Mono-allelic 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 (TALEN) 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 the CC2D1A gene. Seven occurrences of CC2D1A mutations were found to affect four highly conserved amino acid residues of the protein. Functional analyses in the TALEN-mediated zebrafish knock-out models and identified heterotaxy phenotypes of the cardiovascular and gastrointestinal systems in both somatic and germline mutants. Defective cilia were identified by whole-mount immunostaining of acetylated α-tubulin. These abnormalities were rescued by wild-type cc2d1a mRNA, but not cc2d1a mutant mRNA, strongly suggesting a loss-of-function mechanism. On the other hand, over-expression of cc2d1a orthologous mutations cc2d1a P559L and cc2d1a G808V (orthologous to human CC2D1A P532L and CC2D1A G781V) did not affect embryonic development. Conclusions - Using a zebrafish model, we were able to establish a novel association of CC2D1A 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 of CC2D1A in left-right patterning and ciliary dysfunction.