Functional analysis on mutations in POU3F4 in DFNX2 patients

Abstract

Introduction: Most X-linked nonsyndromic hearing loss is caused by various types of mutations of the POU domain class 3 transcription factor 4 (POU3F4) gene. Therefore, how various mutations of POU3F4 lead to hearing loss needs to be investigated.

Methods: For POU3F4 mutations found in DFNX2 patients, required constructs for further assays were cloned. RT-PCR and polysome fraction analysis were carried out for testing the expression of POU3F4 at RNA level. Western blot and immunofluorescence staining were performed for confirming protein level and localization, and finally luciferase assay was carried out for testing activity of protein.

Results: Five unique missense or frameshift truncation and extension mutations were found in Korean patients and the information was given. Two missense mutations (p.Thr211Met and p.Gln229Arg) disturbed transcriptional activity. Two frameshift extension mutations (p.Thr354GlnfsX115 and p.X362ArgextX113) were located outside of POU domain and nuclear localization signal at the C-terminus. POU3F4 protein levels were low and could be restored by MG132, a proteasome inhibitor in vitro. These mutant POU3F4 proteins were exclusively localized to the cytoplasm and did not have transcriptional activity. Frameshift mutation (p.Leu317PhefsX12) in POU3F4 leads to the truncation of the C-terminal 44 amino acids spanning the POU domain and nuclear localization signal. This frameshift truncation mutant protein was located in both the nucleus and cytoplasm and was present at a low protein levels. This mutant was also transcriptionally inactive, even in presence of MG132.

Conclusions: From these results, I conclude that frameshift truncation and extension mutations in the C-terminus of POU3F4 lead to cytoplasmic localization and subsequent proteosomal degradation due to structural aberrations, which cause transcriptional inactivity and thus nonsyndromic hearing loss.