Introduction
Hearing loss is an extremely common problem worldwide, and it is one of the most genetically heterogeneous disorders occurring in humans. Approximately half of the cases have a genetic etiology, including nonsyndromic hearing loss (NSHL) and syndromic hearing loss (SHL). NSHL as the sole defect accounts for seventy percent or more of deafness cases, and different modes of inheritance have been observed. To date, 65 genes with more than 1000 discrete deafness-causing mutations have been identified [
1]. SHL comprises the remaining 30% of hearing loss cases, and it appears accompanied by other medical or physical findings. Similar to NSHL, SHL is associated with a growing list of causative genes and hearing loss syndromes [
2]. Thus, the discovery of a causative gene (mutation) of hereditary hearing loss is necessary to resolve the clinical and genetic heterogeneity of deafness.
Previously, most deafness genes (mutations) have been identified through traditional positional cloning (Sanger sequencing), which is an expensive and time-consuming process. More recently, next-generation sequencing, which is also known as massively parallel sequencing (MPS), has been introduced as an alternative approach to more traditional methods [
3]-[
5]. Whole-exome sequencing (WES) allows for the targeted enrichment and resequencing of nearly all exons of protein-coding genes and identifies genetic variation at a single base-pair resolution. Accordingly, WES uses next-generation technologies to provide a transformational approach for identifying causative mutations of Mendelian disorders. Different targeted genomic capture methods and MPS have been successfully applied to detect gene mutations in relatively small sets of deafness families [
6]-[
9]. In the present study, we performed targeted genomic capture and MPS to screen 104 genes and three microRNA regions that are known to be responsible for hereditary hearing loss in 23 unrelated probands of Chinese families with NSHL. We identified six causative variants in DFNA genes, including four novel mutations, and a novel combination of two monoallelic mutations in
GJB2 and
USH2A. This study provides a reliable strategy for the routine genetic diagnosis of hearing loss.
Discussion
In the present study, we confirmed the presence of seven non-synonymous variants in 7 of the 23 deafness families. Six families likely had causative mutations in DFNA genes [WFS1 (n = 2), ACTG1, POU4F3, COCH and TMC1], and one showed two monoallelic and most likely causative mutations in the GJB2 and USH2A genes, respectively. With the exception of the p.R653C mutation in WFS1 and the p.D572N mutation in TMC1, the other five variants have not been previously reported to be associated with hereditary hearing loss. The causative genes detected in the remaining 16 families require further examination, presumably by linkage analysis and/or WES. It is probable that hearing loss in these families is due to mutations in unidentified deafness-related genes. Alternatively, pathogenic mutations might exist in those regions not covered in our sequencing analysis, including intronic regulatory sequences.
In the JSNY-021 family, a novel in-frame indel mutation caused by c.2036_2038delAGG (p.E680del) was detected in
WFS1, which is responsible for DFNA6/14/38 hearing loss. This gene encodes wolframin, which is a membrane glycoprotein predominantly located in the endoplasmic reticulum (ER). This protein is essential for maintaining correct levels of Ca
2+ and other charged particles necessary for hearing, and its lack of function induces apoptotic input signaling in the ER [
16],[
17]. Although it remains unknown whether wolframin is expressed in the human cochlea, mutations in the
WFS1 gene, such as c.511G > A (p.D171N), c.2005 T > C (p.Y669H) and c.2590G > A (p.E864K), have been identified as frequent causes of autosomal dominant low-frequency hearing loss in different ethnicities [
18]-[
20]. The heterozygous 3-bp deletion (c.2036_2038delAGG) identified in the present study is expected to cause the loss of the E680 codon, which might affect the three-dimensional shape or properties of the wolframin protein and consequently interfere the normal function of the wolframin tetramer. Considering previous studies and the low-frequency NSHL phenotype found in the JSNY-021 family in this study, we hypothesize that this mutation likely has a pathogenic effect.
ACTG1 encodes cytoskeletal actin gamma 1, which is known to be the building block of hair cell stereocilia. These stereocilia are constantly undergoing actin polymerization at their tips and depolymerization at their bases [
21]. In auditory hair cells of the cochlea and intestinal epithelial cells, actin gamma 1 has a predominant and unique expression pattern. Mutations in the
ACTG1 gene have been mainly associated with autosomal dominant progressive sensorineural deafness 20/26 (DFNA20/26), and some have been linked to Baraitser-Winter syndrome, which is a rare autosomal recessive disorder characterized by developmental delay, facial dysmorphology, brain malformations, coloboma, and variable hearing loss [
22]-[
24]. To date, a total of 19
ACTG1 mutations have been reported in patients with DFNA 20/26 and Baraitser-Winter syndrome, of which 12 have been identified in NSHL families (
http://www.hgmd.org/, designed by P.D.Stenson HGMD®). In this study, we found the novel missense mutation p.E316K, which was caused by a c.946 G > A transition in
ACTG1, in the JSNY-043 family. p.E316K is located in subdomain 3 of actin gamma 1, which is a highly conserved actin domain of ACTG1. Until now, all reported
ACTG1 missense mutations have been located in this domain.
The POU4F3 protein is a well-known transcription factor encoded by
POU4F3. This protein belongs to the POU-domain class IV transcription factor family and plays an important role in the maturation, differentiation and survival of hair cells [
25]. Mutations in the
POU4F3 gene have been described in patients with nonsyndromic sensorineural deafness autosomal dominant type 15 (DFNA15). To date, more than five different mutation types, including the deletion of the entire
POU4F3 gene sequence, have been reported worldwide in different ethnic groups [
26],[
27], but none have been reported in the Asian population. In the present study, the
POU4F3 p.P164R missense mutation caused by c.491C > G was identified as a novel mutation in the JSNY-056 Chinese family.
The
COCH gene encodes a 550-amino acid protein with multiple domains, including a signal peptide (SP), an LCCL module and two von Willebrand factor A (vWFA) domains [
28]. The COCH protein, cochlin, is abundantly expressed in the cochlea and vestibular system of the inner ear. Mutations in the
COCH gene lead to autosomal dominant nonsyndromic sensorineural deafness 9 (DFNA9), which has been clinically characterized by progressive late-onset hearing loss with or without vestibular dysfunction [
29]. Presently, 18
COCH mutations have been identified in DFNA9 families (
http://www.hgmd.org/, designed by P.D.Stenson HGMD®), most of which are located in the LCCL region. Here, we identified a novel missense mutation (c.113G > A, p.G38D) in exon 3 of the
COCH gene, which is the LCCL domain of cochlin. Mutations in this domain are expected to cause misfolding and protein aggregation in a dominant-negative manner, leading to cytotoxicity of the inner ear fibrocytes [
30]. The clinical features associated with this novel mutation in the JSNY-027 family lends support to the pathogenic nature of the p.G38D variant.
In the present study, we also identified one novel mutation of
USH2A c.5051C > A (p.P1684L) in the JSNY-045 family.
USH2 mutations result in an autosomal recessive disorder characterized by retinitis pigmentosa and mild-to-moderate sensorineural hearing loss, and the
USH2A gene is most commonly mutated. This gene is located at 1q41 and encodes a protein with a predicted size of 171.5 kDa [
31],[
32]. To date, more than 120 different disease-causing mutations have been reported in the
USH2A gene, which are widely distributed over the coding regions of all 72 exons. Interestingly, the novel p.P1684L mutation, which is located in exon 25 of the
USH2A gene, was identified together with the
GJB2 c.235delC mutation in two deaf siblings of the JSNY-045 family, while their hearing parents each carried only one monogenic recessive mutation. All subjects exhibited normal vestibular and visual functions. We therefore deduced that the
USH2A monogenic recessive mutation failed to cause the Usher phenotype but may have contributed to the pathogenesis of the
GJB2 c.235delC mutation, resulting in phenotypic hearing loss in the two patients. In fact, digenic inheritance of nonsyndromic deafness caused by mutations in the
GJB2 gene and other connexin genes, such as
GJB3,
GJB6,
GJB4, or
GJA1, have been previously reported in several deaf patients [
33]-[
35]. In addition, a Japanese family with comorbid
GJB2 and
WFS1 mutations was also described in 2012. In this family, one individual with mutations in both
GJB2 and
WFS1 presented with a
GJB2 phenotype [
36]. Our results together with previous findings suggest that monoallelic
GJB2 mutations may contribute to NSHL as a result of their co-inheritance with other deafness-causing genes. However, the pathogenic mechanisms underlying the cooperation of these genes with
GJB2 require further research.
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Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XC and GX conceived and designed the study. QW, HZ, XQ, ZC, JY and YL performed the experiments and analysis. QW and HZ wrote the original manuscript. XC and GX contributed to revisions of the manuscript. All authors read and approved the final version.