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Heterotaxy (HTX) is a congenital disorder characterized by abnormal left-right organ placement, often leading to severe congenital heart disease (CHD). Despite advances in sequencing, many CHD and 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 and HTX, 3 with recessive, and 2 with damaging heterozygous variants 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 and HTX.
Figure 1. Flnb depletion results in LR patterning defects(A) Flowchart showing the HTX gene prioritization process.(B) Abnormal heart looping in Xenopus embryos is indicative of an LR patterning defect leading to HTX and CHD. White arrows indicate the direction of the outflow track. Black stars and arrowheads indicate the position of the gallbladder and gut in normal and abnormal conditions.(C) Eighteen predicted CHD- and HTX-associated genes with varying previous associations with cilia were selected from whole-exome sequencing data of 2,871 patients for a CRISPR-mediated LOF screen in Xenopus. Flnb was the top candidate for the LR patterning defects identified in the screen. Cilia db, ciliary database; %ab, % abnormal. (D) Cilia in the LRO generate a leftward flow that sets off a gene cascade, repressing coco, and in turn activates pitx2 in the left lateral plate, which alters organogenesis. Normal heart looping is a result of left-sided pitx2 expression.(E and F) Control, CRISPR, and 10- or 20-ng morpholino-injected embryos were scored for (E) abnormal heart looping and (F) abnormal pitx2 expression (20 ng MO only). Loss of Flnb causes both abnormal heart looping and abnormal pitx2 expression. The number of embryos (n) scored for each experiment is indicated in the bar graph. The p value is shown above each bar graph.
Figure 2. Flnb depletion leads to defects in the left-right organizer and in ciliogenesis(A) LRO in control and morphant Xenopus embryos at embryonic stage 16. White boxes indicate the enlarged region of the LRO. Note that cells in the LRO can occasionally exhibit 2 cilia arising from mother and daughter centrioles and that precise cilia-per-cell quantification would require basal body labeling.(B) Statistical comparison of the length-to-width ratio of LROs in control and morphants.(C) Statistical comparison of cilia number between control and morphants.(D) Histogram showing cilia length distribution in control and morphants.(E) Interquartile range (25%–75%) showing distribution of cilia length in controls and morphants.(F) Table showing number of cilia in each category of cilia length: short, medium, and long. n = 19 and 21 LROs for controls and morphants, respectively, from 2 trials. The p value is shown above each bar graph.
Figure 3. Flnb depletion results in dysfunctional multiciliated cells with aberrant gross morphology(A) Control and flnb morpholino-injected embryos were stained with anti-acetylated α-tubulin antibody (magenta) and phalloidin (green) to label cilia and F-actin, respectively.(B) Mucociliary clearance in control and Flnb-depleted embryos was evaluated by measuring the rate of flow of beads over the surface of the embryos. The flow rate was categorized as either normal, slow, or none. Flnb morphants showed significantly slow or no flow.(C) Higher magnification of MCC morphology showing loss of apical F-actin, reduced cilia, and decreased α-tubulin intensity in Flnb morphants.(D) Quantification of MCC morphology parameters: apical area, thinness ratio, acetylated α-tubulin signal, and cortical and medial F-actin intensity. Cortical and medial F-actin intensity was also assessed in non-MCCs. The thinness ratio measures the cell shape (circularity). MCCs are round in shape early in development but become more polygonal (closer to 1) as they undergo apical expansion and mature. Scale bar, 20 μm in all images. n = 30–80 cells from 9 embryos across 3 trials.
Figure 4. h-FLNB localizes to both the apical and subapical pool of F-actin and rescues the flnb morphant phenotype of MCCs(A and B) hFLNB-GFP was injected into wild-type fertilized eggs, and embryos were grown to stage 28, fixed, and stained to analyze the localization of hFLNB in MCCs. Scale bar, 10 μm in (A). hFLNB-GFP (cyan) localizes to the apical meshwork and subapical pool of F-actin in MCCs. Scale bar, 2 μm in (B).(C–E) To assess the functionality of the hFLNB-GFP construct in Xenopus, flnb morphants were injected (rescued) at the 4-cell stage with hFLNB-GFP, grown to stage 28, fixed, and stained. Rescued cells were marked by GFP expression (cyan). Scale bar, 20 μm in (D). Flnb expression, medial F-actin, and acetylated tubulin intensity were quantified in morphant and rescue cells. hFLNB-GFP rescued medial F-actin and cilia intensity. n = 16–17 MCCs from 7–9 embryos across 3 trials.
