Genetic findings of Sanger and nanopore single-molecule sequencing in patients with X-linked hearing loss and incomplete partition type III

Abstract

Background POU3F4 is the causative gene for X-linked deafness-2 (DFNX2), characterized by incomplete partition type III (IP-III) malformation of the inner ear. The purpose of this study was to investigate the clinical characteristics and molecular findings in IP-III patients by Sanger or nanopore single-molecule sequencing. Methods Diagnosis of IP-III was mainly based on clinical characteristics including radiological and audiological findings. Sanger sequencing of POU3F4 was carried out for these IP-III patients. For those patients with negative results for POU3F4 Sanger sequencing, nanopore long-read single-molecule sequencing was used to identify the possible pathogenic variants. Hearing intervention outcomes of hearing aids (HAs) fitting and cochlear implantation (CI) were also analyzed. Aided pure tone average (PTA) was further compared between two groups of patients according to their different locations of POU3F4 variants: in the exon region or in the upstream region. Results In total, 18 male patients from 14 unrelated families were diagnosed with IP-III. 10 variants were identified in POU3F4 by Sanger sequencing and 6 of these were reported for the first time (p.Gln181*, p.Val215Gly, p.Arg282Gln, p.Gln316*, c.903_912 delins TGCCA and p.Arg205del). Four different deletions that varied from 80 to 486 kb were identified 876–1503 kb upstream of POU3F4 by nanopore long-read single-molecule sequencing. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutations. Among these 18 patients, 7 had bilateral HAs and 10 patients received unilateral CI. The mean aided PTA for HAs and CI users were 41.1 ± 5.18 and 40.3 ± 7.59 dB HL respectively. The mean PTAs for patients with the variants located in the exon and upstream regions were 39.6 ± 6.31 versus 43.0 ± 7.10 dB HL, which presented no significant difference (p = 0.342). Conclusions Among 14 unrelated IP-III patients, 28.6% (4/14) had no definite mutation in exon region of POU3F4. However, possible pathogenic deletions were identified in upstream region of this gene. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutation. There was no significant difference of hearing intervention outcomes between the IP-III patients with variants located in the exon region and in the upstream region.

Considering gene therapy to protect from X‐linked deafness DFNX2 and associated neurodevelopmental disorders

“…DFNX2 deafness was originally considered as a nonsyndromic hearing loss 2,3 . However, further observations of DFNX2 patients revealed additional disorders such as motor and cognitive developmental delays, mental retardation, autism spectrum disorders, learning disabilities, hyperactivity and attention deficit disorders, and oppositional‐provocative behaviors 9,12,19,33,38,39,42,47,50,52,61–67 . Recently, an explorative study was carried out focusing on neurodevelopmental symptoms in 10 children with incomplete partition type III.…”

“…A recent article reviewed the outcomes after cochlear implantation in patients with DFNX2 deafness 83 . In most studies, cochlear implantation led to significant improvements in audiometric thresholds and speech recognition compared to preoperative performance 12,62,82,84–88 . However, several authors report that auditory perception and language development remain globally limited in DFNX2 patients with cochlear implantation 11,14,22,28,33,38,39,67,89–91 .…”

“…X‐linked deafness type 2 (DFNX2, locus Xq21.1), caused by POU3F4 mutations, accounts for nearly 50% of all cases of X‐linked hearing loss. Although most cases are inherited from the carrier mother, a significant proportion (up to 20% in Eastern Asia) of POU3F4 mutations also occur de novo 9,12,17,24,27,33,34,36,40,47,48 . Jang et al 24 recently observed a significantly higher de novo occurrence of large genomic deletions within the DFNX2 locus in Korean patients than that of point mutations in the POU3F4 gene.…”

“…To date, over 80 deafness‐causative mutations in the coding sequence of POU3F4 have been identified in some 20 countries, including missense, nonsense, deletion, frameshift, and extension mutations (Figure 1). 6–46 Moreover, deletions of the entire gene as well as deletions, paracentric inversions, and duplications upstream of the gene (containing the putative regulatory elements of POU3F4 transcription) were also reported 12,13,24,47–52 . X‐linked deafness type 2 (DFNX2, locus Xq21.1), caused by POU3F4 mutations, accounts for nearly 50% of all cases of X‐linked hearing loss.…”

“… 5 , 6 To date, over 80 deafness‐causative mutations in the coding sequence of POU3F4 have been identified in some 20 countries, including missense, nonsense, deletion, frameshift, and extension mutations (Figure 1 ). 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 Moreover, deletions of the entire gene as well as deletions, paracentric inversions, and duplications upstream of the gene (containing the putative regulatory elements of POU3F4 transcription) were also reported. 12 , 13 , 24 , 47 , 48 , 49 , 50 ...…”