Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families

Abstract

Autosomal dominant non-syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next-generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three-generation Chinese families with late-onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co-segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI-OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild-type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.

Keywords: Chinese family; POU4F3; autosomal dominant; hearing loss; identification; novel mutation.

FIGURE 1

POU4F3 mutations identified in the two Chinese families suffering from autosomal dominant hearing loss. A, Pedigrees of the Chinese families. Black squares and circles represent members with symptoms of DFNA15. The proband is indicated by an arrow. M and—indicate the mutant and wild‐type alleles, respectively. Asterisks indicate the families with POU4F3 mutations identified in the present study. B, Sanger sequencing showing the c.704_705del (p.T235fs) and the c.593G>A (p.R198H) mutations in POU4F3

FIGURE 2

Audiograms of some members participating in this study in the two Chinese families. Blue crosses and red circles represent the air conduction hearing threshold levels of left and right ears, respectively. Asterisks indicate the families with POU4F3 mutations identified in this study. Gender and age are shown below the audiogram of each individual

FIGURE 3

Genomic structure, conservation and 3D molecular model of POU4F3 mutations. A, Genomic structure of POU4F3 based on the open reading frame (NM_002700) containing two exons (grey rectangles). The positions of the c.704_705del (p.T235fs) and the c.593G>A (p.R198H) mutations are arrowed both at the gene (top) and the protein level (bottom). B, Protein alignment showing POU4F3 p.R198H occurred at evolutionarily conserved amino acids (in black box) across eight species. The mutation of c.704_705del (p.T235fs) caused a truncated protein. C, Three‐dimensional molecular models revealed the abnormal incomplete structure of mutant‐type protein

FIGURE 4

The POU4F3 c.704_705del and c.593G>A mutations altered the subcellular localization and protein expression of transcription factor POU4F3. A, Immunofluorescence staining was performed after transient transfection in HEK 293 cells and HEI OC1 cells. Images display DAPI in blue, Flag‐tagged protein in green, and merged pictures. Both in HEK 293 cells and HEI OC1 cells, mutant protein located mostly in cytoplasm while the wild‐type protein was exclusively located in nuclei. The white arrows indicate POU4F3 outside the nuclei. Scale bars = 30 µm. B, The expression of POU4F3 protein and its mutants in transfected HEK 293 cells were detected by immunoblotting using anti‐Flag and anti‐β‐actin (serving as an internal control) antibodies