signaling pathway. It is a regulator that belongs to the TGF-β family, hence it plays a significant role in the determination of left–right asymmetry in vertebrates.62 The expression of NODAL is primarily symmetry but progresses to asymmetrical toward the left side of the LPM, and the gene expression is highly conserved.92 Multiple studies have identified missense variants, in-frame insertion/deletion, and splice site variants in the NODAL gene in patients with heterotaxy and/or isolated cardiovascular malformations (CVM).62 Functional analyses have shown that these NODAL variants exhibit impaired signaling, leading to decreased activation of artificial reporters and reduced induction of SMAD2 phosphorylation and nuclear import.93 Additionally, a study on mouse mutants with a mutation in the MEGF8 gene, which is involved in left–right patterning, showed that Nodal signaling failed to be propagated to the left LPM, resulting in heterotaxy and CHD.94 These findings suggest that mutations and rare deleterious variants in NODAL can contribute to the development of sporadic human heterotaxy.95 A study by Mohapatra et al. (2009) found seven variants where four are missense, while one is in-frame insertion/deletion and two are splice site variants.62 Previously reported NODAL variants are listed in Table 5.

There are two LEFTY genes (LEFTY1 and LEFTY2), which are involved in Nodal signaling from node to the LPM.91LEFTY1 and LEFTY2 genes also belong to the TGF-β superfamily.93 They are NODAL antagonists in the Nodal signaling pathway, where they negatively regulate NODAL expression by blocking receptor complex activation.60 LEFTY1 is expressed in the midline of LPM while LEFTY2 is expressed within the left LPM, where both retain the NODAL signaling suppression.78 In one study, rare copy number variants (CNVs) were identified in patients with heterotaxy, and functional testing in zebrafish models revealed that downregulation of LEFTY disrupted cardiac looping and abnormal expression of left–right patterning markers.62 A nonsynonymous heterozygous variant pair of LEFTY1 (c.A841G) and TTC40 candidate gene were discovered in a CNV study.12 However, most LEFTY1 gene studies focus on gene expressions. Animal studies have revealed that the absence of Lefty1 affects both Nodal and Lefty2 genes expressions in which they are expressed on both sides of the lateral plates instead of only on the left side.64,91 Apart from LEFTY1, a study by Kosaki et al. (1999) found two sporadic variants (c.1025G>A and c.989C>T) of LEFTY2 cases that phenotypically fit the heterotaxy description.96 The variants were discovered to be in the cysteine knot region, which is critical as it directly implicates the binding of receptors.96 Two other LEFTY2 variants (c.-225C>T, ClinVarID: 295978, and c.-222C>A, ClinVarID: 295977) associated with HTX have been reported in the ClinVar database by Illumina, nevertheless no publications have reported these variants.97,98LEFTY2 is transcriptionally regulated by a complex which includes p300, SMAD2, and CITED2. Su et al. (2013), suggested that downregulation of LEFTY2 might play a role in the onset of VSD partly by triggering abnormal activation of the NODAL-PITX2C pathway.99 Additionally, the data reveal that the histone acetyltransferase p300 contributes to the activation of LEFTY2 by promoting hyperacetylation of histone H4 at the promoter region of LEFTY2. Previously reported LEFTY1 and LEFTY2 variants are listed in Table 6.

Based on NCBI NIH Genetic Testing Registry, HTX gene panel for diagnosis includes 10 genes, including NKX2-5, GDF1, GJA1, NODAL, CFC1, LEFTY2, ZIC3, CRELD1, FOXH1 and ACVR2B.100DNAI1, MMP21, PKD1L1, and HYDIN, are other known PCD genes associated with HTX besides those discussed above, while ROCK2, ISL1 and SMAD2 are newly found candidate genes.85

According to Litonjua and Celedón (2006), the definition of candidate gene is a gene that is suggested to have a link to a particular illness or trait by the discovery of either the protein products or chromosomal location.101 Further investigations are required to understand these genes better.

A study conducted using CNVs analysis suggested two candidate genes which are dynein axonemal heavy chain 10 (DNAH10) and ring finger protein 115 (RNF115), where DNAH10 was proposed to disrupt ciliary function as it affects notochord, while RNF115 affects the laterality signaling pathways.12 Six novel candidate genes were found in a study of 25 HTX patients, i.e., FLNA, ITGA1, PCNT, KIF7, GLI1, and KMT2D.102KIF7 and FLNA are indirectly involved in the ciliary movement as KIF7 encodes for cilia associated protein in the Shh signaling pathway, while FLNA encodes for actin-binding proteins involved in cytoskeleton function.102 Apart from the genes discussed in the review, homozygous missense mutation of SHROOM3 gene (c.G179T) was reported in a study; however, the variant cannot be found in the databases.103 In addition, PFKP is among the top HTX candidate genes with proof of its effect in an animal model, Xenopus laevis.13PITX2 expression is important for the formation of the heart, and its abnormal expression has been linked to CHDs.104 Studies in animal models further support the connection of abnormal expression of PITX2 with HTX.105,106