Background
Deafness is one of the most common clinical otological diseases, and most cases are caused by genetic factors [
1,
2]. Hereditary hearing loss has high genetic heterogeneity, non-syndromic hearing loss (NSHL) has only the symptom of deafness and accounts for 70% of all hereditary deafness, while syndromic deafness accounts for 30% of all cases [
1,
2]. The majority of NSHL has been confirmed to have Mendelian inheritance from single gene mutations, and these cases mainly include autosomal recessive or autosomal dominant transmission and X-linked or mitochondrial inheritance [
3]. More than 90 genes related to NSHL have been identified. Earlier studies have shown mutations in several genes that are closely related to NSHL in China, such as mtDNA 12S-rRNA,
SLC26A4,
GJB2,
GJB3, and
GJB6 [
4], while the causes of disease in other deaf individuals remain unknown.
Whole-exome sequencing (WES) is a novel technique that has offered a breakthrough for studying rare Mendelian diseases that allows for sequencing of all expressed genes in the genome, which is substantial considering the protein-coding regions covers approximately 85% of human disease-causing mutations [
5]. WES is a novel technique of genome analysis that has the advantages of being simple, economical and accurate, and it has become the most efficient way to identify genetic variants in all genes for an individual [
6].
Auditory neuropathy spectrum disorder (ANSD) is considered as a type of autosomal recessive non-syndromic hearing loss in which the transmission of sound signals from the inner ear to the auditory nerve and brain is impaired. In the present study, we applied whole-exome sequencing of two affected siblings with ANSD and their healthy sisters from a Chinese family in Guangxi Zhuang Autonomous Region. For the first time, two compound heterozygous mutations, c.1273C > T (R425X) and c.4994 T > C (L1665P), in the OTOF gene were identified as disease-causing mutations in the Chinese deaf population.
Methods
Study design
A total of 127 patients who were diagnosed as NSHL were enrolled at The People’s Hospital of Guangxi Autonomous Region. Peripheral blood samples were collected for genomic DNA. Audiologic tests were performed as necessary. The audiologic tests include auditory brainstem response (ABR), auditory steady-state response (ASSR), distortion product otoacoustic emission (DPOAE) and cochlear microphonic (CM) tests.
Hereditary deafness gene mutation screening and whole-exome sequencing
All the 127 cases enrolled in our study were subjected to a preliminary screening using a microarray method with a Hereditary Deafness Gene Mutation Detection Kit (CapitalBio Technology Co., Ltd. Beijing, China). The kit was designed to detect fifteen well-documented mutations in four deafness-associated genes, including GJB2: 35delG, 176_191del16, 235delC, 299_300delAT; GJB3: 538C > T; 12S rRNA: 1555A > G, 1494 T > C; and SLC26A4: IVS7-2A > G, 2168A > G, 1174A > T, 1226G > A, 1229C > T, 1975G > C, 2027 T > A, and IVS15 + 5G > A. Furthermore, The subsequent whole-exome sequencing was applied to the cases with singularity of its phenotype as following standard protocol by CapitalBio Technology Co., Ltd. Genomic DNA was extracted using a RelaxGene Blood DNA System (TIAGEN Biotech Co., Ltd. Beijing, China). Libraries were prepared using the Ion AmpliSeq™ Exome RDY Kit (Thermo Fisher Scientific Inc. Shanghai, China). We used the Ion OneTouch™ 2 System (Thermo Fisher) to prepare enriched, template-positive Ion PI™ Ion Sphere™ particles (ISPs) containing clonally amplified DNA. The template was sequenced on the Ion Proton™ System (Thermo Fisher) with the Ion PI™ Chip Kit v3 (Thermo Fisher). The average sequencing depth of 86.1× provided sufficient depth to identify variants for 90.9% of the targeted exome.
Variant analysis
The coverage analysis plugin and variant caller plugin from Life Technologies (Thermo Fisher) were used to analyze the Ion Proton sequencing run. Variant discovery, genotype calling of multi-allelic substitutions and indels were performed on each individual sample using the Torrent Variant Caller (TVC, version 4.6.0.7) (Thermo Fisher). Statistics and graphs describing the level of sequence coverage produced for targeted genomic regions were provided by the Torrent Coverage Analysis (version 4.6.0.3). The variants were annotated by the Annotate variants 5.0 of Ion Reporter (Thermo Fisher).
Direct Sanger sequencing
The potential causative variants in the family were confirmed by Sanger sequencing of amplified PCR products. Primer sequences for the pathogenic variant in the OTOF gene (NM_194248.2) were designed using Primer Premier 5.0 as follow: c.1273-F 5′- GGGAATCAATGAATCCTGTCT-3′ and c.1273-R 5′- TCCACGAGGTCCTTGTTTT-3′, c.4994-F 5′- GGTAGACAGGTGATGGCATAG-3′ and c.4994-R 5′- TGTCAAGGACCCAGTTCATC-3′.
Discussion
Although the majority of NSHL cases have confirmed Mendelian inheritance caused by single gene mutations, many deaf individuals do not have identified genetic contributions. WES provides a highly efficient strategy to identify the genetic variation that is responsible for Mendelian diseases. This study identified two disease-causing mutations in the OTOF gene in a Chinese family with ANSD by WES. The mutations in this gene were observed in the Chinese deaf population for the first time.
Auditory neuropathy is viewed as a variety of hearing loss in which the outer hair cells within the cochlea are usually functional, but the sound signals are unable to be accurately transmitted to the auditory nerve and brain also known as auditory neuropathy/auditory dys-synchrony (AN/AD) or auditory neuropathy spectrum disorder (ANSD). Audiology characteristics of ANSD contain severely abnormal or absent ABRs that begin with the VIII nerve component of wave I and preserve normal or near-normal otoacoustic emissions. ANSD can occur independently or be accompanied by other neurological symptoms, such as Charcot-Marie-Tooth disease [
9]. The causes of ANSD are complex, which mainly include specific damage to the inner hair cells, synaptopathies and VIII nerve disorders. Genetic factors nearly account for 40% of all ANSD cases [
10]. Four loci have been identified to account for non-syndromic ANSD; the mutations in the
OTOF and
PJVK genes cause autosomal recessive ANSD, the mutations in the
AUNA1 causes autosomal dominant ANSD, and the mutations in the
AUNX1 causes X-linked ANSD [
10‐
15].
OTOF was first identified using a candidate gene approach and a positional cloning strategy in 1999. This 48-exon gene, encoding otoferlin, is located on Chromosome 2p23.1 [
11].
OTOF encodes four transcript isoforms as different translation start sites combined with alternative splicing of some exons. Long isoforms with six C2 domains can be detected in humans and mice, whereas the short isoforms with three C2 domains are only detected in humans [
16,
17]. Otoferlin is highly expressed in the inner ear hair cells (IHCs) and vestibule. Otoferlin functions for hearing has been well-characterized. To trigger exocytosis of neurotransmitter in a calcium-dependent manner, Otoferlin interacts with two members of presynaptic SNARE complex (Syntaxin1 and SNAP25) at ribbon synapses of cochlear IHCs, which are incorporated into the membranes of transport vesicles during budding and associated with nerve terminal membranes [
18,
19]. These findings revealed that Otoferlin is essential for the synaptic vesicle exocytosis and may acts as a sensor that triggers membrane fusion at the IHC ribbon synapse.
Mutations in the
OTOF gene have been reported as the main cause of non-syndromic recessive ANSD. To date, more than 100 pathogenic mutations related to subjects with NSHL, including ANSD, have been found in different populations [
20]. The c.2485 C > T (p.Q829X) mutation was frequently found in Spanish populations and has also been observed in French, Mexicans, Argentinians and English populations [
21‐
24]. Varga et. al. observed 4
OTOF mutations in three non-syndromic recessive auditory neuropathy (NSRAN) families; two nonsense mutations, c.6141G > A and c.6285C > G; and two splice site mutations, IVS39 + 1G > C and 1778delG [
25]. There were eleven probable pathogenic variants were found among 160 unrelated ANSD patients in Japan [
26]. In China, more than 40 mutations of the
OTOF gene have been identified. Zhang’s team found 15 novel mutations in 14 congenital ANSD patients [
27].
In this study, for the first time, two mutations in the
OTOF gene were observed in a Chinese family with ANSD by whole-exome sequencing. The otoferlin isoform a is encoded by
OTOF and consists of 1997 amino acids (aa), including six C2 domains, C2A (2–97 aa), C2B (255–353 aa), C2C (418–529 aa), C2D (961–1068 aa), C2E (1493–1592 aa), and C2F (1733–1863 aa) (Fig.
3a). The c.1273C > T mutation, results in a premature stop codon from arginine to a terminator (p.R425X). It may lead to abnormal or nonfunctional protein products, suggesting it is responsible for the pathogenicity of deafness. Previously, this mutation was reported in a deaf population in Pakistan [
28], and this is the first time the mutation was identified in the Chinese population. The novel missense mutation c.4994 T > C, located between the C2E and C2F domains, results in a single amino acid substitution, leucine to proline (p.L1665P). Three pathogenic mutations (E1661K, R1676C, and E1700Q) between C2E and C2F domains have been reported. The missense mutation c.4994 T > C (p.L1665P) was assumed to be pathogenic because 1) mutations in
OTOF have been reported to be the main cause of autosomal recessive non-syndromic deafness DFNB9 that associated with ANSD [
11,
17]; 2) the region is highly conserved across mammalian species (Fig.
3b); 3) it was found in compound heterozygosity with p.R452X; 4) some pathogenic mutations have been found near the mutation site; and 5) no other recessive variants related to deafness could be identified by WES.
In this study, we found two mutations of the OTOF gene in a Chinese family with ANSD located in the Guangxi Zhuang Autonomous Region. The findings indicate that there may be rare mutations or genes associated with NSHL in the Zhuang Autonomous Region, which is one of the ethnic minority regions in China. The mutation frequency and hot spots of the deafness gene may differ among disparate ethnic regions and peoples. The findings provide new evidence for investigations of the mutation characteristics of the OTOF gene in NSHL in the Zhuang population in China.
Acknowledgements
We are grateful to the cooperation of all members of the enrolled family. We are also truly thankful for Dr. Jiazhang Wei (Dept. Otolaryngology & Head and Neck Oncology, The People’s Hospital of Guangxi Zhuang Autonomous Region, China) for his insights on our study and the valuable suggestions for the manuscript revision.