Molecular characterization of FAM222B as a novel disease gene for dominant cardiovascular laterality defects

Cardiovascular laterality defects occur with an estimated birth prevalence of 1.1/10,000 live births, associated with congenital heart defects (CHD) and situs abnormalities. Known disease genes explain about 20% of all cases often correlated with primary ciliary dyskinesia (PCD). We aimed to identify disease genes beyond PCD-related aetiologies using exome sequencing in 16 case-parent trios followed by exome survey in 2,109 individuals with situs inversus totalis, heterotaxy, or isolated CHD. We identified six different variants in FAM222B, previously discussed as a candidate gene for cerebral cavernous malformations. The variant c.899G > A (p.300Arg > His) was found de novo in two unrelated families. FAM222B has been described as a substrate of Nemo-like kinase (NLK), associated with left-right body axis determination. Structural modelling suggests a function of FAM222B Arg300 in NLK recognition. We investigated whole-mount in situ hybridization (WISH) expression pattern and genomic context of the zebrafish (zf) FAM222B homologues, fam222ba/bb, and fam222aa. Using a double-knockout (dd-KO) fam222ba/bb zf line, we identified aberrant cardiac looping in these larvae and enlarged atrium and ventricle in adult zf. We further tested the c.899G > A variant using human mRNA injections in Tg(kdrl:EGFP) wildtype (wt) reporter-zf, which led to perturbed cardiogenesis. Together, we propose FAM222B as a novel candidate gene for cardiovascular laterality defects.