Targeted High-Throughput Sequencing Identifies Pathogenic Mutations in KCNQ4 in Two Large Chinese Families with Autosomal Dominant Hearing Loss

Autosomal dominant non-syndromic hearing loss (ADNSHL) is highly heterogeneous, among them, KCNQ4 is one of the most frequent disease-causing genes. More than twenty KCNQ4 mutations have been reported, but none of them were detected in Chinese mainland families. In this study, we identified a novel KCNQ4 mutation in a five generation Chinese family with 84 members and a known KCNQ4 mutation in a six generation Chinese family with 66 members. Mutation screening of 30 genes for ADNSHL was performed in the probands from thirty large Chinese families with ADNSHL by targeted region capture and high-throughput sequencing. The candidate variants and the co-segregation of the phenotype were verified by polymerase chain reaction (PCR) amplification and Sanger sequencing in all ascertained family members. Then we identified a novel KCNQ4 mutation p.W275R in exon 5 and a known KCNQ4 mutation p.G285S in exon 6 in two large Chinese ADNSHL families segregating with post-lingual high frequency-involved and progressive sensorineural hearing loss. This is the first report of KCNQ4 mutation in Chinese mainland families. KCNQ4, a member of voltage-gated potassium channel family, is likely to be a common gene in Chinese patients with ADNSHL. The results also support that the combination of targeted enrichment and high-throughput sequencing is a valuable molecular diagnostic tool for autosomal dominant hereditary deafness.


Introduction
Hereditary hearing loss can be inherited in many patterns, such as autosomal dominant audosomal recessive, X-linked dominant, X-linked recessive, Y-linked pattern, among which ADNSHL has strikingly genetic heterogeneity. To date, more than 60 loci for ADNSHL have been mapped and only 30 corresponding genes have been identified (http://hereditaryhearingloss.org). During the past twenty years, linkage analysis and candidate gene sequencing has been proved to be a powerful tool to identify responsible genes for ADNSHL. However, limited number of samples in the clinical part, the large number of genes in the mapped region, and the large size of many genes restrained the application of this method. Recently, high-throughput sequencing, also known as next-generation sequencing (NGS) has been proved to be an ideal tool to decipher the genetic heterogeneity of deafness. More than ten deafness genes have been identified using NGS including TPRN, GPSM2, HSD17B4, MASP1, CACAM1, HARS2, SMPX, DNMT1, ABHD12, TSPEAR, TNC and P2RX2 [1,2,3].
Among ADNSHL genes, KCNQ4 (MIM*600101), one of the most frequent genes [4], was firstly identified as the causal gene for ADNSHL at DFNA2 by Kubisch and colleagues [5]. As a member of voltage-gated potassium channel family, KCNQ4 plays a crucial role in potassium recycling in the inner ear. KCNQ4 has six predicted transmembrane domains encoded by six exons (exon 2 to 7) and a P-loop between transmembrane domains S5 and S6. The P-loop domain forms a channel pore, containing a potassium ion-selective filter, whose function is eliminated by mutations in the pore region [5]. To date, 20 mutations in KCNQ4 have been reported (Table 1) and it is identified as a common gene with a frequency up to 6.62% in ADNSHL in Japan, predicted to be the most prevalent gene responsible for Japanese ADNSHL patients [6]. However, these mutations have not been found in Chinese mainland populations before this study. Almost all reported cases showed a similar phenotype characterized by post-lingual, progressive, high-frequency hearing impairment (one mid-frequency predominant hearing loss caused by the p.V230E mutation was reported) [6].
Recently, we performed targeted capture and NGS to analyze a cohort of 30 hearing loss probands from Chinese families with ADNSHL (data not shown). Among these families, we identified a novel KCNQ4 mutation p.W275R in exon 5 in family 025 and a known KCNQ4 mutation p.G285S in exon 6 in family 727.

Ethics Statement
The study was approved by the Committee of Medical Ethics of Chinese People's Liberation Army (PLA) General Hospital. We obtained written informed consents from all the participants in this study. Written informed consents were obtained from the next of kin on the behalf of the minors/children participants involved in this study.

Family Recruitment and Clinical Evaluations
A six-generation family (025) with 66 members segregating ADNSHL and a five-generation family (727) with 84 members segregating ADNSHL were ascertained from the Department of Otolaryngology, Head and Neck Surgery, at the Institute of Otolaryngology of PLA, Chinese PLA General Hospital (Figure 1A, 1B). Either personal or family medical evidence of hearing loss, tinnitus, vestibular symptoms, use of aminoglycosides, and other clinical abnormalities of the participants were identified by a team of experienced doctors and audiologists. Audiometric evaluations included pure tone audiometry, auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE). High resolution computed tomography (HRCT) was also performed on some subjects to verify whether the family members had other complications other than hearing disorders.

Targeted capture and NGS
Genomic DNA (gDNA) was extracted from the whole blood samples using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China), and 1 ug of purified gDNA fragmented to 200-300 base pairs using an ultrasonoscope (Covaris S2, Massachusetts, USA). End-repair, adenylation and adapter ligation were performed for library preparation following the Illumina's protocol. The same amount of library were pooled then hybridized to the customized capture array (NimbleGen, Roche) including exons, splicing sites and immediate flanking intron sequences of 29 genes for non-syndromic autosomal dominant hearing loss and TNC, a novel causative gene for ADNSHL identified in our previous research (Table S1). Sequencing was carried out on Illumina HiSeq2000 to generate paired end reads (90 bps at each end) [7].
Raw image files were processed by Illumina Pipeline (version 1.3.4) for base-calling with default parameters. Reads were aligned     to NCBI37/hg19 assembly using the BWA (Burrows Wheeler Aligner). SNPs and indels (inserts and deletions) were detected using the GATK software [8].

Sanger sequencing
After filtering against multiple databases, sanger sequencing was used in all available members from family 025 and 727 to determine whether the potential mutations in causative genes cosegregated with the disease phenotype in these families or not. Direct PCR products were sequenced using Bigdye terminator v3.1 cycle sequencing kits (Applied Biosystems. Foster City, CA) and analyzed using a ABI 3700XL Genetic Analyzer.

Clinical description
For family 727, a total of 41 family members, composed of 15 clinical affected and 26 unaffected individuals were ascertained in this study. While in family 025, 11 patients and 26 control individuals were ascertained. Age of onset in family 727 ranged from 5 to 30 years old, with average onset age 13.08 years old, while in 025 the onset age ranged from 2 to 30 years old, with average onset age 16.45 years old. For both two autosomal families, affected members showed a post-lingual, symmetrical, and bilateral non-syndromic sensorineural hearing loss. The hearing loss was initially presented as high frequencies with subsequent gradual progression to severe level involving all frequencies at later ages. Some patients had associated tinnitus, but no vestibular symptoms or signs were reported (Table 2,  Table 3, Figure 1A, 1B, Figure 2). High resolution computed tomography (HRCT) of the temporal bone in the probands showed normal middle ears structure, including normal vestibular aqueduct and internal auditory canal. None of the affected members had a history of exposure to aminoglycosides, noise, or other causes that may account for the hearing impairment.

Targeted high-throughput sequencing
Approximately 133K bp of exons and adjacent intronic regions of the 30 genes known to be responsible for ADNSHL were captured and sequenced. The average sequencing depth for target region is about 4156, and 99.44% of the average coverage for targeted region is more than 206, which is satisfied with the requirements for calling SNPs and InDels (Table 4). For the proband of family 025, a total of 484 variants were identified, 47 of which were nonsynonymous variants, splice acceptor and donor site mutations and coding indels that were more likely to be pathogenic mutations; only 3 of the 47 variants were with the allele frequency which were less than 0.01 in dbSNP137, HapMap, 1000 human genome and local dataset; and a missense variant, c.823T.C (p.W275R) in exon5 of KCNQ4 (NM_004700), predicted to be ''Damaging'', ''Probably Damaging'', ''Deleterious'', ''Disease_causing'', ''Conserved'' and ''Conserved'' by SIFT, Polyphen2, LRT, Mutation Taster, GERP++, and PhyloP respectively (Tables 5 and 6). For this site, 45% (114/254) reads supported for C vs. 55% (140/254) reads supported for T, which means it is a heterozygote (Het) ( Figure S1). This indicated that this novel mutation may be the cause of the hearing loss in this Chinese family. In the proband of family 727, another missense mutation, c.853G.A (p.G285S) in KCNQ4, a previously reported mutation (rs28937588) was identified ( Figure S2).

Mutation detection and analysis
Sanger sequencing confirmed the co-segregation of p.W275R and p.G285S with the disease phenotype in Family 025 and 727 respectively ( Figure 1C and D and E). The two mutations were not detected in other 28 probands from Chinese families with ADNSHL. Both of the mutations occurred at highly conserved amino acids ( Figure 1F), and are predicted to be deleterious by the SIFT, Polyphen2, LRT, Mutation Taster, GERP++, and PhyloP programs (IS IT NOT IDENTICAL TO WHAT HAVE MENTIONNED JUST ABOVE (Table 6). Based on these results and the phenotypes of these two families, we concluded that these two mutations in the KCNQ4 are responsible for the hearing loss in the family 025 and 727.

Discussion
As is known, mutations in gene KCNQ4 have been associated with ADNSHL, recognized as one of the most frequent causes of ADNSHL, is characterized by post-lingual autosomal dominant non-syndromic progressive sensorineural hearing loss, first affected the high frequencies according to GeneReviews. In this present study, we identified a known mutation and a novel mutation in the P-loop region of the KCNQ4 potassium channel which yielded dominant non-syndromic hearing loss by high-throughput sequencing as well as conventional genetic testing. It is noteworthy that this is the first report of heterozygous mutations in the KCNQ4 genes as a cause of ADNSHL in Chinese mainland families. In family 727, the G285S mutation in exon6 affects the first glycine in the GYG signature sequence of K+ channel pores, which has been identified in three affected members of a small French family with DFNA2. This mutation exert a strong dominant-negative effect on the wild type and render heteromeric channels nonfunctional [5]. By using adenoviral delivery of KCNQ4 channels carrying G285S, Holt JR and colleagues demonstrated that KCNQ4 channels contributed to the M-like conductances: G K,n of the cochlear outer hair cells and G K,L of the vestibular type I hair cells [9]. As far as we know, this is the second report about this mutation. Recurrent mutations in KCNQ4 also include c.211delC (p.Q71fs), c.821T.A (p.L274H), c.827G.C (p.W276S), having been reported in more than one family. Among these recurrent mutations, mutation W276S is a hot spot mutation in Belgian, Dutch and Japanese families [10]. Together with specific audiogram configuration, recurrent mutations may promote genetic testing for ADNSHL with a particular phenotype [6]. In family 025, we found a novel missense mutation W275R in exon 5 of KCNQ4. The mutation c.823T.C (W275R) is immediately adjacent to the previously reported mutation c.827G.C (W276S). These two adjacent tryptophan residues, located with the pore helix, are highly conserved across different potassium channel families and play a important role in K + channel function, presumably holding the pore open at a correct diameter [11]. Mutations in these conserved tryptophan residues result in a complete loss of function of K + channel [12]. Previous studies have demonstrated that the W276S mutation lead to a dramatic decrease in KCNQ4 surface expression with strong dominant-negative effects on the wild type (WT) KCNQ4 subunit. We presume that the W275R mutation may have the similar functional mechanism [13,14].
It is noteworthy that this is the first report of KCNQ4 mutation in Chinese mainland families. Before this report, there was a report of KCNQ4 missense mutation P182L in a Taiwan family. The mutation locates in the S3 domain of KCNQ4. However, it is not conserved in all KCNQ family and unlikely to be pathogenic according to some prediction program, such as SIFT, Polyphen Phylop, LRT, etc. In this study, we found a P182L mutation in one of the 30 probands from ADNSHL families which did not cosegregate with the disease phenotype in the family members. It was also found in a Japanese control sample with normal hearing [4,15].
To date, 16 missense mutations and 4 indels in KCNQ4 have been reported (Table 1). Phenotype-genotype correlation of KCNQ4 has been summarized, most of the patients with missense mutation are younger-onset and pure all-frequency hearing loss, while patients with deletion mutations are later-onset and pure high-frequency hearing loss, so the two families in this study are Table 6. A novel variant in the proband of family 025.  [16,17]. KCNQ4 is also predicted to be a candidate gene for agerelated hearing loss (ARHI) since KCNQ4 mutation families have similar pattern of hearing loss with ARHI, especially a unique pattern of hearing loss with striking resemblance to ARHI, in which only the high frequencies were progressively affected while the lower frequencies remained intact until an older age [18]. To investigate the association of KCNQ4 with ARHI, Van Eyken et al. examined KCNQ4 and detected a significant association between KCNQ4 and ARHI in two independent Caucasian populations. All SNPs are located in the same 13-kb region in the middle of the KCNQ4 gene, indicating that the pathogenic variants for ARHI may locate in this region [19].
KCNQ4 is likely to be a common gene in Chinese ADNSHL: i) KCNQ4 is one of the more frequent genes in ADNSHL in comparison to the other reported genes [4]; ii) In a large Japanese cohort, KCNQ4 is found to be the most prevalent gene responsible for Japanese ADNSHL. In their study, 19 families with 7 different mutations were identified in 287 probands from ADNSHL families [6]. Mutations of KCNQ4 were also found in other east Asian region, such as Korea and other Japanese population (Table 1). iii) Our group found a copy variation of 47 base pairs insertion or deletion in the exon2 and exon3 intron sequence, supposing to be a specific marker for the hearing loss of the pedigree [20]. Then we performed mutation screening of KCNQ4 in 71 patients with high frequency hearing loss and 40 unaffected individuals of matched geographical ancestry, and found the deletion of the second intron 47 bp in 5 patients as well as 2 males with normal hearing, the insertion of 47 bp in 11 patients [21].
Despite an increasing number of pathogenic KCNQ4 mutations have been identified, the molecular aetiology of DFNA2 still unknown. The missense mutations are believed to exert a dominant-negative effect by interfering with the normal channel subunit. The two deletions, c.211delC and c.211_223del13, are proposed to exert a pathogenic effect through haploinsufficiency [3]. In the mouse models, loss of KCNQ4 function leads to progressive sensorineural hearing loss, paralleled by selective degeneration of outer hair cells and spiral ganglion neurons [22]. Among the missense mutations, L274H, W276S, L281S, G285S, G296S and G321S are loss of function mutations and eletrophysiological studies have shown that these mutations lead to loss of KCNQ4 currents [5,13,22,23,24]. However, the molecular mechanisms about how these mutations lead the loss of KCNQ4 currents remain unknown. Recently Gao YH et al. reported two mechanisms underlying DFNA2, the decreased cell surface expression detected by immunofluorescent microscopy and Western blot and the impaired conductance of KCNQ4 demonstrated by electrophysiological studies [14]. Because of the restriction of the lack of the understanding of the molecular aetiology, no therapeutic methods to prevent progressive hearing loss are available for now. Further functional studies regarding mutations in these residues in KCNQ4 may help clarify the molecular mechanism, which in turn, will facilitate informative genetic counseling, early diagnosis and even treatment of hearing impairment [25]. It is anticipated that future management of these genetic hearing disorders will be more targeted to the cellular processes involved and improve the likelihood of hearing recovery.
In conclusion, we have shown two mutations in Chinese ADNSHL families using targeted high-throughput sequencing. This is the first report of KCNQ4 mutation in Chinese mainland families, providing more information for discovering the molecular mechanism of KCNQ4 mutation-induced hearing loss. The results also support that the combination of targeted capture and NGS is a valuable molecular diagnostic tool for autosomal dominant hereditary deafness.  File S1 Data S1-S4. Data S1. Next generation sequencing results of proband of family 025 (including all SNP and INDEL results). Data S2. Next generation sequencing results of proband of family 727 (including all SNP and INDEL results). Data S3. Sanger sequencing results of family 025. This file shows the sanger sequencing results of 36 members ascertained in this study. Data S4. Sanger sequencing results of family 727. This file shows the sanger sequencing results of 41 members ascertained in this study. (RAR)