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A POU3F4 Mutation Causes Nonsyndromic Hearing Loss in a Chinese X-linked Recessive Family

Background: The molecular genetic research showed the association between X-linked hearing loss and mutations in POU3F4. This research aimed to identify a POU3F4 mutation in a nonsyndromic X-linked recessive hearing loss family. Methods: A series of clinical evaluations including medical history, otologic examinations, family history, audiologic testing, and a high-resolution computed tomography scan were performed for each patient. Bidirectional sequencing was carried out for all polymerase chain reaction products of the samples. Moreover, 834 controls with normal hearing were also tested. Results: The pedigree showed X-linkage recessive inheritance pattern, and pathogenic mutation (c.499C>T) was identified in the proband and his family member, which led to a premature termination prior to the entire POU domains. This mutation co-segregated with hearing loss in this family. No mutation of POU3F4 gene was found in 834 controls. Conclusions: A nonsense mutation is identified in a family displaying the pedigree consistent with X-linked recessive pattern in POU3F4 gene. In addition, we may provide molecular diagnosis and genetic counseling for this family.


Clinical evaluation
The proband, a 10-year-old boy, presented to the otolaryngology clinic with genetic severe deafness and speech delay. His family history was obtained by his mother. He was subjected to detailed clinical evaluations including medical history, family history, otologic examinations, audiologic testing (pure-tone audiometry, tympanometry, acoustic reflex, auditory brainstem response, and 40 Hz auditory events-related potential), and high-resolution axial computed tomography (CT) scan of temporal bones. This research was approved by the collectors' Institutional Review Board of the Ethics Committee at Chinese People's Liberation Army General Hospital.

Molecular genetic analysis
After getting informed consent from all participants and parents of persons <18 years, genomic DNA was extracted from peripheral blood leukocytes of the proband (V1) and other members of family (IV1, IV2, IV3, IV4, IV5, IV6, III1) using standard procedures. Moreover, 834 controls with normal hearing were also tested. The coding region of POU3F4 was amplified by polymerase chain reaction (PCR) with two pairs of primers. The first pair primers were a forward primer (5′-TAACCCGTGCTAGCGTCTTT-3′) and a reverse primer (5′-GAACCTGCAGATGGTGGTCT-3′) and the second pair primers were a forward primer (5′-CAACCTCTGATGAGTTGGAACA-3′) and a reverse primer (5′-AAAGGAAGAGATGGAAGGGAAG-3′). The PCR procedure was as follows: 95°C for 5 min, followed by 32 cycles of denaturation at 94°C for 45 s; annealing at 56°C for 30 s, extension at 72°C for 30 s, and a final elongation at 72°C for 7 min. All the PCR products were purified and subjected to sequencing in both directions using a DNA sequencer (ABI 3730).

Clinical investigations
The five-generation pedigree presented with an X-linkage recessive inheritance pattern of hearing loss. Otomicroscopical examination showed intact tympanic membranes and no middle ear effusion in proband (V1). Moreover, pure tone audiometry indicated bilateral severe to profound deafness, but bone hearing thresholds were normal at 250 Hz. Further, high-resolution axial CT scan revealed a dilatation of the lateral end of the internal auditory meatus and a deficit or absence in the basal turn of the cochlea [ Figure 1].

Identification of the mutation in POU3F4 gene
Variant analysis of the POU3F4 was performed for the radiologic results. Sequence screening of all the family members showed a nonsense mutation at nucleotide position c.499C>T in the proband (V1) and his uncle with hearing loss phenotype (IV3). The proband's mother (IV1), one aunt (IV2), and grandmother (III1) carried the c.499C>T mutation without hearing loss phenotype. The proband's other two aunts (IV4, IV5) and another uncle (IV6) showed normal hearing without any mutation. No mutation of POU3F4 gene was found in 834 controls. The location of POU3F4 gene on the X chromosome and the variations found on this gene were shown in Figure 2.

DisCussion
In recent years, studies have performed on POU3F4 gene mutations in different countries. Until now, mutations in this gene have been identified containing missense mutations, in-frame deletions, and truncating mutations [ Table 1]. This is the first study to identify a nonsense mutation c.499C>T in POU3F4 gene in Asians. So far, the most identified mutations of POU3F4 gene are located in nucleotide poison-encoding POU domain. [4,5,9,18] The  a c.499C>T mutation changes an arginine (CGA) to a stop code (ATG), which leads to a premature termination prior to the entire POU domains in amino acid level. [17] This mutation co-segregates with hearing loss in this family. Male members carried c.499C>T mutation. However, females with this mutation are normal hearing. Therefore, we consider that this mutation may be the cause of disease etiology of the pedigree. By molecular testing, we provided definitive diagnosis and genetic counseling for this family and further enriched pathogenic mutation spectrum of POU3F4 gene.
Pedigree analysis is an important approach to search the underlying cause of hearing loss. However, more patients with hearing loss were sporadic and there is a lack of characteristic phenotype in clinical practice. Hence, using all the available phenotypic clues to direct molecular testing would be cost-effective. The clinical features of CT images of the temporal bones are now well recognized through reported cases. [11,21,23,24] Besides the characteristics of CT images, audiological phenotype of DFNX3 is defined as profound deafness with or without a conductive element. [7,14,19,25] The conductive component in  Choi et al. [22] the audiogram could also be concerned about because of an atypical communication between an internal auditory canal and the inner ear leading to a pathologic third window which deteriorates air-conducted thresholds and increases bone-conducted thresholds. The supposed improvement in bone conduction sensitivity could be concealed by a true sensorineural deafness following the disorder. [15,26] The proband had a profound deafness but displayed normal bone conductive hearing at 250 Hz. Based on other authors' records and our experience, it was revealed that patients with this type of audiogram of low-frequency air-bone gap increased suspicion for inner malformation. Subsequent axial CT images of the temporal bones showed the defects of the bony labyrinth in proband such as a symmetric bulbous dilatation of the internal auditory canals in both ears and partial separation of the cochlea with the fundus of internal auditory canals, which was consistent with DFNX3. It was the above clinical information that guided us to reveal the causative mutation of this family. Last but not the least, DFNX3 has already been diagnosed in molecular level, so POU3F4 gene should be screened in the outpatient persons who have the clinical phenotypes of DFNX3. [27][28][29] Our study also has some limitations. On one hand, more pedigree and sporadic people should be recruited. On the other hand, new and rapid technique should be adopted to detect the patients.
In conclusion, a nonsense mutation is identified in a Chinese family displaying the pedigree consistent with X-linked recessive pattern in POU3F4 gene. Furthermore, we may provide molecular diagnosis and genetic counseling for this family.

Conflicts of interest
There are no conflicts of interest.