Patient-informed CRISPR Screen Identifies FLNB as a Novel Congenital Heart Disease and Ciliopathy Gene
Angelo Arrigo1,2, Venkatraman Rao1, Aakrosh Ratan3
1Department of Cell Biology, University of Virginia, Charlottesville, VA 22903.
Insights
Filamin B (FLNB) mutations cause heterotaxy and congenital heart defects by disrupting cilia function. This study validates FLNB as a key gene in these developmental disorders.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- Heterotaxy (HTX) syndrome involves abnormal organ placement and severe congenital heart defects (CHD).
- Many genes linked to CHD/HTX lack functional validation, complicating diagnosis and treatment.
- The role of actin-binding proteins in ciliopathies and HTX remains largely unexplored.
Purpose of the Study:
- To functionally validate candidate genes for CHD/HTX using a high-throughput screening approach.
- To investigate the role of Filamin B (FLNB) in ciliogenesis and left-right patterning.
- To establish a link between FLNB mutations and human CHD/HTX phenotypes.
Main Methods:
- Utilized CRISPR/Cas9 screening in *Xenopus* to evaluate candidate genes from human CHD patient exome data.
- Identified and characterized FLNB mutations in human probands with CHD/HTX.
- Disrupted FLNB in *Xenopus* to model HTX phenotypes and performed rescue experiments.
Main Results:
- Filamin B (FLNB) was identified as a novel gene associated with CHD/HTX.
- Five human probands with CHD/HTX harbored pathogenic mutations in FLNB.
- *Xenopus* models with disrupted FLNB exhibited cardiac defects and impaired cilia function, mirroring human phenotypes.
- Rescue experiments confirmed the functional conservation of human FLNB.
Conclusions:
- FLNB dysfunction is directly implicated in human ciliopathies and HTX.
- Disruption of the actin cytoskeleton contributes to ciliogenesis and left-right patterning defects.
- This study validates FLNB as a critical gene in congenital heart development and organ positioning.
Abstract:
Heterotaxy (HTX) syndrome is a congenital disorder characterized by abnormal left-right organ placement, often leading to severe congenital heart defects (CHD). Despite advances in sequencing, many CHD/HTX-associated genes remain functionally unvalidated, hindering effective clinical diagnosis and management. Here, we leveraged a high-throughput CRISPR/Cas9 screening approach in the Xenopus model to rapidly evaluate candidate genes identified from whole-exome sequencing of human CHD patients. Our screen identified Filamin B (FLNB), an actin-binding protein previously linked to skeletal disorders but not to ciliopathies or CHD. We identified 5 probands with CHD/HTX, 3 with recessive, and 2 with damaging heterozygous mutations in FLNB. Disrupting FLNB in Xenopus reproduced key features of the human HTX phenotype, including defects in cardiac development and impaired motile cilia function. Rescue experiments confirmed the functional conservation of human FLNB, directly implicating actin cytoskeletal disruption in ciliogenesis and left-right patterning defects. Our results provide crucial evidence linking human FLNB dysfunction to ciliopathies and CHD/HTX.
