Acrofacial Dysostosis, Cincinnati Type, a Mandibulofacial Dysostosis Syndrome with Limb Anomalies, Is Caused by POLR1A Dysfunction

Abstract

We report three individuals with a cranioskeletal malformation syndrome that we define as acrofacial dysostosis, Cincinnati type. Each individual has a heterozygous mutation in POLR1A, which encodes a core component of RNA polymerase 1. All three individuals exhibit varying degrees of mandibulofacial dysostosis, and two additionally have limb anomalies. Consistent with this observation, we discovered that polr1a mutant zebrafish exhibited cranioskeletal anomalies mimicking the human phenotype. polr1a loss of function led to perturbed ribosome biogenesis and p53-dependent cell death, resulting in a deficiency of neural-crest-derived skeletal precursor cells and consequently craniofacial anomalies. Our findings expand the genotypic and phenotypic heterogeneity of congenital acrofacial disorders caused by disruption of ribosome biogenesis.

Figure 1

Individual 1A1 (A) Newborn photo demonstrates extensive craniofacial malformations. (B and C) Frontal and profile images were taken at age 18 months after multiple reconstructive surgeries. (D) 3D reformatted image demonstrates severe maxillary and zygomatic hypoplasia (black open dashed arrow) and severe micrognathia and retrognathia (white block arrow). (E) Axial CT of the temporal bones demonstrates severe microtia with absent pinnae (white arrows), external auditory atresia (white open dashed arrows), and severe middle-ear hypoplasia and ossicular dysplasia (black open arrows). (F) X-ray of individual 1A1 demonstrates bilateral hip dysplasia and anterior bowing deformity of the femurs.

Figure 2

Individuals 1A2 and 1A3 (A) Individual IA2 at 6 years of age. (B) 3D bone image of individual IA2 demonstrates moderate zygomatic hypoplasia (white block arrow), midface hypoplasia with absent nasal bones (white open arrow), and midline alveolar process hypoplasia (open white dashed arrow). (C) Axial CT image of individual IA2 demonstrates bilateral choanal atresia (white arrows) and left maxillary and ethmoid sinus hypoplasia (black arrows). (D and E) Individual 1A3 at 52 years of age. Profile and frontal photos demonstrate subtle craniofacial dysmorphism including malar hypoplasia, micrognathia, and dysplastic ears. (F) Short, broad fingers of individual IA3.

Figure 3

In Situ Hybridization for polr1a at Various Zebrafish Stages Reveals a Dynamic Expression Pattern Maternal expression of polr1a was present at the 2-cell stage (A) but was not detected at 6 hpf (C). polr1a was ubiquitously expressed at 12 hpf (E), coincident with early NCC migration, and was expressed at 18 hpf in regions of the brain, eye, and somites (G). At 24 hpf, expression was present in the eye, midbrain-hindbrain boundary, otic vesicle, and somites (I). Beyond 36 hpf, expression was much reduced and was present in the lens of the eye and the midbrain-hindbrain boundary (K). The same expression was seen at 48 hpf (M), at which time additional expression was observed in the developing liver, which was clearly present at 72 hpf (O). The sense probe (B, D, F, H, J, L, N, and P) showed no signal at each stage examined. Scale bars represent 200 μm.

Figure 4

Phenotype of polr1a^{hi3639Tg/hi3639Tg} Zebrafish from 15 hpf to 4 dpf At 15 hpf, compared to wild-type siblings (A), mutant embryos first appeared with a grainy and irregular shape to the anterior region (B). This persisted through 24 hpf, when the cranial phenotype was more pronounced, the eyes were smaller, and the somites were wider (D) than those in wild-type siblings (C). At 34 hpf, pigment formation was clearly slower in mutant embryos than in wild-type siblings (E), and a bit of pericardial edema began to appear in mutant embryos (F). By 72 hpf, there was a clear lack of the ceratohyal and ceratobranchial cartilage (arrows point to cartilage in G and to the absence of these elements in H). Mutant embryos were smaller than wild-type siblings at 3 dpf (I and J) and 4 dpf (K and L). They showed much smaller eyes and otic vesicles and very small pectoral fins, failed to inflate their swim bladder, and had varying degrees of pericardial edema. Some mutants died by 4 dpf, and others died at 5 dpf, which was most likely due to cardiovascular defects. Scale bars represent 200 μm.

Figure 5

polr1a^{hi3639Tg/hi3639Tg} Embryos Show Reduced Formation of NCC-Derived Elements Alcian blue staining at 5 dpf (A–D) shows that whereas elements of the neurocranium (such as the trabeculae) were present in both wild-type and mutant embryos (red arrows), very little of the viscerocranium was present in mutant embryos (D). Some cartilage in the region of the jaw was faintly present in mutant embryos. It is possible that this could be Meckel’s cartilage (black arrows). A smaller region of Alcian blue staining posterior to the black arrow could potentially be the ceratohyal. There was also a small remnant of staining in the pectoral fin (green arrows in A and B) in the mutant embryos. Scale bars in (A)–(D) represent 200 μm. Immunostaining for HuC at 82 hpf (E and F) shows reduced and delayed neuronal development in mutant embryos. All cranial ganglia were present in mutant embryos (F), but they were smaller than those in control siblings (E). Abbreviations are as follows: Tg, trigeminal ganglion; aLLG, anterior lateral line ganglion; F, facial ganglion complex; SA, statoacoustic ganglion; pLLg, posterior lateral line ganglion; G, glossopharyngeal ganglion; V, vagal ganglia. Scale bars in (E) and (F) represent 100 μm.

Figure 6

In Situ Hybridization for Markers of NCC Development In situ hybridization for sox2 (A–D) and sox10 (E–H) at 12 hpf shows that levels of NCC induction were relatively similar between mutant embryos and wild-type siblings. At 17 hpf, soon after the onset of a visible mutant phenotype, the level of sox10 staining was lower in mutant embryos (J and L) than in wild-type controls (I and K), indicating a reduced migratory NCC population. By 24 hpf, the population of cartilage precursors labeled by sox9a (M–P) showed a strong reduction throughout mutant embryos, especially in the pharyngeal arches (N and P). The population of NCCs in the pharyngeal arches (shown by dlx2a in situ at 36 hpf in Q–T) was also reduced in mutant embryos (R and T). The mutant embryos showed overall diminished staining and a lack of the fifth arch. Scale bars represent 200 μm.

Figure 7

TUNEL Staining Reveals Increased Cell Death in Mutant Embryos Cell death was present throughout the polr1a^{hi3639Tg/hi3639Tg} embryos at both 14 and 24 hpf and was especially high within the neural tube. At 14 hpf (A–D), TUNEL staining did not significantly co-localize with the migratory NCC population, as shown by sox10:gfp expression. Scale bars in (A)–(D) represent 200 μm. At 24 hpf (E–J), cross-sections through the embryos showed cell death in the dorsal portion of the neural tube in mutant embryos (H and J), whereas control embryos did not show cell death in this location (G and I). Scale bars in (E)–(J) represent 100 μm.

Figure 8

qRT-PCR for rRNA Transcripts Shows a Significant Reduction in polr1a^{hi3639Tg/hi3639Tg} Embryos, whereas tp53 Levels Are Increased (A) The level of ITS1 in mutant embryos was 23% of that in wild-type siblings, the level of ITS2 was 41%, and the level of the 5′ externally transcribed sequence (ETS) was 24%. The 18S levels also tended to be lower in mutants (71%) than in wild-types (100%), but this difference was not significant (p = 0.095). qRT-PCR showed a 4-fold increase in the transcription of tp53 in polr1a mutant embryos at 24 hpf, which is when rRNA transcription diminished. Error bars represent 95% confidence intervals. (B) Immunoblot analysis was used to determine levels of Tp53. Lane 1 shows the control, lane 2 shows the polr1a^{hi3639Tg/hi3639Tg} embryo, and lane 3 shows the negative control. The red signal is α-tubulin, and the green signal is Tp53. (C) Quantification of immunoblots in ImageJ revealed that the levels of Tp53 were significantly higher (p = 0.007) in mutant embryos than in wild-type siblings at 4 dpf. ^∗p < 0.01. Error bars represent 95% confidence intervals.