The expression of cardiac transcription factors occurs in highly specified temporal-spatial patterns throughout development [3] (see Chap. 12). Transcription factors orchestrate heart development, and many of them are associated with isolated CHD [3, 31] (see, e.g., Chap. 30). Transcriptional focal points include NK2 homeobox 5 (NKX2-5) and GATA binding protein 4 (GATA 4) which are known to be involved in situs defects.

Watanabe et al. identified a deletion frameshift mutation in NKX2-5 in a familiar CHD case with heterotaxy as well as atrial septal defect (ASD) [7]. Izumi et al. described a further deletion frameshift mutation in NKX2-5 (along with three other variants of unknown significance in genes associated with ciliary disease; see Sect. 38.3.4) in a patient with complex CHD including heterotaxy using clinical exome sequencing [8]. In GATA4, a frameshift mutation was identified by Hirayama-Yamada et al. in a family with multiple ASD cases of which one showed additional dextrocardia [9]. However, affection of other contributing genes might be likely in this case.

The X-linked form of heterotaxy is caused by mutations in the zinc finger transcription factor ZIC3 (Zic family member 3) and affects approximately 1 % of sporadic heterotaxy cases [1]. Mutations often cause loss of function and, in some cases, result in abnormal subcellular localization and trafficking [1]. ZIC3 was the first gene unequivocally associated with human situs abnormalities [32, 33]. To date, a number of different ZIC3 point mutations (missense, nonsense, and frameshift) have been described in X-linked familial heterotaxy cases as well as in sporadic heterotaxy and isolated CHD cases [10–12, 32, 34]. Patients from these studies show the whole spectrum of situs defects such as situs inversus, heterotaxy (situs ambiguus including asplenia and polysplenia), and dextrocardia. In summary, ZIC3 is the most frequent disease gene for laterality defects.

Heart development involves coordination of a number of signaling pathways [31]. Several mutations in signaling molecules have been detected in different forms of CHD associated with situs defects.

Activins and their receptors are members of the transforming growth factor beta (TGFβ) family of signaling molecules. Two missense mutations in ACVR2B encoding the activin receptor type II B were found in three patients showing heterotaxy and complex CHD [13]. Kaasinen et al. described a family with right atrial isomerism associated with situs inversus, situs ambiguus, dextrocardia, and asplenia showing mutations in the growth differentiation factor 1 (GDF1) [14]. Two truncating mutations of GDF1 were observed to segregate with the phenotype in an autosomal recessive manner [14]. Of note, this study also identified 11 carriers of heterozygous truncating mutations in GDF1 in control subjects without CHD indicating a high frequency and compatibility with normal development and health.

CFC1 encoding CRYPTIC protein is a member of the EGF (epidermal growth factor)-CFC (Cripto, Frl1, and Cryptic) family encoding extracellular proteins important for intercellular signaling pathways during vertebrate embryogenesis. Bamford et al. were the first to describe loss-of-function mutations in human CFC1 in patients with heterotaxic phenotypes [15]. They identified nine patients carrying four different missense mutations and one deletion with various forms of heterotaxy associated with CHDs such as d-transposition of the great arteries (d-TGA), ventricular septal defect (VSD), and ASD; five of the patients showed dextrocardia. The mutant proteins had aberrant cellular localization in transfected cells and showed functional effects in a zebrafish model [15]. Selamet Tierney et al. found three non-synonymous variants in CFC1 in patients with laterality defects and CHD and suggested that these may act as susceptibility alleles in conjunction with other genes and/or environmental factors [16]. Further, Roessler et al. screened CFC1 in a cohort of 251 patents with laterality defects and identified two mutations [17].

A further member of the TGFβ family, the left-right determination factor 2 (LEFTY2, also known as LEFTY A), was shown to be mutated in heterotaxy patients and is well known for its role in left-right patterning during mouse development [18]. Kosaki et al. found one nonsense and one missense mutation in LEFTY A in two patients with LR-axis malformations and CHD [18]. However, they stated that the LEFTY A mutant alleles may be necessary, but not sufficient, to give an LR phenotype in these affected individuals, because each mutation was found to be carried by one of the parents [18].

Analysis of a cohort of 269 patients with heterotaxy and/or isolated cardiovascular malformations revealed four different missense mutations in NODAL (Nodal growth differentiation factor) [19]. NODAL mutations were found in 14 unrelated subjects consisting of one in-frame insertion/deletion and two conserved splice site mutations. About one third of these patients showed dextrocardia and situs inversus (as well as asplenia in some cases) associated with a wide spectrum of CHD including pulmonary atresia, d-TGA, ASD, and VSD.