Novel damaging mutations in CC2D1A suggest a role in human heterotaxy and ciliary dysfunction

Abstract

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, but cilia have been suggested to have a central role, especially in breaking the L-R symmetry. 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. 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. We further evaluated the novel roles of CC2D1A by 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. To determine the role of cilia in these abnormal phenotype, 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 cc2d1aP559L and cc2d1aG808V (orthologous to human CC2D1AP532L and CC2D1AG781V) did not affect embryonic development. 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.