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.


| INTRODUC TI ON
Hearing loss is the most frequent sensory impairment in humans, with a morbidity of 1/1000 in newborns. 1 Based on available evidence, approximately 60% of all cases are because of genetic factors. According to clinical characteristics, hearing loss can be divided into syndrome hearing loss and non-syndrome hearing loss (NSHL).
Non-syndrome hearing loss accounts for about 70% of all hereditary cases. 2 The hereditary modes of NSHL include autosomal recessive, autosomal dominant and X chromosome-linked or mitochondrial inheritance, the incidences of which are, respectively, 80%, 20% and <1%. 3  POU4F3, characterized by two DNA-binding POU domains, was expressed exclusively in cochlear, vestibular hair cells and sensory. 4,5 Function studies have demonstrated that POU4F3 was required for the differentiation and maturation of hair cells and establishment of ear neural network, but not in fate determination of inner ear hair cells. 6 For example, hair cells experienced a rapid, progressive apoptosis in POU4F3 knockout mice during the late gestation and early postnatal period, which further reduced innervation and loss of sensory neurons and caused severe hearing loss and balance impairment. 7 Other studies show that POU4F3 is involved in the regulation of downstream targets, such as BDNF, NT-3, Lhx3, Gfi1 et al, which play important roles in inner ear development. [8][9][10][11] POU4F3 might be a candidate for hereditary hearing impairment because of its important role in hair cells.
Clinicians have made great efforts for the identification of pathogenic mutations for hearing loss. POU4F3 is the earliest discovered deafness-related gene to cause ADNSHL DFNA15. In

1998, Vahava et al first identified an 8-base pair deletion in POU4F3
leading to progressive hearing loss. 12 Until now, more than 30 mutations of POU4F3 were found to be associated with the pathogenesis of DNFA15, most of which occurred in DNA-binding domains.
According to previous studies, POU4F3-related DFNA15 manifested as late-onset bilateral, progressive hearing loss with down-sloping audiometric configurations. [13][14][15] For example, in a five-generation Dutch family linked with p.L289F on POU4F3, the progression rate for DNFA15 was 0.8-1.4 dB/year. 16 Here, we performed mutation analysis on two Chinese families with ADNSHL by using NGS and Sanger sequencing. A missense mutation c.593G>A (p.R198H) and a frameshift mutation c.704_705del (p.T235fs) were identified as the causative factor for DNFA15 with a late-onset progressive symptom. Furthermore, we also investigated the effects of these two mutations on the microscopic structure and subcellular location of POU4F3 in cells.

| Clinical features
Two three-generation Chinese families, respectively, named as F052* and F493*, have been recruited through Shandong Provincial ENT Hospital Affiliated to Shandong University. The pedigrees of the families with autosomal dominant pattern of inheritance are shown in Figure 1A. Due to patients' privacy or other reasons, there were only six members (II-2, II-4, III-1, III-2 with hearing loss and II-3, II-5 with normal hearing) in F052* and 5 members (II-1, II-2, II-4, III-1 with hearing loss and III-2 with normal hearing) in F493* participating in our study ( Figure 1).
The clinical examinations were performed in the Otologic Center, Shandong Provincial ENT Hospital Affiliated to Shandong University.
After physical and otoscopic examinations, pure tone audiometry (PTA) was performed. Other diseases or ototoxic medication history that may cause hearing loss were ruled out. The study was approved by the ethics committee of the Shandong Provincial ENT Hospital and written informed consents were obtained.

| NGS of deafness gene
For mutation analysis, genomic DNA of the probands was extracted from peripheral blood using DNA extraction kit (Axygen) and subjected to a targeted NGS including 127 deafness-related genes (see Table S1). Data were analysed in accordance with NGS standard process. Sequence alignment was performed by using BWA 0.6.2-r126 software. UCSC hg19 Feb.2009 was used as reference genome.
Mutation identification was performed by using GATK. dbSNP (snp137) as a reference. The pathogenicity of novel mutations was predicted by using 1000 genome database (phase I), HapMap database (combined data from phases II and III) and own databases as references. Guideline of American College of Medical Genetics and Genomics was used as the reference of data interpretation. 17

| Cell culture
The culture condition was as follow: MEM medium (Gibco) contain-

| Clinical features
Two three-generation Chinese families with ADNSHL were enrolled in this study. The pedigree and disease statements of all participants were shown in Figure 1A.  Based on self-description of patients with c.704_705del in F052* and c.593G>A in F493*, the hearing loss associated with these two mutations was typically post-lingual, late onset and progressive (Table 1).
For example, the proband III-2 in F052* recalled that the onset age of bilateral NSHL was 10 and symptoms became worse after then.

| Identification of pathogenic POU4F3 mutations in two Chinese ADNSHL families by targeted NGS
Here, we aimed to screen possible pathogenic mutations in probands of another two Chinese ADNSHL families (F052*-III-2 and F493*-II-2, Figure 1A) by using targeted NGS of 127 deafness-related genes. As shown in Figure 1A, two novel mutations on POU4F3, c.704_705del (p.T235fs) and c.593G>A (p.R198H), were, respectively, identified as potential pathogenic variants in F052*-III-2 and F493*-II-2. These deleterious and pathogenic aspects of those two mutations are listed in Table 2.
Next, we used Sanger sequencing to confirm the two POU4F3  Figure 3B.
The three-dimensional structures of wild-type and mutant-type POU4F3 were simulated according to the crystal structure. As shown in Figure 3C, compared with the wild-type structure, p.T235fs protein exhibited an abnormal truncated structure, resulted from the c.704_705del frameshift mutation. While, in p.R198H protein, the c.593G>A missense variant was predicted to perturb protein structure because of the substitution of arginine by histidine acid. In this respect, our prediction study revealed that these two novel mutations possibly lead to protein dysfunction.

| Variations of p.T235fs and p.R198H alter the subcellular localization of POU4F3
Accumulated evidence demonstrated that POU4F3 functions as an important transcription factor for the functions of hair cells. 13,20 So the subcellular localization of POU4F3 in nuclei is necessary for its function, because POU4F3 needs to combine to target DNA sequence. To investigate whether the pathogenicity of POU4F3 mutations was mediated by the alteration of protein subcellular localization, we overexpressed wild-type and mutant-type POU4F3 by using pCMV-Tag2B plasmids in cell lines of HEK 293 and HEI OC1, an immortalized model of Corti-derived epithelial cell line.
As a FLAG-tag was fused to the N-terminal of POU4F3 protein, the subcellular localization of POU4F3 was detected by using anti-FLAG immunofluorescence analysis. As shown in Figure 4A, wildtype POU4F3 protein was exclusively located in the cell nuclei in both cell lines, while mutant-type POU4F3 exhibited a strong fluorescence signal in cytoplasm and a much weaker in nuclei. These results suggested that variations of p.R198H and p.T235fs caused a retreat of POU4F3 from nuclei, which might explain the pathogenicity of screened mutations.  The result ( Figure 4B) showed that the wild-type and the p.R198H

| Mutations of p.T235fs and p.R198H affect POU4F3 protein expression
POU4F3 proteins were expressed as a single protein of the correct molecular weight, meaning that the staining observed in the immunocytochemical analysis is specifically derived from the tagged POU4F3 proteins. While a weak band of p.R198H POU4F3 protein was detected, indicating that the stability of p.R198H POU4F3 differed from that of the wild-type protein. In addition, the p.T235fs POU4F3 protein was detected with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein.  In terms of molecular biology, dominant inheritance involved three mechanisms: haploinsufficiency, function-gaining and dominant-negative effect. 26 As a dominant-negative effect have been ruled out, haploinsufficiency is the most likely mechanism so far. 13,27 Although mice with Brn3c −/− behaved healthily, several studies in humans supported the haploinsufficiency mode. 28 Freitas, E. L. et al reported that a complete deletion of POU4F3 caused ADNSHL. 29 Mutai, H. et al reported a frameshift variant of POU4F3 in a Japanese family, which would produce an extended mRNA because of the absence of in-frame stop codon. 30 The overlong mRNAs might be degraded in cells. In other words, these two mutations make POU4F3 a haplotype, which could only be explained by haploinsufficiency. Furthermore, the altered protein localization of POU4F3 in this study also supports haploinsufficiency mechanism.

| D ISCUSS I ON
In summary, by using a targeted NGS-base genetic test, we identified two novel variants of POU4F3, c.704_705del (p.T235fs) and c.593G>A (p.R198H), in two Chinese families with ADNSHL, respectively. Function analysis suggested that these two mutations caused an abnormal, incomplete protein structure and impaired nuclear localization of POU4F3. This study provided new knowledge of POU4F3 mutation spectrum associated with hearing loss and helps to identify the pathogenic mechanism of POU4F.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
XB and LX were in charge of the idea, project design and concept of the study; XB performed the in vivo experiments; FZ, YX and YJ performed DNA extraction, PCR amplification, sequencing and data analysis; HW and LX recruited the clinical patients and were in charge of the clinical assessments; XB wrote the manuscript; QZ and LX revised the manuscript. All authors read and approved the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.