A novel missense variant in the nuclear localization signal of POU4F3 causes autosomal dominant non-syndromic hearing loss

Abstract

Autosomal dominant non-syndromic hearing loss (ADNSHL) is genetically heterogeneous with more than 35 genes identified to date. Using a massively parallel sequencing panel targeting 159 deafness genes, we identified a novel missense variant of POU4F3 (c.982A>G, p.Lys328Glu) which co-segregated with the deafness phenotype in a three-generation Taiwanese family with ADNSHL. This variant could be classified as a "pathogenic variant" according to the American College of Medical Genetics and Genomics guidelines. We then performed subcellular localization experiments and confirmed that p.Lys328Glu compromised transportation of POU4F3 from the cytoplasm to the nucleus. POU3F4 p.Lys328Glu was located within a bipartite nuclear localization signal (NLS), and was the first missense variant in bipartite NLS of POU4F3 validated in functional studies. These findings expanded the mutation spectrum of POU4F3 and provided insight into the pathogenesis associated with aberrant POU4F3 localization.

Figure 1

(a) The pedigree and segregation pattern of the family, which harbored POU4F3 p.Lys328Glu. (b) The audiogram of both ears in the proband of the family revealed profound hearing loss with down-sloping shape. Hearing levels of the right and the left ear are marked using red and blue lines, respectively. (c) MPS-based panel identified a POU4F3 c.982A>G, p.Lys328Glu variant in the proband and this variant was validated by Sanger sequencing.

Figure 2

(a) The POU4F3 p.Lys328 indicated in red shading was located within the bipartite nuclear localization signal (NLS) in the POU homeodomain. The bipartite NLS was conserved across nine species. (b) The location of six reported POU4F3 missense variants and p.Lsy328Glu. POU-specific domain and POU homeodomain are indicated by black boxes. Two NLSs were indicated by red lines. (c) The predicted protein products of five reported POU4F3 frameshift variants. Boxes with slash indicate the new amino acid sequences after the frameshift.

Figure 3

The POU4F3 c.982A>G mutation altered the subcellular localization of transcription factor POU4F3. Nuclei stained with DAPI (blue) and POU4F3 detected by Anti-DYKDDDDK-Tag antibody (red) were visualized by confocal microscopy. The white arrows indicate POU4F3 outside the nuclei.