Clinical and zebrafish studies of truncating SF3B2-variants in craniofacial microsomia.

Craniofacial microsomia (CFM) exhibits significant phenotypic variability and degree of severity. While loss-of-function variants in SF3B2 have recently emerged as a genetic etiology, the molecular basis underlying this clinical heterogeneity remains poorly understood. Here, we report two probands harboring novel truncating SF3B2 variants and presenting with distinct clinical phenotypes. Proband 1, with a heterozygous p.(Gln60*) variant, exhibited characteristic CFM features, including mandibular hypoplasia, cleft palate, bilateral tragal abnormalities, microtia, external auditory canal stenosis with hearing impairment, and an epibulbar dermoid. In contrast, Proband 2, carrying a p.(Lys507*) variant, exhibited a milder craniofacial phenotype, although he had hearing loss and developmental delay that could not be explained with certainty by the SF3B2-variant. Western blot analysis demonstrated complete loss of p.(Gln60*) protein, whereas the p.(Lys507*) variant-despite lying outside the predicted nonsense-mediated decay (NMD) escape region-retained 15.5% residual expression of truncated protein. To investigate functional consequences, we performed CRISPR/Cas9-mediated sf3b2 knockout in zebrafish. Mutant larvae showed a 25.33% malformation rate and recapitulated human craniofacial features, including a reduced head-to-body length ratio (p = 0.0013), hypoplastic mandibular cartilage-evidenced by shortened Meckel's cartilage (p = 0.0104) and Palatoquadrate (p = 0.0174)-and impaired skeletal mineralization (p = 0.0028). Together, these findings suggest that truncating variants at different positions may be associated with differential protein expression levels and variable clinical presentations. Our study reinforces SF3B2's role as a loss-of-function disease gene and highlights the importance of variant-specific molecular characterization in understanding CFM.