Genetics of atrioventricular canal defects

Down syndrome is the most frequent genetic condition associated with AVCD. CHDs are diagnosed in 40–50% of these patients [24]. In this syndrome AVCD is frequently complete, showing a “simple type”, since rarely associated with other cardiac anomalies, with the exception of tetralogy of Fallot [25, 26]. In particular, left-sided obstructions are significantly more rare in patients with DS and AVCD in comparison with patients with AVCD and normal chromosomes [11, 24, 27]. Clinical studies on surgical prognosis of AVCD have shown that corrective surgery in patients with DS results in lower mortality and morbidity rates, compared to the children without trisomy 21 [28, 29].

From the molecular point of view, several genes located in the “CHD critical region” on chromosome 21 have been long investigated as a cause of AVCD, including DSCAM, COL6A1, COL6A2, and DSCR1 [30, 31]. Additional genes mapping on different chromosomes including CRELD1, FBLN2, FRZB, and GATA5 have been studied [32]. Particularly, the interaction between trisomic genes and modifiers on different chromosomes has been supported in experimental studies using mouse models of DS with high prevalence of CHD, in which loss-of-function alleles of Creld1 or Hey2 genes have been crossed with the trisomic background [33]. In addition, mouse models have evidenced the involvement of the Shh signaling pathway also in DS, since it has demonstrated that cerebral, skin, liver and intestine mice trisomic cells have a defective mitogenic Shh activity with cell proliferation impairment due to a higher expression of Ptch1, a receptor normally repressing the Shh pathway, located on Cr9 [34].

Deletion of the terminal part of the short arm of chromosome 8 (del 8p23) is the second chromosomal anomaly associated with AVCD [13]. Cardiac malformations are diagnosed in two third of the patients and AVCD is detected in about 40% of the cases [35]. AVCD is generally complete, with a frequent association with pulmonary valve stenosis and Tetralogy of Fallot [36, 37]. Heart defects as dextrocardia, abnormalities of the pulmonary and systemic venous returns, common atrium, single ventricle and transposition of the great arteries are also found in a group of patients with del 8p23 [35]. Some of these malformations are also characteristic of laterality defects. The candidate gene for CHD in this syndrome is GATA4, which maps to the 8p23.1 region and is expressed in the developing heart [38]. GATA4 interacts with other transcriptional factors to drive DMP progression via SHH signaling [39].

Deletion 3p25 syndrome is also often associated with AVCD [40‐42]. Cardiac malformations are diagnosed in about one-third of patients with deletion 3p25 patients [42]. In this syndrome AVCD is usually complete and CRELD1 gene is the “critical “gene, based on its map position on chromosome 3p25 and considering that it is known to be causally related also to non-syndromic AVCD [43, 44]. The study of Burnicka-Turek et al. suggested that CRELD1 mutations can cause AVCD acting on SHF Hh signalling [45].

Alveolar capillary dysplasia is a congenital pulmonary vascular abnormality, often associated with misalignment of the pulmonary vessels. The disease is associated with CHD in about 10% of the cases, prevalently consisting in partial or complete AVCD and various degrees of left heart obstruction (small left ventricle with or without aortic coarctation) [79].

Alveolar capillary dysplasia is caused by FOXF1 gene mutations. Several studies demonstrated that FOXF1 gene is activated by Sonic Hedgehog signaling [80].

The term RASopathies includes the Noonan Syndrome and similar related syndromes (i.e., the LEOPARD syndrome or “Noonan syndrome with Multiple Lentigines”, the cardio-facio-cutaneous syndrome, the Costello syndrome, the Mazzanti syndrome and others) caused by mutations in genes encoding proteins with a role in the RAS/MAP kinase (MAPK) signalling pathway [81, 82].

The RASopathies are characterized by distinctive facial features, growth retardation, CHD, skeletal anomalies and variable neuropsychological deficits [81]. CHD occurs in about 65–85% of cases, depending on the mutated genes. Although pulmonary valve stenosis with dysplastic leaflets and hypertrophic cardiomyopathy of left ventricle are the most frequent cardiac defects, AVCD was also described. PTPN11 and RAF1 gene mutations have been prevalently detected in patients with AVCD associated with RASopathies [83‐85]. AVCD is usually partial and may be associated with systemic obstructions including subaortic stenosis or aortic coarctation [85]. Structural abnormalities causing congenital subaortic stenosis include accessory fibrous tissue and/or anomalous insertion of mitral valve and anomalous papillary muscle of left ventricle [83‐85].

Normal SHP2/PTPN11 function seems to act as IHH suppressor, and experiments in mice have documented decreased IHH levels in Noonan syndrome caused by germline activating mutations in PTPN11 [86].

CHARGE syndrome is characterized by ocular coloboma, choanal atresia, growth and developmental delay, genital anomalies and hearing loss. CHD is detectable in about 85% of patients with CHARGE syndrome [87] and AVCD is the second most frequent cardiac malformation, often in association with tetralogy of Fallot [88, 89].

The syndrome is caused by mutations in the CHD7 gene in the majority of the patients [90].

CHDs including septal defects have been described also in patients with holoprosencephaly [91]. Holoprosencephaly (HPE) is a severe congenital forebrain disorder usually associated with a broad spectrum of facial anomalies ranging from single axillary dental incisor and hypotelorism to extreme features such as cyclopia, proboscis and cleft lip with or without cleft palate. Shh role on commitment of the midline of neural structures is well known. Until now, at least 10 HPE loci have been identified (Shh [92, 93], DKK1 [94],GLI [95], SIX3 [96], PTCH1 [97], TDGF1 [98], TGIF [99] and ZIC2 [100]). All the genes previously mentioned functionally interact or regulate the Shh concentration to drive forebrain development and ventral midline cell induction during different embryonic stages. In fact, the Shh −/−(null) mouse embryo displays a severe form of HPE [46, 92, 93, 101]. A correct regulation of Shh concentration is therefore crucial for the correct brain septation. However, Shh signaling pathway is deeply implicated also in ciliary function and acts on the DMP to drive the proper development of the cardiac AVC. In fact, in human beings, Shh pathway dysregulation has a well known impact on different types of AVCD [23]. This molecular considerations are supported by the striking phenotypical similarities between sonogram images of HPE (due to SHH deficiency in brain development) (Fig. 2a) and echocardiographic images of AVCD (Fig. 2b). Images (and phenotypes), indeed, support the unifying role of Sonic Hedgehog signalling on the commitment of midline structures of both brain and heart.

In different ethic population AVCD can show distinct prevalence also in the context of the same syndrome supporting the multiple genetic origin of this CHD. In particular, in the context of DS, several studies highlight the effect of sex and ethnic factors in addition to trisomy 21 to determine different prevalence of AVCD.

It is notable that in oriental and native-American DS patients the most frequent CHD is represented by VSD whereas in Caucasian DS populations AVCD are prevalent [102‐104]. Freeman et al. reported significant ethnic differences in the prevalence of AVCD in DS patients. The study demonstrated that blacks with DS were twice as likely to be born with a complete AVCD whereas Hispanics DS patients showed a trend toward fewer AVCD [105].

The majority of AVCD not related to trisomy 21 occur as sporadic cases [13] and non-syndromic patients with visceroatrial situs solitus (without heterotaxy) account for about 25% [13]. Indeed, AVCD prevalence decreases to 0.97–1.32 per 10,000 livebirths looking at non-syndromic cases (Fig. 1). In this population of non-syndromic patients, only 3–5% show familial recurrence. The autosomal dominant pattern of inheritance is prevalently involved, sometimes with incomplete penetrance. There is emerging evidence that maternal risk factors (genetic and environmental) can confer a major risk for non-syndromic CHD [106, 107].

The first gene mapped for AVCD was CRELD1, located inside the “CHD critical region” on 3p25, known as the AVCD2 locus. CRELD1 gene acts as a regulator of calcineurin/NFATc1 signaling which is crucial for the regulation of cardiac development. In fact, NFATc signaling determines valve initiation and maturation, regulating the activity of VEGF to undergo endomesenchimal transition (EndoMT) [108]. CRELD1 the most frequently AVCD associated gene, since heterozygous mutations have been shown to occur in about 6% of non-syndromic partial AVCD [109]. In addition, some CRELD1 gene mutations, including the c.985C > T (p.Arg329Cys) as recurrent one [110], have been reported to be a risk factor for CHD also in patients with DS [111]. Experimental studies in mice have shown that the introduction of a null allele of Creld1 in theDs65Dn mouse can increase the prevalence of CHDs [112]. Interestingly, a link between CRELD1 and ciliary dysfunction through disruption of Shh signaling has been suspected [45, 113].

The fact that defective NFATC1 function could contribute to isolated AVCD was also demonstrated by a recent work by Ferese et al. [114]. The authors reported missense rare variants in NFATC1 gene in two patients with non-syndromic AVCD and in one syndromic patient with AVCD in the context of heterotaxia and polysplenia with left isomerism. Experimental studies in zebrafish have demonstrated that NFATC1 variants have a great impact on cardiogenesis, affecting specifically cardiac looping process. Interestingly, a link between NFATC1 and CRELD1 genes has been noted, since CRELD1 has been shown to be a master regulator of calcineurin/NFATC1 signaling [114].

Several studies highlight the importance of testing “syndromic” genes when investigating patients with isolated CHDs. Some genes causative or contributory for specific syndromes with cardiac involvement can play a role also in isolated AVCD. In fact, linkage studies of familial AVCD first excluded chromosome 21 loci in the pathogenesis of isolated sporadic AVCD [115, 116].

Weissman et al. [117] reported a non-synonymous mutation of PTPN11 in a subject with isolated complete AVCD. Missense mutation of this gene account for approximately 50% of Noonan syndrome, an autosomal dominant disorder presenting with atrioventricular septal defects in almost 15% of cases.

Recently, D’Alessandro et al. [118] performed a NGS (exome sequencing) analysis in a large cohort of unrelated AVCD probands and in a replication cohort of unrelated, non-syndromic, Caucasian AVCD probands. Data for replication analysis were obtained from population databases. The authors found rare damaging non-synonymous variants in six genes (NIPBL, CHD7, CEP152, BMPR1a, ZFPM2, MDM4) all known for their association with some syndromes with CHDs. In humans there is a considerable phenotypic heterogeneity in AVCD whereby different genes can contribute to the same phenotype. For these reasons, NGS is a powerful tool that has the potential to increase the specificity and accuracy of the observed results.

One of the most robustly CHD-associated gene is GATA4, mapping on the “CHD critical region” 8p23.1 [38]. GATA4 is a developmental transcription factor associated with atrial septal defects and ventricular septal defects but also with non-syndromic AVCD.

GATA4 is required for proliferation of SHF atrial septum progenitors and for the progression of the DMP via Hedgehog signaling. The role of GATA4 in cardiac AVC septation is therefore deeply dependent on Shh signaling [39].

Thanks to the wide spread of NGS techniques additional locus for isolated AVCD have been found. Rare de novo missense variants in NR2F2 were described by Al Turky et al. in 13 trios and 112 unrelated individuals with non-syndromic AVCD [119]. The role of NR2F2 gene on cardiogenesis was postulated on the basis of a previously published mutant mouse that shows defective endothelial mesenchymal transformation and hypocellularity of the atrioventricular canal, strongly suggesting a role for NR2F2 in cardiac developmental in a dosage-sensitive fashion [120].

Priest et al. in a recent study confirmed that de novo mutations may account for a small fraction of isolated CHDs [121]. The authors found rare de novo variants in multiple genes (NR1D2, ADAM17, RYR1, CHRD, PTPRJ, IFT140, ATE1, NOTCH1, NSD1, ZFPM2, MYH6, VCAN, SRCAP, KMT2D, NOTCH2, BBS2, EHMT1) surveying a multi-institutional cohort, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons). The study was ruled out combining both exome-sequencing and array-CGH, suggesting a locus heterogeneity and a oligogenic inheritance of isolated AVCD.

The possible role of genomic structural variants such as copy number variants (CNV) in the etiology of non-syndromic AVCD has only been studied in a minority of cases. Priest et al. [122] identified two sub-chromosomal deletions occurring in cr20p12.3 and in cr3q26.1 respectively, previously not directly linked to AVCD. However, the deletions found at these loci contain some genes that can be linked to cardiac morphogenesis. The authors, indeed, conclude that large CNV might confer a minor risk for isolated AVCD.

The studies cited above indicate that isolated non-syndromic AVCD is a highly genetically heterogeneous malformation that probably requires an unknown combination of factors to break the theoretical disease threshold. Noteworthy, specific genes implicated in different steps of cardiogenesis can have a contributory role in different CHD. This observation provides additional evidence of the wide molecular heterogeneity in establishing cardiac phenotype and highlights the fact that CHDs are not to be considered monogenic disorders.

The Baltimore Washington Infants Study revealed that among non-syndromic children showing CHDs, only 3–5% presented familial recurrence. Studies on several pedigrees showed that the recurrence risk for CHD among siblings of patients with AVCD was about 3.6% [123], similarly to the mean recurrence risk reported in previous studies [124].

Traditionally, segregation analysis in families with AVCD suggested an autosomal dominant pattern of inheritance related to a major gene. The hypothesis that AVCD shared a monogenic or oligogenic pattern of inheritance agreed with the clinical observation that CHDs in the offsprings were concordant with cardiac defects in parents [123]. Nevertheless, recent studies on large pedigrees highlight low concordance ratios in families and importance of sex and ethnical drive as risk factor for recurrence rates. These observations support the multigenic origin of familial AVCD that often shows complex traits of inheritance with incomplete penetrance [125, 126].

Molecular basis of familial AVCD are largely unknown. Due to the fact that AVCD represent the major CHD among DS patients, candidate genes on chromosome 21 were firstly investigated with linkage analysis studies. The results, however, excluded the involvement of chromosome 21 “critical region” loci [115, 116, 127]. Exclusion of linkage with chromosome 21 in families with recurrence of non-syndromic AVCD was also consistent with previous observations on anatomic differences between Down and non-Down AVCD [13].

Some genes deeply implicated in cardiogenesis have been found in pedigrees with AVCD. Missense mutation in CRELD1 gene, mapping on cr3p, has been described in the context of familial AVCD [128, 129] as well as mutations in PTPN11 [117],GATA4 [130] and the p93 gene, mapping on chromosome 1 p [131].

A recent work of Demal et al. reported a family with multiple cardiac defects including AVCD and found out that every affected family member carries a BMPR1A missense mutation. BMPR1A is required to ensure the correct development of endocardial cushions via EndoMT regulating the Wnt/ß-catenin signalling. The reported BMPR1A variant leads to reduced atrioventricular valve area and ectopic valvular tissue in experimental studies in zebrafish and is to be considered a potential candidate gene in the development of non-syndromic AVCD [132].

Familial and isolated cases of AVCD sometimes show variants in genes encoding for transcriptional factors deeply implicated in cardiogenesis such as TBX20 and Tbx2. Tbx20 is a T-box transcription factor that interacts with Tbx2 to promote EndoMT and proliferation of the AVC tissue. Therefore this gene directly acts on endocardial cushion formation [133].

Mutations in well known genes account only for a small percentage of familial AVCD, whereas the majority of isolated AVCD with familial recurrence seems to have a complex etiology based on a variety of genes. Combination of traditional linkage analysis techniques with genome and exome sequencing represent a powerful tool to evaluate complex trait of recurrence of this CHDs.

A better understanding of the molecular basis of familial AVCD could have a significant impact on clinical outcome driving a correct genetic counseling based on a focused family history.

The knowledge of genetic basis of AVCD can be useful for prenatal and postnatal clinical management of affected patients.

Information about the prevalence and type of genetic syndromes possibly associated with AVCD can be useful for clinicians involved in prenatal controls and for targeted screening for extracardiac defects. The link between anatomic types of AVCD and specific genetic syndrome could be a marker in diagnostic work. The large genetic heterogeneity of AVCD associated with the possible limits of prenatal genetic testing should be known in prenatal counseling.

In postnatal management of syndromic patients with AVCD it is important to try to perform an early and precise genetic diagnosis. This can lead to knowledge of risk factors, early monitoring and treatment of extracardiac defects, the use of specific multidisciplinary protocols and guidelines.

Genetic counseling to families is also important. Molecular diagnosis in the proband gives the possibility to test the parents and other relatives, in order to precise the possible familial genetic risk. Based on the present genetic knowledge, the molecular approach is more suitable for syndromic rather than non-syndromic AVCD.