The development of the heart is orchestrated by transcription factor (TF) networks including members of the NK2 homeobox, T-box, and GATA binding families (see Chap. 12) [26]. GATA binding protein 4 (GATA4) is a transcriptional activator found to be affected in sporadic and familial cases of isolated VSD. Three different missense mutations in the GATA4 gene (p.Pro407Val, p.Ser175Cys, and p.Ala411Val) have been reported in sporadic cases [12–14]. In addition, two families carrying GATA4 missense mutations (p.Arg43Trp and p.Gly296Arg) were discovered in a follow-up analysis of index patients that participated in a screen of unrelated individuals (see also Sect. 23.3.2.1) [15, 16]. In the follow-up of affected family members, two and four additional cases of isolated VSD were found in the studies by Wang et al. [15] and Yang et al. [16], respectively.

So far, mutations affecting three members of the T-box family, namely, TBX1, TBX5, and TBX20, have been associated with isolated VSDs. Pan et al. screened 230 CHD cases and observed a heterozygous nonsense mutation (p.Gln277X) in the DNA-binding domain of TBX1 in one patient with double outlet right ventricle who had one affected relative with isolated VSD carrying the same mutation (see also Sect. 23.3.2.1) [19]. A missense mutation (p.Ile152Met) in TBX20 causing impaired DNA binding was found in a family affected by multiple septal defects including isolated VSD, atrial septal defect (ASD), and a large patent foramen ovale in different relatives [21]. In the case of TBX5, a non-coding variant in one of its enhancers was suggested to impact on the development of VSD [20]. The variant was found homozygous in a case of isolated VSD with unaffected heterozygous parents and supported by functional evidence based on transgenic expression studies in mouse and zebrafish [20].

A study focusing on the analysis of PITX2 (paired-like homeodomain 2) in a cohort of 170 unrelated neonates with CHD found two missense mutations (p.Arg91Gln and p.Thr129Ser) in two affected families [18]. Four mutation carriers presented with isolated VSD whereas two other relatives showed transposition of the great arteries (TGA) with VSD (see Sect. 23.3.2.1) [18].

Mutations have also been identified in the TF genes NKX2-6, CITED2, and IRX4 [11, 17, 27]. Screening a CHD cohort including 66 isolated VSD cases, Wang et al. identified a missense mutation (p.Lys152Gln) in the homeodomain of NKX2-6 (NK2 homeobox 6) [27]. Subsequent analysis identified the mutation in two further family members with isolated VSDs [27]. In a cohort of nearly 400 sporadic CHD cases, a nine amino acid deletion (p.Ser170_Gly178del) in CITED2 (Cbp/P300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2), resulting in impaired activity, was detected by us in one patient with isolated perimembranous VSD [11]. For the ventricle-specific TF IRX4 (Iroquois homeobox 4), two missense mutations (p.Asn85Tyr and p.Glu92Gly) were reported in two unrelated patients with isolated VSD by direct sequencing of the gene in a cohort of about 700 CHD patients [17]. The two mutations affected the interaction with retinoid X receptor alpha, a nuclear receptor of the vitamin A signaling pathway important in cardiac morphogenesis.

Various cellular processes in both the embryonic and adult organism are regulated via transforming growth factor beta (TGFβ) signaling pathways. Important developmental steps, such as the establishment of left–right asymmetry, are driven by the NODAL signaling pathway, which is named after the TGFβ superfamily member of the same name (see Chap. 7). Two genes encoding cofactors of the NODAL signaling pathway, TDGF1 (teratocarcinoma-derived growth factor 1 also known as CRIPTO) and CFC1 (Cripto, FRL-1, Cryptic family 1 also known as CRYPTIC), have been analyzed in a cohort of 500 CHD cases [22, 24]. Three missense mutations, p.Arg41Gly in TDGF1 [24] as well as p.Leu219Phe and p.Gly169Val in CFC1 [22], were identified in three patients with isolated VSD (see also Sect. 23.3.2.2). Another member of the NODAL signaling pathway, GDF3 (growth differentiation factor 3), was analyzed by Xiao et al. [23]. Direct sequencing of GDF3 in a cohort of 200 CHD patients led to the identification of a missense mutation (p.Ser212Leu) in a patient with isolated muscular VSD [23].

The gene HAS2 encodes hyaluronan synthase 2, an enzyme that synthesizes hyaluronic acid (a major component of the extracellular matrix) during embryogenesis [25]. Among 100 non-syndromic VSD cases, Zhu et al. detected a HAS2 missense mutation (p.Glu499Val) in one patient. The synthesis of hyaluronic acid was significantly impaired in the mutant enzyme as shown by in vitro assays [25].

Two microduplications and two microdeletions were described in four cases of VSD with additional cardiac malformations (see Table 23.3) [6, 9]. Tomita-Mitchell et al. analyzed a cohort of several hundred CHD cases and identified among others one patient with VSD and pulmonary atresia who carried a duplication at the locus 1q21.1 comprising candidate genes such as CHD1 (chromodomain helicase DNA binding protein 1) and GJA5 (gap junction protein alpha 5, 40 kDa; also known as connexin 40) [9]. Screening a cohort of 105 CHD patients by array CGH, Erdogan et al. found a deletion at the 22q11.2 locus (including TBX1) in a case of VSD and aortic coarctation [6]. Further, they detected a duplication at chromosome 4q32.3 in one patient with VSD and PDA and a large 4 megabase deletion at chromosome 17p11.2 in a patient with VSD and ASD. No evident candidate genes have been identified in either of these regions [6].

Mutations have been reported in transcription factors, signaling molecules, and sarcomeric proteins in patients and families with non-syndromic VSD (see Table 23.4).

Chromosomal aneuploidy is the presence of an abnormal number of chromosomes in the cell leading to various syndromic genetic disorders. Aneuploidy syndromes can occur with almost any cardiac malformation [38]. CHD occurs in about 45–50 % of patients with Down syndrome (trisomy 21), the most common aneuploidy syndrome [39, 40]. Källen et al. showed in a large epidemiologic study with more than 5000 patients with trisomy 21 that VSD was present in 28 % of individuals with cardiac malformation [41]. In the National Down Syndrome Project cohort, Freeman et al. found a VSD rate of 19 % regarding all registered infants (65 % were membranous and 35 % were muscular VSDs) [40]. A retrospective cohort study including about 4300 Down syndrome patients undergoing CHD surgery was performed by Fudge et al. to examine postoperative outcomes [42]. VSD closure of any type was the second most common procedure (19 %) performed for patients with Down syndrome [42].

Further, CHD occurs in about 35 % of cases of Patau syndrome (trisomy 13) and about 45 % of cases with Edwards syndrome (trisomy 18) as Pont et al. described in a large epidemiologic study of hospitalizations of live-born infants with chromosomal abnormalities [43]. VSD was the most common heart defect in trisomy 13 (18 %) and in trisomy 18 (31 %) [43]. Pallister–Killian syndrome, a sporadic multisystem developmental disorder, is typically caused by the presence of a supernumerary isochromosome composed of the short arms of chromosome 12 resulting in tetrasomy 12p [44]. Tilton et al. evaluated 30 patients with this syndrome and CHD and found a VSD in 10 % of those [44].

A number of studies have shown the importance of CNVs, mainly microdeletions and microduplications, in syndromic CHD [38, 45]. Those studies reported subjects with well-known syndromes as well as so far undefined syndromic forms.

In Table 23.5, clinically delineated microdeletion and microduplication syndromes are listed in which a specific association to VSD was described. Of note, the most frequent genomic disorder associated with CHD is DiGeorge syndrome (DGS; 22q11 deletion or velocardiofacial syndrome) (see, e.g., Chap. 38). Cardiovascular anomalies are present in about 80 % of neonates with DGS [54]. Ryan et al. evaluated a cohort of 545 DGS patients and could show that VSDs were observed in 14 % of these patients (among other heart defects) [55]. Momma described in his review of several studies of DGS cohorts (ranging from 100 up to 222 patients) quite similar VSD rates [54]. All other syndromes show lower VSD rates. For example, in Williams–Beuren syndrome (caused by a deletion of about 1.5 megabases in chromosome 7q11.23), VSDs, mainly muscular ones (75 %), are present in 4–9 % of all patients [49]. A similar VSD rate (8 %) was described by Jefferies et al. in patients with Potocki–Lupski syndrome whereby most individuals harbor a common 3.7 megabase duplication within chromosome 17q11.2 [53].

Besides well-known syndromes, there are a number of studies describing syndromic patients with VSD who present different unspecific symptoms (see Table 23.6). Using array-based CGH, Syrmou et al. screened a cohort of 55 syndromic CHD patients and detected CNVs in 37 of them [57]. They found five patients with VSD showing either one CNV or a combination of up to three different deletions and duplications (ranging from 0.023 to 6.6 megabases) [57]. Through a genome-wide survey of two independent cohorts of CHD subjects with extracardiac abnormalities (700 subjects in total), Lalani et al. identified 16 CNV regions, present in two or more cases and absent in about 3000 controls [60]. Interestingly, one of the most frequent CNVs they found was a de novo copy number loss of 16q24.3 (affecting ANKRD11 encoding ankyrin repeat domain 11) in five subjects of whom four presented with VSD together with other CHDs (two with perimembranous and one each with muscular and conoventricular VSD) [60]. Screening a cohort of 60 syndromic CHD cases by array CGH, Thienpont et al. identified among others one patient with muscular VSD and various extracardiac manifestations showing a 3.8 megabase duplication at locus 19p13.12-13.11 [61]. Using the same detection method, a similar cohort of 90 patients was analyzed by Breckpot et al. [56]. They found two deletions at locus 1p36.33 (ranging from 3.5 to 5.9 megabases) in two patients with VSD and minor extracardiac malformations, one subject with additional hypertrophic cardiomyopathy and one with microcephaly [56]. Goldmuntz et al. analyzed 58 syndromic CHD cases and reported six different CNVs in six patients characterized by VSD and further congenital abnormalities and dysmorphic features such as cleft palate [59]. They detected copy number gains at loci 5q21.1-21.2 and 18p11.32, whereas losses were detected for the loci 9p23, 10p12.1-11.21, 15q26.1, and 22q11.21 (ranging from 0.4 to 7.1 megabases in length) [59]. Analyzing several hundred CHD trios (non-syndromic and syndromic cases) by SNP arrays and whole exome sequencing, a paternally inherited deletion of 35 kilobases at chromosome 4q34.1 was detected in a patient with VSD, TGA, and extracardiac manifestations [7]. Fakhro et al. genotyped a cohort of more than 250 heterotaxy cases by SNP arrays: they identified a heterozygous 1.5 megabase deletion at locus 3p24.1-23 (affecting TGFBR2 encoding TGFβ receptor II) in a patient characterized by VSD, ASD, partial anomalous pulmonary venous return, and situs inversus (see Chap. 38) [58].