Phenotype prediction of Mohr-Tranebjaerg syndrome (MTS) by genetic analysis and initial auditory neuropathy

Three probands, from Family 1, Family 2 and Family 3, who were diagnosed with auditory neuropathy and their parents were recruited (Fig. 1a, Fig. 2a and Fig. 3a), written informed consents were obtained from the next of kin on the behalf of the minors/children participants involved in this study. This study was approved by the Ethics Committee of Chinese PLA General Hospital.

Their medical histories were collected by a questionnaire. Otological examinations, including otoscopy, immittance testing, pure-tone audiometric examination (PTA, Madsen Astera², DENMARK), speech recognition score (SRS, Madsen Astera², DENMARK), distortion product otoacoustic emission (DPOAE, Madsen Capella, DENMARK), click-evoked auditory brainstem response (ABR), cochlear microphonics (CM) and electrocochleography (ECochG) tests (SmartEp, USA or Neuro-Audio, Russia), were performed to evaluate the auditory conditions. The diagnosis of auditory neuropathy was made according to the results of DPOAE/CM and ABR tests. The hearing level was assessed at 125, 250, 500, 1000, 2000, 4000 and 8000 Hz by PTA. The brain magnetic resonance imaging (MRI) examination for the temporal bone of both ears was also performed on the proband. Vestibular function evaluation included vestibular-evoked myogenic potentials (ocular VEMP, oVEMP and cervical VEMP, cVEMP), oculomotor function tests, positional nystagmus tests, positioning nystagmus tests and bithermal caloric tests. The ocular examinations included visual acuity, perimetry, flash and pattern visual-evoked potential testing, and detailed stereoscopic fundoscopy. The neurological examination included regular physical examinations and electromyography.

Peripheral blood samples of these families were collected with consent. Genomic DNA was extracted from the whole blood of the probands and their parents (TIANGEN BIOTECH, BEIJING, CHINA). After the examination of DNA quality, Beijing Genomics Institute built the DNA libraries by following Illumina’s protocol, and then 127 known deafness-related genes, including exons, splicing sites and their flanking introns, were captured by using a custom probe and sequenced by the Illumina HiSeq2000 [10]. The paired-end reads generated by sequencing were aligned to the NCBI37/hg19 assembly by the BWA software, and variant calling was performed by GATK. The bioinformatics analysis method had been described in detail previously. To obtain rare variations, common variants and low-frequency variants in the 1000 Human Genomes Project database, HapMap Project database, ExAC database, EVS database and BGI in-house databases were excluded (0.5%). All remaining variants were considered to be rare variations and were annotated using Ensembl VEP, OMIM, MGI, Gene Ontology, HGNC gene annotation database and so on. Candidate variants were validated by Sanger sequencing.

Genome-wide chromosome copy number anomalies were detected using Illumina’s InfiniumOmniZhongHua-8 DNA Analysis Bead Chip (200925710145_R03C01), with a resolution of 20 kb.

Variants with allele frequencies higher than 5% in the 1000 Genomes Project and the local database were excluded. Splicing-site, frameshift and nonsense variants were taken into further consideration. Moreover, SIFT and PolyPhen2 software were used to evaluate the pathogenic possibility. Sanger sequencing was performed to establish the co-segregation of the candidate gene variations with the phenotype in the family members. A three-dimensional structure of TIMM8A was built by SWISS-MODEL (https://​swissmodel.​expasy.​org/​) and then visualized by Swiss-PdbViewer 4.1 (version 4.1, http://​spdbv.​vital-it.​ch/).

The EMBASE and PUBMED databases were searched for literature review. The clinical phenotypes and associated genotypes of these MTS cases were summarized. Then, comparisons of the types of genotype and onset times of different clinical characteristics were analysed.

The proband of Family 1 (Fig. 1a) was found to have hearing impairment from age three. Otological examinations, including DPOAE, ABR, CM, PTA and Phmax, were performed on this 7-year-old boy, and the typical auditory neuropathy was illustrated. His parents had normal hearing, while the audiogram of the proband showed severe hearing impairment with profoundly damaged SRS (16 and 12% for the left and right ears, respectively). PTA audiogram on the left side was low-frequency ascending and on the right was mid-frequency U-shaped (Fig. 1b). As shown in Fig. 1c, DPOAE for the proband was normal, and no waves could be detected in ABR testing bilaterally, while CM waves from both ears existed (Fig. 1d). ABR testing on patient II:4 (mother of the proband) showed no abnormal latency or amplitude of ABR waves I, III and V (Additional file 1: Figure S1), as well as normal DPOAE, indicating the presence of outer hair cells in the cochlea (Additional file 2: Figure S2). In addition, waves of oVEMP and cVEMP testing showed normal amplitude and latencies, but decreased function of the horizontal semicircular canal was indicated by the bithermal caloric test (details recorded in Additional file 3: Figure S3 & Additional file 4: Table S1). MRI of the proband (Additional file 5: Figure S4) demonstrated an abnormally small cochlear nerve symmetrically. Regarding dystonia, no abnormal manifestation was observed by neurological examination, nor was any found on electromyography. Regarding optic atrophy, both the proband and his mother had normal visual acuity. Flash and pattern visual-evoked potential testing showed a normal latency and amplitude of P100 in both eyes, and detailed stereoscopic fundoscopy showed normal results (Additional file 6: Figure S5). Perimetry was not available for this young child. The γ-globulin level of the proband was normal.

The probands from Family 2 (Fig. 2a) and Family 3 (Fig. 3a), who were diagnosed with auditory neuropathy (Fig. 2b, and c and Fig. 3b, c, d, e, and f), had similar audiological phenotypes as the proband from Family 1, as well as similar MRI findings and visual acuity. The major difference in the proband from Family 3 was the abnormal γ-globulin level, showing agammaglobulinaemia when tested. The serum immunoglobulins of the proband from Family 2 were 0.30, 1.22 and 0.13 g/l of IgA, IgG and IgM, respectively.

We identified a hemizygous variation, c.232_233insCAAT (p.Leu78SerfsX21), in the TIMM8A gene (NM_004085) in Family 1 (Fig. 4a and Table 1). This novel variation in the second exon of TIMM8A caused one amino acid substitution, leucine to serine at position 78, and a frame shift after that position. That position is highly conserved across species (Fig. 4b). Co-segregation of this variation with the disease in the family was confirmed by using Sanger sequencing, as shown in the Fig. 4c. The c.232_233insCAAT variant was also detected in the proband’s mother with normal hearing, but it was absent in his father. This variation was not found in the 1000 Genomes Project, ExAC 65,000 exome allele frequency data or 1751 ethnicity-matched controls, which further supported its pathogenicity. By using SWISS-MODEL, a three-dimensional molecular structure was built to locate the mutated scope on the second exon of TIMM8A (Fig. 4d).

To further determine the frequency of TIMM8A variations in the auditory neuropathy population, we analysed the targeted gene capture and NGS from 167 cases initially diagnosed with auditory neuropathy, with or without other clinical symptoms. We found another two variations: one deletion c.133_135delGAG was identified in Family 2 (Fig. 2d), and a two-exon deletion in the TIMM8A gene without a precise location was identified in Family 3 (Fig. 5a). To further confirm the deletion region, a SNP array was performed on the members from Family 3, and the molecular cell karyotype of Xq22.1(100,593,213-100,609,547) × 0 was shown (Fig. 5b), including the TIMM8A and BTK genes. Thus, the proportion of TIMM8A variation in the auditory neuropathy population was 1.8% (3/168).

Genotype and phenotype correlation analysis for MTS cases is summarized in Table 1. Worldwide, the 14 previously reported TIMM8A abnormalities associated with MTS included missense/nonsense variations, deletions and splicing variants [7]. Therefore, the new insertion variation in this study presented a new genotype for MTS. Regarding phenotype, we observed that three variations in exon 2 did not result in visual loss, whereas other clinical manifestations showed poor correlations with genotypes.