Introduction

Cardiovascular laterality defects are rare congenital anomalies of embryonic left–right axis patterning with a reported birth prevalence of about 1.1 in 10,000 live births1. Despite decades of progress in both pre- and postnatal diagnostic approaches and surgical treatment of cardiovascular laterality defects, the condition remains challenging for clinicians2. While individuals with situs inversus totalis do not experience physical impairments despite completely mirrored visceral organs, individuals with heterotaxy, particularly situs ambiguous, are in 90% associated with complex CHD, which remain challenging in terms of surgical reconstruction3. The affected individuals may also present with pulmonary situs abnormalities (bilateral bi-lobed lungs) or abdominal situs abnormalities (e.g. mal-positioned liver, poly- or asplenia). Besides complex cardiovascular laterality defects, estimates suggest that about 3–7% of all isolated congenital heart defects (CHD) are due to abnormal embryonic left–right axis patterning comprising double outlet right ventricle (DORV), atrioventricular septal defect (AVSD) or transposition of the great arteries (TGA)4.

Determination of left–right body axis during embryogenesis commences at the early somite state. To this point the embryonic development is completely symmetric. The break in this symmetric development is primarily steered by a leftward flow generated by rotating primary cilia of the primitive node. Nodal cilia play a major role in forming asymmetry and organogenesis of unpaired organs as heart, liver or spleen generating a leftward flow of extracellular fluid, also called nodal flow5,6. Through clockwise rotation, nodal cilia produce a leftward nodal flow, which triggers a downstream signal cascade in the lateral plate mesoderm. Disruption of genetic drivers that govern determination of left–right body axis during embryogenesis may cause complex laterality defects or isolated CHD only7. The known genetic background of laterality defects explains about 20% of cases and comprises (de novo) dominant monoallelic, recessive biallelic, and X-linked variants4,8,9. So far, primary ciliary dyskinesia (PCD) represents a main underlying cause of laterality defects10 .

In order to identify new disease genes for cardiovascular laterality defects without PCD background, we analysed the exomes of 16 case-parent trios with cardiovascular laterality defects in which PCD was excluded prior to exome analysis. Here, we previously identified and described two novel variants in laterality defects associated disease genes PKD1L1 and ZIC3 as well as ultra-rare biallelic variants in LMBRD1 and DNAH17 and one ultra-rare de novo variant in WDR47 suggesting all three as novel candidate genes11. In two case-parent trios we found an identical de novo ultra-rare missense variant (dbSNP rs753661690, c.899G > A, p.Arg300His) in the FAM222B gene.

Single exome survey of 2,109 individuals with situs inversus totalis, heterotaxy, or isolated CHD identified five additional variants in FAM222B in five independent families, including one novel variant. FAM222B is localised on chromosome 17 and encodes a ~ 60 kDa nucleoplasmic and mitochondrial protein of yet unknown function (UniProt database). Computational analysis of the predicted protein sequence so far did not reveal any typical signal-peptide, transmembrane nor membrane-anchor regions12. FAM222B has been discussed as a candidate gene for cerebral cavernous malformations before12, still its protein function and potential role in embryonic development remains elusive and has not been described in the context of CHD.

To characterize the role of FAM222B on cardiac development, we examined its expression, function and genetic context in the zebrafish (zf) model. In previous analyses, expression of both zf fam222b paralogues and fam222aa could be confirmed in the cardiac region of the zebrafish larvae (zfl), especially at 48 h post fertilization (hpf), the time period of cardiac looping (daniocell). In single cell transcriptional analysis of mice embryos between 9.5 and 13.5 days of gestation, expression of Fam222b could clearly be detected in cardiac muscle lineages (Mouse Organogenesis Cell Atlas (MOCA)). To gain further insights into the effect of zf fam222b paralogues on cardiogenesis, we further examined heart looping in zfl as well as cardiac morphology and histology in adult zf’ hearts using a TALEN-generated double-knockout (dd-KO) zf line for fam222ba/bb. Here, we focused on the de novo variant, FAM222B SNV: 17–28,759,060-G-A; GRCh38, dbSNP rs753661690, c.899G > A, p.300Arg > His, which occurred de novo in two unrelated families and is in close spatial proximity to the FAM222B phosphorylation-site Ser296. Using structural modelling we find that p.300Arg is predicted to be involved in an interaction with the β-sandwich region of the N-lobe of Nemo-like kinase (NLK). NLK though has been associated to human embryonic heart development and left–right axis determination before. It has been shown that NLK regulates the Wnt signalling pathway13 which plays a role in several steps of left–right determination in development14. Furthermore, Nlk is proposed to be an essential co-activator of Wnt signalling during early zf development, too15. It is also involved in human phosphorylation and ensuing inhibition of CREB binding protein (CBP)16 and NOTCH17 which both as well take part in cardiac development (CBP18; NOTCH19). In order to advance the understanding of a possible pathogenic effect of the missense variant c.899G > A in our affected patients, we performed poly(A) mRNA injections for overexpression in fluorescent wildtype (wt) reporter-zf embryos. Our results suggest FAM222B to be involved in cardiac development and in human cardiovascular laterality defects.