Ascl5 variant alters gene expression in postnatal maxillary molars through potential gain-of-function effect

Lobodontia is a rare dental anomaly characterized by supernumerary cusps and a single, pyramid-shaped molar root resembling those of carnivorous mammals. Through genetic analysis of six unrelated families, we identified a novel heterozygous missense variant in the ASCL5 gene (c.274G>A; p.Glu92Lys) that segregates fully with the phenotype. To investigate the functional impact of this variant, we generated Ascl5 knock-in mutant mice using CRISPR/Cas9. Heterozygous (Ascl5Mut/WT) mice displayed dental anomalies consistent with human lobodontia, whereas homozygous (Ascl5Mut/Mut) mice exhibited more severe craniofacial abnormalities, highlighting the essential role of ASCL5 in tooth and jaw development. To explore the molecular basis of the dental phenotype, we performed transcriptomic analysis of maxillary first molars from postnatal day 1 (P1) Ascl5Mut/WT and Ascl5Mut/Mut mice, each compared to wild-type controls. Based on a gain-of-function hypothesis for the p.Glu89Lys variant, we focused on genes upregulated in both mutant genotypes. This analysis identified 87 commonly upregulated genes. Pathway enrichment analysis using Metascape revealed 11 significantly enriched biological pathways, including Dmp1, Phex, and Atg4b genes associated with biomineral tissue development. Notably, the regulatory region of Phex contains a conserved E-box motif, a known binding site for bHLH transcription factors such as ASCL5, suggesting it may be a direct transcriptional target.