Identification and Functional Characterization of a Novel POU3F4 Frameshift Mutation in a Chinese Family

Hereditary sensorineural hearing loss (SNHL) represents a significant global public health burden. DFNX2, an X-linked form of non-syndromic SNHL, is caused by pathogenic variants in the POU3F4 gene. This study aimed to identify a novel POU3F4 mutation and characterize its functional consequences to elucidate the molecular pathogenesis of DFNX2. A three-generation Chinese family with X-linked deafness was recruited. Targeted next-generation sequencing was used to screen candidate variants, which were validated by Sanger sequencing for co-segregation analysis. Functional assays, including subcellular localization, dual-luciferase reporter assay, Western blotting, and homology modeling, were performed to assess the mutation's effects. A novel frameshift mutation, c.670_673dupGGTA (p.(Asn225Argfs*2)), was identified and showed complete co-segregation with the deafness phenotype. The mutant protein exhibited cytoplasmic mislocalization, and dual-luciferase assays revealed a severe reduction in transcriptional activation capacity, whereas Western blot confirmed stable expression of the truncated protein. Structural modeling predicted the loss of both the POUS and POUH DNA-binding subdomains. Collectively, this study expands the mutational spectrum of POU3F4 and supports previously reported mechanisms underlying DFNX2 pathogenesis.