Mandibulofacial dysostosis (MFD) is a developmental disorder characterized by hypoplasia of neural crest- derived craniofacial bones, particularly the maxilla, mandible, and zygomatic complex, as well as malformation of the outer ears and middle ear ossicles. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects, typically hypoplasia or absence of the thumbs. Mutations in SF3B4 (splicing factor 3b, subunit 4), a component of the pre-mRNA spliceosomal complex located on chromosome 1, were recently identified as a cause for Nager syndrome, accounting for approximately 60% of the cases. Interestingly, mutations in others component of the spliceosome, EFTUD2 and SNRPB, also cause MFD in two related but distinct syndromes known as mandibulofacial dysostosis with microcephaly and cerebro-costo-mandibular syndrome, respectively, suggesting that mutations in components of the spliceosome may underlie the etiology of MFD. The proposed experiments will test the hypothesis that components of the spliceosomal complex are required for neural crest progenitors formation and cause MFD when their function is altered. We have designed three specific aims to specifically address this question. (1) We will characterize the molecular mechanisms underlying MFD in Nager syndrome, by analyzing neural crest development in Sf3b4-depleted Xenopus embryos using morpholino antisense oligonucleotides and the CRISPR/Cas9 genome editing technology. We will ask whether the expression of neural crest specific genes and the pattern of cell proliferation and apoptosis are affected in these embryos. (2) We will characterize the pre-mRNA splicing activity of Sf3b4 by RNA-seq to identify transcripts that show intron retention in Sf3b4-depleted embryos, and determine whether these transcripts encode factors important for neural crest formation. (3) A region of chromosome 9 (9q32) has also been linked to Nager syndrome. This region contains two genes involved in pre-mRNA processing, PRPF4 (pre-mRNA processing factor 4) and PTBP3 (Polypyrimidine tract binding protein 3). We will analyze the function of these genes and determine their possible involvement in neural crest and craniofacial development, as potential novel candidate genes for Nager syndrome. The studies proposed in this application will identify and characterize genes critically required for neural crest and craniofacial development, and will provide important novel insights into the etiology and pathogenesis of Nager syndrome.
Nager syndrome is a developmental disorder characterized by hypoplasia of the facial bones and a variety of limb defects. Mutations in SF3B4 gene, which encodes a protein involved in the maturation of pre-mRNA, were recently identified as a cause for Nager syndrome, accounting for 60% of affected individuals. In this application we propose to develop an animal model to characterize the molecular mechanisms underlying the craniofacial defects observed in Nager syndrome patients, and identify novel candidate genes as potential causes of the disease.
