Mutations in TP73 cause impaired mucociliary clearance and lissencephaly

Abstract

TP73 belongs to the TP53 family of transcription factors and has therefore been well studied in cancer research. Studies in mice, however, have revealed non-oncogenic activities related to multiciliogenesis. Utilizing whole-exome sequencing analysis in a cohort of individuals with a mucociliary clearance disorder and cortical malformation, we identified homozygous loss-of-function variants in TP73 in seven individuals from five unrelated families. All affected individuals exhibit a chronic airway disease as well as a brain malformation consistent with lissencephaly. We performed high-speed video microscopy, immunofluorescence analyses, and transmission electron microscopy in respiratory epithelial cells after spheroid or air liquid interface culture to analyze ciliary function, ciliary length, and number of multiciliated cells (MCCs). The respiratory epithelial cells studied display reduced ciliary length and basal bodies mislocalized within the cytoplasm. The number of MCCs is severely reduced, consistent with a reduced number of cells expressing the transcription factors crucial for multiciliogenesis (FOXJ1, RFX2). Our data demonstrate that autosomal-recessive deleterious variants in the TP53 family member TP73 cause a mucociliary clearance disorder due to a defect in MCC differentiation.

Keywords: PCD; RGMC; TP73; cilia; ciliogenesis; lissencephaly; motile ciliopathy; primary ciliary dyskinesia; reduced generation of multiple motile cilia.

Figure 1

Homozygous loss-of-function variants in TP73 found in seven individuals originating from five non-related families (A) Schematic overview of chromosome 1. TP73 is located on chromosome 1p36 (red mark). (B) In healthy respiratory epithelial cells, two different transcripts are expressed. TAp73 (CCDS49) consists of 14 exons resulting in a 5,192 bp long transcript. The shorter variant ΔNP73 (CCDS44049) encodes for 12 exons yielding in a 5,120 bp long transcript. All LoF variants in the seven individuals from five unrelated families reported here (OP-1693, OP-3039, KI-645, 19DG2776, 18DG0963, 19DG0120, and 20DG1336) affect both transcript variants. Based on TAp73, the identified variants are marked on top. The deletion spanning exon 7–14 identified for OP-1693 II1 is marked below. (C) Overview of TAp73 (636 amino acid). Functional domains are marked in the protein structure including the transactivation (TA-), p53-, p53 tetram-, and sterile alpha motif (SAM-) domain.

Figure 2

Individuals affected with mutant TP73 variants suffer from a ciliary clearance disorder and cortical malformation (A) Chest computed tomography scans of OP-3039 II1, OP-1693 II1, and 18DG0963 II1 display chronic airway disease with atelectasis, pneumonia, mucus plugging, and bronchiectasis. (B) Cranial magnetic resonance imaging of KI-645 II1, OP-3039 II1, and OP-1693 II1 show frontoanterior pachygyria consistent with lissencephaly and malformation of the corpus callosum. Additional dysplasia of the hippocampus but a preserved fornix and commissura anterior was observed for OP-3039 II1. (C) Summary of clinical findings in the affected individuals.

Figure 3

The overall number of MCCs is reduced in spheroids of TP73 mutant respiratory epithelial cells (A) Ciliary axonemes of respiratory epithelial cells cultured as spheroids (control and OP-1693 II) were stained with antibodies targeting acetylated α-tubulin (acet. tub.; green). Spheroids of OP-1693 II1 display a reduced number of multiciliated cells (MCCs) compared to healthy control subjects. Nuclei were stained with Hoechst33342. (B) Transmission electron microscopy photographs of spheroids from a healthy control subject and OP-1693 II. TP73-affected cells show an overall reduction of the number of MCCs. While the number of cilia seems to be slightly reduced, the few cilia observed seem to be reduced in length (magnification III). Basal bodies (specialized centrioles) attached to the apical membrane anchoring the cilia in healthy control subjects are occasionally mislocalized within the cytoplasm in individuals affected by mutant TP73 (magnification IV).

Figure 4

Ciliary length and number are reduced in ALI cultures of TP73-affected respiratory epithelia (A) Respiratory epithelial cells were cultured under air-liquid interface (ALI) condition (control, 18DG0963 II1, OP-3039 II1). Ciliary axonemes were stained with antibodies directed against acetylated α-tubulin (acet. tub.; green) after full differentiation. Nuclei were stained with Hoechst33342. ALI cultures of individuals affected by mutant TP73 show a marked reduction in MCCs compared to healthy control subjects. MCCs are exemplary highlighted by red arrows. Grey boxes represent 3D confocal images of the ALI-cultured cells. Statistical analyses of the respiratory epithelia or cilia are performed as described in supplemental information. (B) Cilia length was measured of ALI-cultured cells and is significantly reduced for individuals affected by mutant TP73 (18DG0963 II1: 1.8 μm; OP-3039 II1: 1.5 μm). Cilia of healthy control subjects show an average length of 3.8 μm (∗∗∗p < 0.0001; 137–145 cilia were analyzed per person). (C) To calculate the correlation of ciliated versus non-ciliated cells, the ALI-cultured cells were counted for cilia bundles and the number of nuclei of the first apical cell layer. Under healthy condition, 72.7% of the apical cell layer is covered by MCCs. For ALI-cultured cells of individuals affected by mutant TP73, the apical cell layers represent a significant reduction in MCC-covering rate by 20.1%–29.9% (∗∗∗p < 0.0001; 266–722 cells were counted in total per person). (D and E) To evaluate the differentiation ability for the individuals affected by mutant TP73, respiratory epithelial cell layers grown on ALI cultures were analyzed by counting the number of nuclei (D) and measuring the distance in μm (E) from basal to apical. TP73-affected cell layers represent a significant reduction in height (18DG0963 II1, OP-3039 II1: two nuclei on average or 7–10 μm, respectively) in comparison to the control. Accordingly, the six-layered control ALI cultures represent an average height of 39.9 μm (∗∗∗p < 0.0001; 66 measuring points per individual were analyzed on average). A detailed description of all statistical data described here can be found in the supplemental information.

Figure 5

Key factors of the mutliciliogenesis pathway are reduced in respiratory epithelial cells of individuals affected by mutant TP73 in vitro Air-liquid interface-cultured respiratory epithelial cells were stained for transcription factors of the NOTCH1-dependent pathway of multiciliogenesis using antibodies directed against FOXJ1 (A; red) and RFX2 (B; red) (control, 18DG0963 II1 and OP-3039 II1). Ciliary axonemes were marked with antibodies directed against acetylated α-tubulin (acet. tub.; green). Nuclei were stained with Hoechst33342. (A) In healthy control subjects, the transcription factor FOXJ1 locates in nuclei of multiciliated cells (MCCs). For TP73-affected a severely reduced number of FOXJ1 positive nuclei was observed. The FOXJ1 staining in OP-3039 II1 below the cell layer is an artifact staining the ALI-Transwell Insert membrane (serving as growing platform while cultivation) (B) RFX2, transcription factor and interaction partner of FOXJ1, locates in nuclei of MCCs from healthy control subjects as well. The number of RFX2-expressing cells in TP73-affected MCCs is reduced compared to healthy control subjects.

Figure 6

TP73 mutant respiratory epithelial cells are not able to transport particles by ciliary beating in vitro To mimic the process of ciliary clearance function in vitro, respiratory epithelial cells from a healthy control subject and OP-3039 II1 cultured at air-liquid interface were used for particle-tracking experiments (A). Tracking videos are represented as z stack projections, while the transport direction of each particle is summarized in polar graphs (B and C). Statistical evaluation was performed for speed (μm/s) and mean square displacement (μm²). Under healthy condition, particles are transported consequently and directed along the cell layer. In contrast, a highly reduced transport of particles was observed for OP-3039 II1, indicating a reduced ciliary clearance capacity (Mann Whitney test; two-tailed: p < 0.0001). In total, thirty 20 s videos were analyzed per individual. Scale bars represent 20 μm, ∗∗∗p value < 0.0001.