Monoallelic Mutations in CC2D1A Suggest a Novel Role in Human Heterotaxy and Ciliary Dysfunction

Background: Human heterotaxy is a group of congenital disorders characterized by misplacement of one or more organs according to the left-right axis. The genetic causes of human heterotaxy are highly heterogeneous. Methods: We performed exome sequencing in a cohort of 26 probands with heterotaxy followed by gene burden analysis for the enrichment of novel rare damaging mutations. Transcription activator-like effector nuclease was used to generate somatic loss-of-function mutants in a zebrafish model. Ciliary defects were examined by whole-mount immunostaining of acetylated α-tubulin. Results: We identified a significant enrichment of novel rare damaging mutations in theCC2D1A gene. Seven occurrences ofCC2D1A mutations were found to affect 4 highly conserved amino acid residues of the protein. Functional analyses in the transcription activator-like effector nuclease–mediated zebrafish knockout models were performed, and heterotaxy phenotypes of the cardiovascular and gastrointestinal systems in both somatic and germline mutants were observed. Defective cilia were demonstrated by whole-mount immunostaining of acetylated α-tubulin. These abnormalities were rescued by wild-typecc2d1a mRNA but notcc2d1a mutant mRNA, strongly suggesting a loss-of-function mechanism. On the other hand, overexpression ofcc2d1a orthologous mutationscc2d1a P559L andcc2d1a G808V (orthologous to humanCC2D1A P532L andCC2D1A G781V) did not affect embryonic development.Conclusions: Using a zebrafish model, we were able to establish a novel association ofCC2D1A with heterotaxy and ciliary dysfunction in the F2 generation via a loss-of-function mechanism. Future mechanistic studies are needed for a better understanding of the role ofCC2D1A in left-right patterning and ciliary dysfunction.