POLR1B and neural crest cell anomalies in Treacher Collins syndrome type 4

Abstract

Purpose: Treacher Collins syndrome (TCS) is a rare autosomal dominant mandibulofacial dysostosis, with a prevalence of 0.2-1/10,000. Features include bilateral and symmetrical malar and mandibular hypoplasia and facial abnormalities due to abnormal neural crest cell (NCC) migration and differentiation. To date, three genes have been identified: TCOF1, POLR1C, and POLR1D. Despite a large number of patients with a molecular diagnosis, some remain without a known genetic anomaly.

Methods: We performed exome sequencing for four individuals with TCS but who were negative for pathogenic variants in the known causative genes. The effect of the pathogenic variants was investigated in zebrafish.

Results: We identified three novel pathogenic variants in POLR1B. Knockdown of polr1b in zebrafish induced an abnormal craniofacial phenotype mimicking TCS that was associated with altered ribosomal gene expression, massive p53-associated cellular apoptosis in the neuroepithelium, and reduced number of NCC derivatives.

Conclusion: Pathogenic variants in the RNA polymerase I subunit POLR1B might induce massive p53-dependent apoptosis in a restricted neuroepithelium area, altering NCC migration and causing cranioskeletal malformations. We identify POLR1B as a new causative gene responsible for a novel TCS syndrome (TCS4) and establish a novel experimental model in zebrafish to study POLR1B-related TCS.

Keywords: POLR1B; Treacher Collins–Franceschetti; apoptosis; neural crest cells.

Fig. 1

POLR1B pathogenic variants in six patients with Treacher Collins syndrome (TCS). (a) Pedigrees of five families with TCS. Solid symbols indicate affected family members (patients 1–6), open symbols indicate unaffected members, gray solid symbol (5^m) indicates patient 5’s father with mosaicism, and squares and circles indicate males and females, respectively. (b) Front and lateral view of patients from this series. (c) Representative electropherograms of pathogenic variants.

Fig. 2

In silico studies of consequences of POLR1B pathogenic variants. (a) Uniprot alignment of POLR1B protein showing interspecies conservation of amino acids affected by pathogenic variants. Position of pathogenic variants and conservation across species are highlighted in bold. (b) Space fill model of POLR1B and its association with the POLR1A subunit of RNA polymerase around the variant residues. (Left) The wild-type (WT) structure: residue found pathologically modified in Homo sapiens. Distance compatible with hydrogen bond is indicated for Ser682-His967 and Glu724-Arg1003. (Right) p.(Ser682Arg) (S682R) and p.(Arg1003Cys) (R1003C) pathogenic variant model with Arg682, which clearly could hamper the ɑ-helix POLR1A wild-type position.

Fig. 3

Polr1b knockdown in zebrafish reduces neural crest cell (NCC) migration and causes severe craniofacial defects similar to Treacher Collins phenotype. (a) At 20 hours postfertilization (hpf) (upper panels) and 48 hpf (lower panels), polr1b morphants present smaller heads, smaller eyes, and developmental delay (larger yolk sac) compared with controls (scale bars, upper panels = 400 µm, lower panels = 200 µm). (b) At 3 days postfertilization (dpf), polr1b morphants present smaller size as well as pigmentation defects (indicated by white arrows) compared with controls, and some morphants also present cardiovascular defects (large cardiac edema) and died at 4 dpf (data not shown). (c) polr1b morphants present altered NCC marker expression. At 30 hpf, injection of Tg(sox10:GFP) (upper panels) and Tg(foxd3:GFP) (lower panels) conferred NCC abnormalities in polr1b morphants compared with controls, in particular where branchial arches normally formed (white arrows, right panels), which indicates reduced migratory NCC population. (d) At 3 dpf, cranioskeletal structures were visualized by using Tg(col2a:mCherry). Compared with control morphants (MO Control), polr1b morphants (MO polr1b) lacked mandibular and branchial arches (1: mandibula, 3: branchial arches), and the otic vesicle was smaller (2). polr1b morphants show impaired pigmentation (pigments indicated by an asterisk) and cardiac edema (white arrow, upper right panel), which indicates that other NCC-derived tissues are affected. Scale bars = 200 µm. Five embryos were used in each condition and representative images of six experiments are shown.

Fig. 4

Polr1b knockdown in zebrafish alters RNA polymerase I (pol I)–mediated ribosome biogenesis in neural crest cell (NCC) precursors. (a) Ribosomal RNA (rRNA) transcript expression profile in control and polr1b morphants by quantitative reverse transcription polymerase chain reaction (qRT-PCR) normalized to EF1a level. 5’ETS, ITS1, ITS2, and 18S depend on RNA pol I activity, whereas 5S is RNA pol III–dependent. (b–e) Confocal images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling (in red) of 20 hpf Tg(sox10:GFP) embryos (lateral views in (b,d), dorsal views in (c,e)). Imaging of apoptotic cells stained with TUNEL in polr1b and control morphants (dorsal part indicated by white arrows in (d,e)). Scale bar: 100 µm. (f) Quantification of TUNEL-positive cells in MO control and MO polr1b (n = 10 zebrafish larvae in MO control and n = 11 zebrafish larvae in MO polr1b). (g) Quantification of p53 messenger RNA (mRNA) level in MO control and MO polr1b (n = 6 groups of 15 pooled zebrafish larvae at 24 hpf). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-tailed unpaired Mann–Whitney nonparametric test.