DROSHA Variant-Mediated miRNA Dysregulation Underlies Nonsyndromic Orofacial Cleft

Introduction and aims Nonsyndromic orofacial clefts (NSOFCs) are among the most common human craniofacial anomalies. Although genetic factors are known to play a critical role in NSOFCs, causal genes have been identified in only a limited proportion of cases. Here, through genetic analysis, a knock-in mouse model and mechanistic experiments, we identify DROSHA as a regulator of craniofacial morphogenesis and elucidate the pathogenic mechanisms underlying DROSHA variant-mediated OFC development. Methods Exome sequencing data from a multiplex family with NSOFCs were analysed to identify potentially pathogenic variants. A knock-in mouse model harbouring the patient-derived DROSHA variant was generated, followed by phenotypic characterisation and miRNA/mRNA sequencing. Luciferase reporter assays were conducted in vitro to validate miRNA-target gene interactions. CellTiter-Glo assays were conducted to assess the effects of the DROSHA variant and dysregulated miRNAs on the proliferation of palatal mesenchymal cells. Results A missense variant (p.P170L) in DROSHA was identified as the most likely disease-associated variant in the affected family. Both heterozygous and homozygous Drosha knock-in mice recapitulated cleft palate phenotypes. Mechanistically, the DROSHA variant dysregulated the expression of a subset of miRNAs, with miR-30a-5p and miR-103-3p upregulated in mutant mice. These miRNAs negatively regulated Snail1 and Ihh expression by binding to the 3’ untranslated regions of their respective target genes. In addition, the DROSHA variant impaired palatal mesenchymal cell proliferation. Conclusion Collectively, our findings establish DROSHA as a key regulator of craniofacial development and suggest that DROSHA variant-mediated miRNA dysregulation represents a potential molecular mechanism underlying NSOFC development. Clinical relevance This study identifies a DROSHA variant associated with human craniofacial anomalies, providing new insights into the genetic basis of NSOFCs and highlighting the potential value of DROSHA -related variants for genetic diagnosis and counselling.