Discussion

Exome analysis of case-parent trios with cardiovascular laterality defects identified an identical de novo ultra-rare missense variant in the FAM222B gene. Survey of 2,109 single exomes of individuals with situs inversus totalis, heterotaxy, or isolated CHD identified five additional variants in FAM222B in five independent families. One of these five FAM222B variants is novel. Family EGAN00001389505 turned out to be a three-generation multiplex family with seven differently affected family members.

The frequencies of the missense variants reported in families 1, 2, 3 and 5 were significantly more common in the presented case cohort compared to the general population. The here reported nonsense indel variant identified in family 6, has not been described in gnomAD at all. In two families (family 4 and 7) the identified variant has the same frequency in our combined cohort here compared to the general population, arguing against its pathogenic effect (Fig. 1B).

As mentioned before, FAM222B has previously been considered a potential candidate gene for cerebral cavernous malformations12, but its embryonic function and protein role are still poorly understood and have not been explored in relation to CHD. Collectively, this data highlights FAM222B as a promising new candidate gene for cardiovascular laterality defects, leading us to investigate its role during zf embryonic development.

Our zf data revealed that the dd-KO of the two fam222b zf-paralogues, fam222ba/bb, has multiple effects on cardiac development. Both zf’ _fam_222 paralogues show a strong gene expression profile during early development in WISH analysis especially in the cardiac region (Fig. 2A). Furthermore, the gene knockout might well enhance the chance of a disturbed cardiac looping in the zfl (Fig. 2B,C). As shown in our data, cardiac looping was significantly more impaired in dd-KO zfl than in wt zfl (Fig. 2C). Other variables such as the zfl heart rate in bpm (Fig. 2E) do not seem to be affected by the knocked out fam222ba/bb genes. Although we focused on zfl heart rate, it is only one of several functional prognostic cardiac measures that might be affected.

We provide further evidence that fam222ba/bb not only affects zf in their larval stadium but that aberrant early cardiac development caused by a fam222ba/bb dd-KO might very well lead to disorders in adult age (≈ two years). This includes a significant enlargement of the dd-KO zf ventricle (Fig. 3B) which has previously been associated with heart failure25. The reduced cell density in dd-KO ventricular heart tissue, in the absence of intercellular gaps, suggests increased average cell size and is consistent with a hypertrophic heart phenotype. In parallel, the increased tissue area per unit mass indicates an expansion of tissue architecture relative to organismal weight. When integrating both parameters, dd- KO heart samples show an increased total cell number per mg despite reduced local cell density. Taken together, these findings indicate that the observed heart phenotype is likely driven by a combination of cellular hypertrophy and hyperplasia. In our studies, a decreased cell density under confluent conditions reflects hypertrophy whereas the increased total cell number per mg heart tissue supports hyperplasia. In many described human cases, a consequent enlargement of the atrium as also observed in our dd-KO adult fish hearts (Fig. 3C) results from ventricular dysfunction26. Other variables analysed in our study, such as the adult heart muscle ultra-structure and assembly do not seem to be affected by the knocked out fam222ba/bb genes. Disruption in sarcomere assembly or function often resulting in diverse cardiomyopathies27, could not have been detected in this study (Fig. 3F). Ventricular enlargement appears to result from hypertrophic growth, which was established through our comprehensive histological analyses, involving targeted nuclear staining and quantification (Fig. 3D,E).

Nonetheless, a limitation of this study arises from the fact that comprehensive analysis of cardiac functions requires analysis beyond zfl heart rate or comparison of heart morphology and histology, including additional parameters like, e.g., adult zf hearts’ ejection fraction, electrocardiogram and zf behaviour such as rapid breathing or exercise intolerance25. In order to percept multidimensional and more precise analysis of the zf heart, additional methods could be implemented. Greater measurement specificity could be achieved for example by including high-resolution echocardiography and electrocardiography25.

As mentioned above, we found an identical de novo variant (variant 1/2 rs753661690) of FAM222B in two independent families with left isomerism additional to complex CHD (Fig. 1A, cases 1&2). Via poly(A) mRNA injection of this missense variant within the human FAM222B gene, we showed that it causes significantly more cardiac harm to the injected zfl than purely the overexpressed wt FAM222B poly(A) mRNA (Fig. 4C,C‘). Based on this observation we conclude that it should not generally be the missing – in the case of KO – or excessive – in the case of overexpression – presence of FAM222B protein itself causing damage to the zf-heart, but rather the specific alteration in its amino acid sequence, in our case Arg > His at position p.300 heading to cardiac disorders. This conclusion was further supported by the structural modelling of FAM222B binding to NLK, which convincingly placed Ser296 of FAM222B at a site readily accessible for phosphorylation through the kinase, and proposed Arg300 to mediate key interactions with the kinase (Fig. 4A). Change of Arg300 to the shorter and uncharged histidine could impair the interaction with the kinase, possibly resulting in the loss of phosphorylation of FAM222B (Fig. 4A‘). However, it must be mentioned that most regions of FAM222B have a rather low confidence in the Alphafold prediction (UniProt), which is due to missing experimental reference structures within our protein of interest.

The various CHDs in the context of laterality defects in human individuals with heterozygous missense variants or deletion in FAM222B could only be recapitulated to a certain degree in the zf model presented here. It remains unclear whether each of our six identified variants individually gives rise to a distinct cardiovascular laterality defect phenotype in humans and zf, and exactly how. It is further questionable whether the cardiac phenotypes found in our mutant mRNA injected zf, e.g. hypotrophic ventricle and concomitant dilated/ hypertrophic atrium as well as no heart looping and no grip, could be precisely compared to the found human phenotypes e.g. VSD, ASD, AVSD and congenital bicuspid aortic valve. Due to fundamental differences in cardiac anatomy between zf and humans, such as the presence of only one atrium and ventricle (two-chambered heart) in zf in comparison to the human four-chambered heart, certain structural defects observed in our patients like the here described VSD or ASD cannot be reconstructed properly in this animal model. A more anatomically comparable model, such as mouse, which possesses two atria and two ventricles, could well provide more meaningful and well-founded evidence to this question. The here identified additional expression pattern of fam222ba/bb in the cloaca, pineal gland, and the cranial head region of the zfl (see Supplementary S3 online) may offer important insights into our candidate gene’s role across different other tissues and diseases and could therefore well hold relevance for further studies. Collectively, the here presented human genetic and zf data strongly support an important role for FAM222B in the pathogenesis of cardiac diseases and potentially in early cardiac axis development as well.