Research ArticleMutation in the Hair Cell Specific Gene POU4F3 Isa Common Cause for Autosomal Dominant NonsyndromicHearing Loss in Chinese Hans
Longxia He123 Xiuhong Pang4 Penghui Chen123 Hao Wu1235 and Tao Yang123
1Department of Otolaryngology-Head and Neck Surgery Xinhua Hospital Shanghai Jiaotong University School of MedicineShanghai China2Ear Institute Shanghai Jiaotong University School of Medicine Shanghai China3Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases Shanghai China4Department of Otorhinolaryngology-Head and Neck Surgery Taizhou Peoplersquos Hospital Jiangsu Province China5Department of Otorhinolaryngology-Head and Neck Surgery Shanghai Ninth Peoplersquos HospitalShanghai Jiaotong University School of Medicine Shanghai China
Correspondence should be addressed to Hao Wu wuhao622sinacn and Tao Yang yangtfxlsinacom
Received 8 September 2016 Revised 26 October 2016 Accepted 2 November 2016
Academic Editor Renjie Chai
Copyright copy 2016 Longxia He et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Autosomal dominant nonsyndromic hearing loss (ADNSHL) is extremely heterogeneous So far the genetic etiological contributionof the gene POU4F3 associated with ADNSHL has been rarely reported In our previous study a c603 604delGG mutation in thehair cell specific gene POU4F3 has been identified as the pathogenic cause in one of the seven Chinese Han ADNSHL families Inthe present study we performed targeted next-generation sequencing of 144 known deafness genes in another nine Chinese HanADNSHL families and identified two more novel mutations in POU4F3 pLeu311Pro and c120+1GgtC as the pathogenic causeClinical characterization of the affected individuals in these three families showed that the three POU4F3 mutations may lead toprogressive hearing loss with variable ages of onset and degrees of severity Our results suggested that mutations in POU4F3 are arelatively common cause (316) for ADNSHL in Chinese Hans which should be routinely screened in such cases during genetictesting
1 Introduction
Hearing loss is one of the most common sensorineuraldefects which may result from a great variety of genetic andenvironmental factors Based on the inheritance patterns thegenetic hearing loss can be classified into autosomal recessiveautosomal dominant and X-linkedmitochondrial account-ing for approximately 80 15 and less than 5 of nonsyn-dromic hearing loss respectively [1] Both autosomal dom-inant (ADNSHL) and autosomal recessive (ARNSHL) non-syndromic hearing loss have an extremely high degree of het-erogeneity For the former 35 causative genes and over 60 locihave been reported for ADNSHL (The hereditary HearingLoss Homepage httphereditaryhearinglossorg) So farmutations in most ADNSHL genes were reported based on
studies of the individual cases or families The etiologicalcontribution of the ADNSHL gene POU4F3 has been rarelystudied In recent years however the development of next-generation sequencing (NGS) has complemented the tradi-tional Sanger sequencing method and made it possible toscreen all deafness-associated genes in a high throughoutmanner [2ndash4]
Mutations in POU4F3 have been reported to lead toADNSHL named as DFNA15 [5] This gene encodes a 338amino acidsrsquo POU family transcription factor with two con-servedDNA-binding domains (the POU-specific domain andthe POU homeodomain amino acids 179minus256 and 274minus333resp) In mouse inner ear Pou4f3 is strongly expressed inboth inner and outer hair cells [6ndash9] It activates the tran-scription of a downstream target gene Gfi1 whose expression
Hindawi Publishing CorporationNeural PlasticityVolume 2016 Article ID 9890827 6 pageshttpdxdoiorg10115520169890827
2 Neural Plasticity
is required for the maintenance of the outer hair cells [10] Todate only eleven different mutations in POU4F3 have beenreported in Israeli Jewish Dutch Korean Japanese and Chi-nese ADNSHL families [5 11ndash17] The hearing loss caused byPOU4F3mutationswas highly variable in the age of onset theprogression course and the severity of hearing impairment
In the previous (119899 = 7) and the present (119899 = 9) studies[18] we preformed targeted NGS of known deafness genes in16 ChineseHanADNSHL families and identified novelmuta-tions in POU4F3 as the pathogenic cause in three of themCharacterization of the hearing phenotype was performed inthe affected family members of these three families in thepresent study Our results expanded the mutation spectrumof DFNA15 and suggested that mutations in POU4F3 are arelatively common cause for ADNSHL in Chinese Hans
2 Materials and Methods
21 Subjects Probands of sixteen Chinese Han families seg-regating ADNSHL were recruited through Xinhua Hospi-tal Shanghai China The pedigrees of the families wereshown in Figure 1(a) and Supplementary Figure S1 in Supple-mentaryMaterial available online at httpdxdoiorg10115520169890827 For Families P1748 PD6 and P59 in whichmutations in POU4F3were identified 8 10 and 13 additionalfamily members were subsequently recruited respectivelyInformed consent was obtained from all subjects This studywas approved by the Ethics Committee of Xinhua HospitalShanghai Jiaotong University School of Medicine ShanghaiChina
22 Clinical Evaluations A detailed physical and medicalhistory examination was performed in all probands of theADNSHL familiesThe hearing levels were measured by puretone audiometry (PTA) and shown as the average thresholdsof 05 1 2 and 4 kHz from the better ear The hearing levelswere classified as normal (lt20 dB) mild (20ndash40 dB) moder-ate (41ndash70 dB) severe (71ndash90 dB) and profound (gt90 dB)
23 Mutation Analysis Targeted NGS of 144 known deaf-ness genes (see complete list in Supplementary Table S1) wasperformed using the MyGenotics gene enrichment system(MyGenotics Boston MD USA) and the Illumina HiSeq2000 sequencer (Illumina SanDiego CAUSA) as previouslydescribed [18] The raw data were first analyzed to filter outthe low quality reads NCBI37hg19 assembly was used as thereference sequences Nonsynonymous on-target variantswith maximumminor allele frequency (MAF) less than 0001in public databases NHLBI Exome Sequencing Project (ESPhttpevsgswashingtoneduEVS) and the Exome Aggrega-tion Consortium (ExAC httpexacbroadinstituteorg)were considered as the candidate pathogenic mutations incompliance with the ADNSHL inheritance The pathogeni-city of the candidate variants was predicted by computationalprograms Mutation Taster (httpwwwmutationtasterorg)PolyPhen-2 (httpgeneticsbwhharvardedupph2) PRO-VEAN and SIFT (httpsiftjcviorg cutoff scores set at minus25and 005 resp) Cosegregation of the pathogenic mutations
and the hearing phenotype was verified in members of Fami-lies P1748 and PD6 by Sanger sequencing
3 Results
31 POU4F3 Mutations Identified in the ADNSHL FamiliesIn our previous studies of 7 Chinese Han ADNSHL familiesa c603 604delGG (pLeu201fslowast12) mutation in POU4F3 hasbeen identified by targeted NGS as the pathogenic causein Family P59 [18] In the present study we performed acomprehensive mutation screening in probands of anothernine Chinese Han ADNSHL families (marked in asterisks inFigure 1(a) and Supplementary Figure S1) by targeted NGS of144 known deafness genes Interestingly two novel heterozy-gous variants pLeu311Pro (c932TgtC) and c120+1GgtC inPOU4F3were identified as the candidate pathogenic variantsin probands P1748-III-1 and PD6-IV-1 respectively (Table 1)alongwith a heterozygousTECTA pVal1830Met and a hetero-zygous TMC1 pSer697X variant in proband P1748-1 (Table 1)Sanger sequencing in the rest of the family members con-firmed that pLeu311Pro and c120+1GgtC were the only twocandidate variants segregating with the hearing phenotypein Families P1748 and PD6 (Figures 1(a) and 1(b)) while theTECTA pVal1830Met and the TMC1 pSer697X variants werenot seen in any other affected family members of P1748 ThepLeu311Pro (c932TgtC) and c120+1GgtC in POU4F3 werenot reported in previous studies not present in the NHLBIESP (119899 = 6503) and ExAC (119899 = 60706) database andnot seen in 300 Chinese Han normal hearing controls Thec120+1GgtC mutation was predicted to abolish the 51015840 splicesite of introns 1 of POU4F3 (Figure 2(a)) The pLeu311Promutation substituted a conserved amino acid Leu311 (Fig-ure 2(b)) and was predicted to be deleterious by computa-tional programs Mutation Taster PolyPhen-2 PROVEANand SIFT (Table 1)
32 Clinical Characteristics of the Three ADNSHL Familieswith POU4F3 Mutations Based on the audiograms andthe self-description of the affected individuals with c603604delGG pLeu311Pro and c120+1GgtC mutations inPOU4F3 the hearing loss associated with the POU4F3muta-tionswas typically progressivewith considerable variability inages of onset and degree of severity both interfamilially andintrafamilially In Family P1748 with the pLeu311Pro muta-tion proband III-1 had notable hearing loss since age 10 yearsThe hearing loss affected high frequency most and graduallyprogressed to moderate hearing loss at age 29 years (Fig-ure 1(c))The affected individuals in the second generation ofFamily P1748 had hearing loss at the onset age of 10 years to 20years and all eventually progressed to profound after age 50years Interestingly all six female patients in the second gen-eration had significantly decreased hearing levels after givingbirth to their children In Family PD6 with the c120+1GgtCmutation proband IV-1 had congenital moderate hearingloss with a relatively ldquoflatrdquo audiometric profile at age 23 yearsaffecting all frequencies (Figure 1(c)) Other affected individ-uals in this family demonstrated progressive moderate-to-profound deafness depending on their agesThe ages of onset
Neural Plasticity 3
POU4F3 pLeu311Pro (c932TgtC)Family P1748lowast
II-1 II-1 II-2
III-1
IV-1
III-2 III-3
II-3II-2 II-3 II-4 II-5 II-6 II-7
III-4 III-5 III-6 III-7
III-1 III-2
POU4F3 c120+1GgtCFamily PD6lowast
minus+
minus+ minus+
minus+ minus+ minus+ minus+ minus+minus+
minus+ minus+ minus+
minus+
minus+ minus+
minus+
++
++
++
++
(a)
POU4F3 c932TgtCFamily P1748T T T TC
C C C C
C C C CG G G G GA
T T T TC G G G G GA A A A A A A A A A
T
T T T
T TC
C C C C C C
C C C C CG
G G G G G G G G
G G G G G GA
A A A A
A A AA A A A A A A AA T
T
C
POU4F3 wild type POU4F3 wild type
POU4F3 c120+1GgtCFamily PD6
(b)
0102030405060708090
100110120
Hea
ring
leve
l (dB
)
Frequency (Hz)250 2K1K500
P1748-III-1PD6-IV-1P59-III-8(54 yo)
(23 yo)(29 yo)
(c)
Figure 1 POU4F3 mutations identified in the Chinese Han ADNSHL families (a) Pedigrees and genotypes of the families with POU4F3mutations Probandswere pointed by arrowsminus and+ indicate themutant andwild type alleles respectively Asterisks indicate the familieswithPOU4F3mutations identified in the present study (b) Chromatograms showing the c932TgtC (pLeu311Pro) and the c120+1GgtC mutationsin POU4F3 (c) Audiograms of the probands of the three families
4 Neural Plasticity
Table 1 Candidate pathogenic mutations identified in probands of Families P1748 and PD6 by targeted NGS
Proband Gene (referencesequence) Mutation MAF
(ExAC)
MAF(NHLBIESP)
MutationTaster
PROVEAN(score) SIFT (score)
PolyPhen-2(HumVarscore)
Intrafamilialphenotype
cosegregation
P1748-III-1
POU4F3(NM 002700)
pLeu311Pro(c932TgtC) 0 0 Disease
causingDeleterious(minus363)
Damaging(0)
Probablydamaging (1) Yes
TECTA(NM 005422)
pVal1830Met(c5488GgtA) 00003871 0 Disease
causingNeutral(minus083)
Damaging(0008)
Probablydamaging(0969)
No
TMC1(NM 138691)
pSer697X(c2090CgtG) 0 0 Disease
causingDeleterious(minus1026) mdash mdash No
PD6-IV-1 POU4F3(NM 002700) c120+1GgtC 0 0 Disease
causing mdash mdash mdash Yes
pLeu223ProDutch
c662_675del14Korean
pGlu232LysKorean
c880_887del8Israeli Jewish
pLeu289PheDutch
Chinese
pLeu311ProChinese
Chinesec1007delC
Japanese
Exon1 Exon2
pArg326LysKorean
POU-specific domain POU homeodomain
pPro164ArgChinese
pPro246ThrJapanese
c602delTChinese
lowast
c120+1GgtClowast c603_604delGGlowast
(a)
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
Leu311
Xenopus laevis
Danio rerio
Mus musculus
Pan troglodytes
Homo sapiens
(b)
Figure 2 Summary and conservation of the POU4F3 mutations (a) Schematic illustration of the thirteen reported POU4F3 mutationsassociated with DFNA15 Asterisks indicated the mutations reported in the present study (b) Multispecies sequence alignment showing theevolutionary conserved amino acid Leu311
ranged from congenital to 40 years In Family P59 withthe c603 604delGG mutation the proband III-8 had onlymoderate hearing loss at 54 years of age (Figure 1(c)) andall affected individuals in this family had a rather late ageof onset around 40s For other auditory symptoms tinnitushas been complained by proband P1748-III-1 No vestibulardysfunction was shown in any affected individuals
4 Discussion
Combined with our present and previous studies we iden-tified mutations in POU4F3 as the pathogenic cause ofdeafness in 3 of the 16 (18) Chinese Han ADNSHL familiessuggesting that it is a relatively common cause for ADNSHLin Chinese Hans Consistently seven of the ten previously
Neural Plasticity 5
reportedPOU4F3mutations fromother research groupswerealso from the East Asians (three in Korean two in Japaneseand two in Chinese Figure 2(a)) suggesting that this geneshould be routinely screened in ADNSHL cases of East Asiandescent In contrast only three (one in Israeli Jewish and twoin Dutch Figure 2(a)) POU4F3 mutations were previouslyreported from regions other thanEastAsiaThedistinguishedmutation spectrum among different ethnical groups has alsobeen reported for other deafness genes such as SLC26A4 inwhich case biallelic SLC26A4 mutations can be identified in884 of deaf patients with nonsyndromic EVA in Chinesebut only 15 in Caucasians [19 20]
Our study also expanded the mutation spectrum ofPOU4F3 Figure 2(a) summarized the type position andassociated ethnicity of the thirteen POU4F3 mutationsreported to date Four of them including the c603 604delGG(pLeu201fslowast12) mutation reported in our previous studywere truncating mutations that were predicted to lead toprematurely stopped protein product or nonsense-mediateddecay of themRNA while another seven POU4F3mutationsincluding the pLeu311Pro mutation reported in the presentstudy were missense mutations leading to single amino acidsubstitutionsNotably these twelvemutationswere all locatedwithin or close to the POU-specific domain or the POUhomeodomain the two conserved DNA-binding domains ofPOU4F3 encoded in exon 2 On the contrary the c120+1GgtCmutation identified in the present study is the only reportedmutation outside exon 2 of POU4F3
Consistent with previous reports [5 11 13ndash16] thePOU4F3mutations identified in the present study were asso-ciated with progressive hearing loss with considerable vari-ability in the ages of onset and the degrees of severity andthis variable hearing phenotype can be seen both interfa-milially and intrafamilially Apparently no simple genotype-phenotype correlation can be drawn based on the positionor the truncatingnontruncating nature of the POU4F3muta-tions In a previous study of the Pou4f3 mutant deaf micedeficiency of Pou4f3 has been found to result in reducedexpression of its hair cell specific downstream target Gfi1which was suggested as the direct cause of the outer hair celldegeneration in the Pou4f3mutant mice [10] In future stud-ies therefore it will be interesting to correlate the presumablyreduced levels of theGfi1 transcriptionwith differentPOU4F3mutations and the severity of the associated hearing loss
5 Conclusions
Mutations in POU4F3 are a relatively common cause forADNSHL in Chinese Hans The hearing loss associated withPOU4F3mutations has considerable variability in the ages ofonset and the degrees of severity
Competing Interests
The authors declare no competing financial interests
Authorsrsquo Contributions
Longxia He Xiuhong Pang and Penghui Chen contributedequally to this work
Acknowledgments
This research was supported by grants from National Sci-ence Foundation of China (81570930 and 81371101 to TaoYang 81330023 to Hao Wu) Shanghai Municipal Scienceand Technology Commission (14DZ1940102 to Tao Yang14DZ2260300 and 14DJ1400201 to Hao Wu) and ShanghaiMunicipal Education Commission-Gaofeng Clinical Medi-cine Grant (20152519 to Tao Yang)
References
[1] R J H Smith A E Shearer M S Hildebrand and G VanCamp ldquoDeafness and hereditary hearing loss overviewrdquo inGeneReviews R A Pagon M P Adam H H Ardinger et alEds University of Washington Seattle Wash USA 1993
[2] A E Shearer A P DeLuca M S Hildebrand et al ldquoCompre-hensive genetic testing for hereditary hearing loss using massi-vely parallel sequencingrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 49 pp21104ndash21109 2010
[3] A E Shearer and R J H Smith ldquoGenetics advances in genetictesting for deafnessrdquo Current Opinion in Pediatrics vol 24 no6 pp 679ndash686 2012
[4] Z Brownstein Y Bhonker and K B Avraham ldquoHigh-through-put sequencing to decipher the genetic heterogeneity of deaf-nessrdquo Genome Biology vol 13 article 245 2012
[5] O Vahava R Morell E D Lynch et al ldquoMutation in transcrip-tion factor POU4F3 associated with inherited progressive hear-ing loss in humansrdquo Science vol 279 no 5358 pp 1950ndash19541998
[6] L Erkman R J McEvilly L Luo et al ldquoRole of transcriptionfactors Brn-31 and Brn-32 in auditory and visual systemdevelopmentrdquo Nature vol 381 no 6583 pp 603ndash606 1996
[7] S W Wang X Mu W J Bowers et al ldquoBrn3bBrn3c doubleknockout mice reveal an unsuspected role for Brn3c in retinalganglion cell axon outgrowthrdquo Development vol 129 no 2 pp467ndash477 2002
[8] M Xiang L Gan D Li et al ldquoEssential role of POU-domainfactor Brn-3c in auditory and vestibular hair cell developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 17 pp 9445ndash9450 1997
[9] MXiangW-QGao THasson and J J Shin ldquoRequirement forBrn-3c inmaturation and survival but not in fate determinationof inner ear hair cellsrdquo Development vol 125 no 20 pp 3935ndash3946 1998
[10] R Hertzano M Montcouquiol S Rashi-Elkeles et al ldquoTran-scription profiling of inner ears from Pou4f3119889119889119897119889119889119897 identifiesGfi1 as a target of the Pou4f3 deafness generdquo Human MolecularGenetics vol 13 no 18 pp 2143ndash2153 2004
[11] R W J Collin R Chellappa R-J Pauw et al ldquoMissense muta-tions in POU4F3 cause autosomal dominant hearing impair-ment DFNA15 and affect subcellular localization and DNAbindingrdquo Human Mutation vol 29 no 4 pp 545ndash554 2008
6 Neural Plasticity
[12] HMutai N Suzuki A Shimizu et al ldquoDiverse spectrumof raredeafness genes underlies early-childhood hearing loss in Japa-nese patients a cross-sectional multi-center next-generationsequencing studyrdquo Orphanet Journal of Rare Diseases vol 8article 172 2013
[13] H-J Kim H-H Won K-J Park et al ldquoSNP linkage analysisand whole exome sequencing identify a novel POU4F3 muta-tion in autosomal dominant late-onset nonsyndromic hearingloss (DFNA15)rdquo PLoS ONE vol 8 no 11 Article ID e790632013
[14] H K Lee H J Park K Y Lee R Park and U K Kim ldquoA novelframeshiftmutation of POU4F3 gene associatedwith autosomaldominant non-syndromic hearing lossrdquo Biochemical and Bio-physical Research Communications vol 396 no 3 pp 626ndash6302010
[15] X Z Cai Y Li L Xia et al ldquoExome sequencing identifiesPOU4F3 as the causative gene for a large Chinese family withnon-syndromic hearing lossrdquo Journal of Human Genetics 2016
[16] Q Wei H Zhu X Qian et al ldquoTargeted genomic capture andmassively parallel sequencing to identify novel variants caus-ing Chinese hereditary hearing lossrdquo Journal of TranslationalMedicine vol 12 article 311 2014
[17] M Miyagawa T Naito S-Y Nishio N Kamatani and S-IUsami ldquoTargeted exon sequencing successfully discovers rarecausative genes and clarifies the molecular epidemiology ofJapanese deafness patientsrdquo PLoS ONE vol 8 no 8 Article IDe71381 2013
[18] T Yang X Wei Y Chai L Li and H Wu ldquoGenetic etiologystudy of the non-syndromic deafness in Chinese Hans by tar-geted next-generation sequencingrdquo Orphanet Journal of RareDiseases vol 8 no 1 article 85 2013
[19] Q-J Wang Y-L Zhao A Q Rao et al ldquoA distinct spectrumof SLC26A4 mutations in patients with enlarged vestibularaqueduct in ChinardquoClinical Genetics vol 72 no 3 pp 245ndash2542007
[20] T Yang H Vidarsson S Rodrigo-Blomqvist et al ldquoTranscrip-tional control of SLC26A4 is involved in Pendred syndrome andnonsyndromic enlargement of vestibular aqueduct (DFNB4)rdquoAmerican Journal of Human Genetics vol 80 no 6 pp 1055ndash1063 2007
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
2 Neural Plasticity
is required for the maintenance of the outer hair cells [10] Todate only eleven different mutations in POU4F3 have beenreported in Israeli Jewish Dutch Korean Japanese and Chi-nese ADNSHL families [5 11ndash17] The hearing loss caused byPOU4F3mutationswas highly variable in the age of onset theprogression course and the severity of hearing impairment
In the previous (119899 = 7) and the present (119899 = 9) studies[18] we preformed targeted NGS of known deafness genes in16 ChineseHanADNSHL families and identified novelmuta-tions in POU4F3 as the pathogenic cause in three of themCharacterization of the hearing phenotype was performed inthe affected family members of these three families in thepresent study Our results expanded the mutation spectrumof DFNA15 and suggested that mutations in POU4F3 are arelatively common cause for ADNSHL in Chinese Hans
2 Materials and Methods
21 Subjects Probands of sixteen Chinese Han families seg-regating ADNSHL were recruited through Xinhua Hospi-tal Shanghai China The pedigrees of the families wereshown in Figure 1(a) and Supplementary Figure S1 in Supple-mentaryMaterial available online at httpdxdoiorg10115520169890827 For Families P1748 PD6 and P59 in whichmutations in POU4F3were identified 8 10 and 13 additionalfamily members were subsequently recruited respectivelyInformed consent was obtained from all subjects This studywas approved by the Ethics Committee of Xinhua HospitalShanghai Jiaotong University School of Medicine ShanghaiChina
22 Clinical Evaluations A detailed physical and medicalhistory examination was performed in all probands of theADNSHL familiesThe hearing levels were measured by puretone audiometry (PTA) and shown as the average thresholdsof 05 1 2 and 4 kHz from the better ear The hearing levelswere classified as normal (lt20 dB) mild (20ndash40 dB) moder-ate (41ndash70 dB) severe (71ndash90 dB) and profound (gt90 dB)
23 Mutation Analysis Targeted NGS of 144 known deaf-ness genes (see complete list in Supplementary Table S1) wasperformed using the MyGenotics gene enrichment system(MyGenotics Boston MD USA) and the Illumina HiSeq2000 sequencer (Illumina SanDiego CAUSA) as previouslydescribed [18] The raw data were first analyzed to filter outthe low quality reads NCBI37hg19 assembly was used as thereference sequences Nonsynonymous on-target variantswith maximumminor allele frequency (MAF) less than 0001in public databases NHLBI Exome Sequencing Project (ESPhttpevsgswashingtoneduEVS) and the Exome Aggrega-tion Consortium (ExAC httpexacbroadinstituteorg)were considered as the candidate pathogenic mutations incompliance with the ADNSHL inheritance The pathogeni-city of the candidate variants was predicted by computationalprograms Mutation Taster (httpwwwmutationtasterorg)PolyPhen-2 (httpgeneticsbwhharvardedupph2) PRO-VEAN and SIFT (httpsiftjcviorg cutoff scores set at minus25and 005 resp) Cosegregation of the pathogenic mutations
and the hearing phenotype was verified in members of Fami-lies P1748 and PD6 by Sanger sequencing
3 Results
31 POU4F3 Mutations Identified in the ADNSHL FamiliesIn our previous studies of 7 Chinese Han ADNSHL familiesa c603 604delGG (pLeu201fslowast12) mutation in POU4F3 hasbeen identified by targeted NGS as the pathogenic causein Family P59 [18] In the present study we performed acomprehensive mutation screening in probands of anothernine Chinese Han ADNSHL families (marked in asterisks inFigure 1(a) and Supplementary Figure S1) by targeted NGS of144 known deafness genes Interestingly two novel heterozy-gous variants pLeu311Pro (c932TgtC) and c120+1GgtC inPOU4F3were identified as the candidate pathogenic variantsin probands P1748-III-1 and PD6-IV-1 respectively (Table 1)alongwith a heterozygousTECTA pVal1830Met and a hetero-zygous TMC1 pSer697X variant in proband P1748-1 (Table 1)Sanger sequencing in the rest of the family members con-firmed that pLeu311Pro and c120+1GgtC were the only twocandidate variants segregating with the hearing phenotypein Families P1748 and PD6 (Figures 1(a) and 1(b)) while theTECTA pVal1830Met and the TMC1 pSer697X variants werenot seen in any other affected family members of P1748 ThepLeu311Pro (c932TgtC) and c120+1GgtC in POU4F3 werenot reported in previous studies not present in the NHLBIESP (119899 = 6503) and ExAC (119899 = 60706) database andnot seen in 300 Chinese Han normal hearing controls Thec120+1GgtC mutation was predicted to abolish the 51015840 splicesite of introns 1 of POU4F3 (Figure 2(a)) The pLeu311Promutation substituted a conserved amino acid Leu311 (Fig-ure 2(b)) and was predicted to be deleterious by computa-tional programs Mutation Taster PolyPhen-2 PROVEANand SIFT (Table 1)
32 Clinical Characteristics of the Three ADNSHL Familieswith POU4F3 Mutations Based on the audiograms andthe self-description of the affected individuals with c603604delGG pLeu311Pro and c120+1GgtC mutations inPOU4F3 the hearing loss associated with the POU4F3muta-tionswas typically progressivewith considerable variability inages of onset and degree of severity both interfamilially andintrafamilially In Family P1748 with the pLeu311Pro muta-tion proband III-1 had notable hearing loss since age 10 yearsThe hearing loss affected high frequency most and graduallyprogressed to moderate hearing loss at age 29 years (Fig-ure 1(c))The affected individuals in the second generation ofFamily P1748 had hearing loss at the onset age of 10 years to 20years and all eventually progressed to profound after age 50years Interestingly all six female patients in the second gen-eration had significantly decreased hearing levels after givingbirth to their children In Family PD6 with the c120+1GgtCmutation proband IV-1 had congenital moderate hearingloss with a relatively ldquoflatrdquo audiometric profile at age 23 yearsaffecting all frequencies (Figure 1(c)) Other affected individ-uals in this family demonstrated progressive moderate-to-profound deafness depending on their agesThe ages of onset
Neural Plasticity 3
POU4F3 pLeu311Pro (c932TgtC)Family P1748lowast
II-1 II-1 II-2
III-1
IV-1
III-2 III-3
II-3II-2 II-3 II-4 II-5 II-6 II-7
III-4 III-5 III-6 III-7
III-1 III-2
POU4F3 c120+1GgtCFamily PD6lowast
minus+
minus+ minus+
minus+ minus+ minus+ minus+ minus+minus+
minus+ minus+ minus+
minus+
minus+ minus+
minus+
++
++
++
++
(a)
POU4F3 c932TgtCFamily P1748T T T TC
C C C C
C C C CG G G G GA
T T T TC G G G G GA A A A A A A A A A
T
T T T
T TC
C C C C C C
C C C C CG
G G G G G G G G
G G G G G GA
A A A A
A A AA A A A A A A AA T
T
C
POU4F3 wild type POU4F3 wild type
POU4F3 c120+1GgtCFamily PD6
(b)
0102030405060708090
100110120
Hea
ring
leve
l (dB
)
Frequency (Hz)250 2K1K500
P1748-III-1PD6-IV-1P59-III-8(54 yo)
(23 yo)(29 yo)
(c)
Figure 1 POU4F3 mutations identified in the Chinese Han ADNSHL families (a) Pedigrees and genotypes of the families with POU4F3mutations Probandswere pointed by arrowsminus and+ indicate themutant andwild type alleles respectively Asterisks indicate the familieswithPOU4F3mutations identified in the present study (b) Chromatograms showing the c932TgtC (pLeu311Pro) and the c120+1GgtC mutationsin POU4F3 (c) Audiograms of the probands of the three families
4 Neural Plasticity
Table 1 Candidate pathogenic mutations identified in probands of Families P1748 and PD6 by targeted NGS
Proband Gene (referencesequence) Mutation MAF
(ExAC)
MAF(NHLBIESP)
MutationTaster
PROVEAN(score) SIFT (score)
PolyPhen-2(HumVarscore)
Intrafamilialphenotype
cosegregation
P1748-III-1
POU4F3(NM 002700)
pLeu311Pro(c932TgtC) 0 0 Disease
causingDeleterious(minus363)
Damaging(0)
Probablydamaging (1) Yes
TECTA(NM 005422)
pVal1830Met(c5488GgtA) 00003871 0 Disease
causingNeutral(minus083)
Damaging(0008)
Probablydamaging(0969)
No
TMC1(NM 138691)
pSer697X(c2090CgtG) 0 0 Disease
causingDeleterious(minus1026) mdash mdash No
PD6-IV-1 POU4F3(NM 002700) c120+1GgtC 0 0 Disease
causing mdash mdash mdash Yes
pLeu223ProDutch
c662_675del14Korean
pGlu232LysKorean
c880_887del8Israeli Jewish
pLeu289PheDutch
Chinese
pLeu311ProChinese
Chinesec1007delC
Japanese
Exon1 Exon2
pArg326LysKorean
POU-specific domain POU homeodomain
pPro164ArgChinese
pPro246ThrJapanese
c602delTChinese
lowast
c120+1GgtClowast c603_604delGGlowast
(a)
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
Leu311
Xenopus laevis
Danio rerio
Mus musculus
Pan troglodytes
Homo sapiens
(b)
Figure 2 Summary and conservation of the POU4F3 mutations (a) Schematic illustration of the thirteen reported POU4F3 mutationsassociated with DFNA15 Asterisks indicated the mutations reported in the present study (b) Multispecies sequence alignment showing theevolutionary conserved amino acid Leu311
ranged from congenital to 40 years In Family P59 withthe c603 604delGG mutation the proband III-8 had onlymoderate hearing loss at 54 years of age (Figure 1(c)) andall affected individuals in this family had a rather late ageof onset around 40s For other auditory symptoms tinnitushas been complained by proband P1748-III-1 No vestibulardysfunction was shown in any affected individuals
4 Discussion
Combined with our present and previous studies we iden-tified mutations in POU4F3 as the pathogenic cause ofdeafness in 3 of the 16 (18) Chinese Han ADNSHL familiessuggesting that it is a relatively common cause for ADNSHLin Chinese Hans Consistently seven of the ten previously
Neural Plasticity 5
reportedPOU4F3mutations fromother research groupswerealso from the East Asians (three in Korean two in Japaneseand two in Chinese Figure 2(a)) suggesting that this geneshould be routinely screened in ADNSHL cases of East Asiandescent In contrast only three (one in Israeli Jewish and twoin Dutch Figure 2(a)) POU4F3 mutations were previouslyreported from regions other thanEastAsiaThedistinguishedmutation spectrum among different ethnical groups has alsobeen reported for other deafness genes such as SLC26A4 inwhich case biallelic SLC26A4 mutations can be identified in884 of deaf patients with nonsyndromic EVA in Chinesebut only 15 in Caucasians [19 20]
Our study also expanded the mutation spectrum ofPOU4F3 Figure 2(a) summarized the type position andassociated ethnicity of the thirteen POU4F3 mutationsreported to date Four of them including the c603 604delGG(pLeu201fslowast12) mutation reported in our previous studywere truncating mutations that were predicted to lead toprematurely stopped protein product or nonsense-mediateddecay of themRNA while another seven POU4F3mutationsincluding the pLeu311Pro mutation reported in the presentstudy were missense mutations leading to single amino acidsubstitutionsNotably these twelvemutationswere all locatedwithin or close to the POU-specific domain or the POUhomeodomain the two conserved DNA-binding domains ofPOU4F3 encoded in exon 2 On the contrary the c120+1GgtCmutation identified in the present study is the only reportedmutation outside exon 2 of POU4F3
Consistent with previous reports [5 11 13ndash16] thePOU4F3mutations identified in the present study were asso-ciated with progressive hearing loss with considerable vari-ability in the ages of onset and the degrees of severity andthis variable hearing phenotype can be seen both interfa-milially and intrafamilially Apparently no simple genotype-phenotype correlation can be drawn based on the positionor the truncatingnontruncating nature of the POU4F3muta-tions In a previous study of the Pou4f3 mutant deaf micedeficiency of Pou4f3 has been found to result in reducedexpression of its hair cell specific downstream target Gfi1which was suggested as the direct cause of the outer hair celldegeneration in the Pou4f3mutant mice [10] In future stud-ies therefore it will be interesting to correlate the presumablyreduced levels of theGfi1 transcriptionwith differentPOU4F3mutations and the severity of the associated hearing loss
5 Conclusions
Mutations in POU4F3 are a relatively common cause forADNSHL in Chinese Hans The hearing loss associated withPOU4F3mutations has considerable variability in the ages ofonset and the degrees of severity
Competing Interests
The authors declare no competing financial interests
Authorsrsquo Contributions
Longxia He Xiuhong Pang and Penghui Chen contributedequally to this work
Acknowledgments
This research was supported by grants from National Sci-ence Foundation of China (81570930 and 81371101 to TaoYang 81330023 to Hao Wu) Shanghai Municipal Scienceand Technology Commission (14DZ1940102 to Tao Yang14DZ2260300 and 14DJ1400201 to Hao Wu) and ShanghaiMunicipal Education Commission-Gaofeng Clinical Medi-cine Grant (20152519 to Tao Yang)
References
[1] R J H Smith A E Shearer M S Hildebrand and G VanCamp ldquoDeafness and hereditary hearing loss overviewrdquo inGeneReviews R A Pagon M P Adam H H Ardinger et alEds University of Washington Seattle Wash USA 1993
[2] A E Shearer A P DeLuca M S Hildebrand et al ldquoCompre-hensive genetic testing for hereditary hearing loss using massi-vely parallel sequencingrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 49 pp21104ndash21109 2010
[3] A E Shearer and R J H Smith ldquoGenetics advances in genetictesting for deafnessrdquo Current Opinion in Pediatrics vol 24 no6 pp 679ndash686 2012
[4] Z Brownstein Y Bhonker and K B Avraham ldquoHigh-through-put sequencing to decipher the genetic heterogeneity of deaf-nessrdquo Genome Biology vol 13 article 245 2012
[5] O Vahava R Morell E D Lynch et al ldquoMutation in transcrip-tion factor POU4F3 associated with inherited progressive hear-ing loss in humansrdquo Science vol 279 no 5358 pp 1950ndash19541998
[6] L Erkman R J McEvilly L Luo et al ldquoRole of transcriptionfactors Brn-31 and Brn-32 in auditory and visual systemdevelopmentrdquo Nature vol 381 no 6583 pp 603ndash606 1996
[7] S W Wang X Mu W J Bowers et al ldquoBrn3bBrn3c doubleknockout mice reveal an unsuspected role for Brn3c in retinalganglion cell axon outgrowthrdquo Development vol 129 no 2 pp467ndash477 2002
[8] M Xiang L Gan D Li et al ldquoEssential role of POU-domainfactor Brn-3c in auditory and vestibular hair cell developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 17 pp 9445ndash9450 1997
[9] MXiangW-QGao THasson and J J Shin ldquoRequirement forBrn-3c inmaturation and survival but not in fate determinationof inner ear hair cellsrdquo Development vol 125 no 20 pp 3935ndash3946 1998
[10] R Hertzano M Montcouquiol S Rashi-Elkeles et al ldquoTran-scription profiling of inner ears from Pou4f3119889119889119897119889119889119897 identifiesGfi1 as a target of the Pou4f3 deafness generdquo Human MolecularGenetics vol 13 no 18 pp 2143ndash2153 2004
[11] R W J Collin R Chellappa R-J Pauw et al ldquoMissense muta-tions in POU4F3 cause autosomal dominant hearing impair-ment DFNA15 and affect subcellular localization and DNAbindingrdquo Human Mutation vol 29 no 4 pp 545ndash554 2008
6 Neural Plasticity
[12] HMutai N Suzuki A Shimizu et al ldquoDiverse spectrumof raredeafness genes underlies early-childhood hearing loss in Japa-nese patients a cross-sectional multi-center next-generationsequencing studyrdquo Orphanet Journal of Rare Diseases vol 8article 172 2013
[13] H-J Kim H-H Won K-J Park et al ldquoSNP linkage analysisand whole exome sequencing identify a novel POU4F3 muta-tion in autosomal dominant late-onset nonsyndromic hearingloss (DFNA15)rdquo PLoS ONE vol 8 no 11 Article ID e790632013
[14] H K Lee H J Park K Y Lee R Park and U K Kim ldquoA novelframeshiftmutation of POU4F3 gene associatedwith autosomaldominant non-syndromic hearing lossrdquo Biochemical and Bio-physical Research Communications vol 396 no 3 pp 626ndash6302010
[15] X Z Cai Y Li L Xia et al ldquoExome sequencing identifiesPOU4F3 as the causative gene for a large Chinese family withnon-syndromic hearing lossrdquo Journal of Human Genetics 2016
[16] Q Wei H Zhu X Qian et al ldquoTargeted genomic capture andmassively parallel sequencing to identify novel variants caus-ing Chinese hereditary hearing lossrdquo Journal of TranslationalMedicine vol 12 article 311 2014
[17] M Miyagawa T Naito S-Y Nishio N Kamatani and S-IUsami ldquoTargeted exon sequencing successfully discovers rarecausative genes and clarifies the molecular epidemiology ofJapanese deafness patientsrdquo PLoS ONE vol 8 no 8 Article IDe71381 2013
[18] T Yang X Wei Y Chai L Li and H Wu ldquoGenetic etiologystudy of the non-syndromic deafness in Chinese Hans by tar-geted next-generation sequencingrdquo Orphanet Journal of RareDiseases vol 8 no 1 article 85 2013
[19] Q-J Wang Y-L Zhao A Q Rao et al ldquoA distinct spectrumof SLC26A4 mutations in patients with enlarged vestibularaqueduct in ChinardquoClinical Genetics vol 72 no 3 pp 245ndash2542007
[20] T Yang H Vidarsson S Rodrigo-Blomqvist et al ldquoTranscrip-tional control of SLC26A4 is involved in Pendred syndrome andnonsyndromic enlargement of vestibular aqueduct (DFNB4)rdquoAmerican Journal of Human Genetics vol 80 no 6 pp 1055ndash1063 2007
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Neural Plasticity 3
POU4F3 pLeu311Pro (c932TgtC)Family P1748lowast
II-1 II-1 II-2
III-1
IV-1
III-2 III-3
II-3II-2 II-3 II-4 II-5 II-6 II-7
III-4 III-5 III-6 III-7
III-1 III-2
POU4F3 c120+1GgtCFamily PD6lowast
minus+
minus+ minus+
minus+ minus+ minus+ minus+ minus+minus+
minus+ minus+ minus+
minus+
minus+ minus+
minus+
++
++
++
++
(a)
POU4F3 c932TgtCFamily P1748T T T TC
C C C C
C C C CG G G G GA
T T T TC G G G G GA A A A A A A A A A
T
T T T
T TC
C C C C C C
C C C C CG
G G G G G G G G
G G G G G GA
A A A A
A A AA A A A A A A AA T
T
C
POU4F3 wild type POU4F3 wild type
POU4F3 c120+1GgtCFamily PD6
(b)
0102030405060708090
100110120
Hea
ring
leve
l (dB
)
Frequency (Hz)250 2K1K500
P1748-III-1PD6-IV-1P59-III-8(54 yo)
(23 yo)(29 yo)
(c)
Figure 1 POU4F3 mutations identified in the Chinese Han ADNSHL families (a) Pedigrees and genotypes of the families with POU4F3mutations Probandswere pointed by arrowsminus and+ indicate themutant andwild type alleles respectively Asterisks indicate the familieswithPOU4F3mutations identified in the present study (b) Chromatograms showing the c932TgtC (pLeu311Pro) and the c120+1GgtC mutationsin POU4F3 (c) Audiograms of the probands of the three families
4 Neural Plasticity
Table 1 Candidate pathogenic mutations identified in probands of Families P1748 and PD6 by targeted NGS
Proband Gene (referencesequence) Mutation MAF
(ExAC)
MAF(NHLBIESP)
MutationTaster
PROVEAN(score) SIFT (score)
PolyPhen-2(HumVarscore)
Intrafamilialphenotype
cosegregation
P1748-III-1
POU4F3(NM 002700)
pLeu311Pro(c932TgtC) 0 0 Disease
causingDeleterious(minus363)
Damaging(0)
Probablydamaging (1) Yes
TECTA(NM 005422)
pVal1830Met(c5488GgtA) 00003871 0 Disease
causingNeutral(minus083)
Damaging(0008)
Probablydamaging(0969)
No
TMC1(NM 138691)
pSer697X(c2090CgtG) 0 0 Disease
causingDeleterious(minus1026) mdash mdash No
PD6-IV-1 POU4F3(NM 002700) c120+1GgtC 0 0 Disease
causing mdash mdash mdash Yes
pLeu223ProDutch
c662_675del14Korean
pGlu232LysKorean
c880_887del8Israeli Jewish
pLeu289PheDutch
Chinese
pLeu311ProChinese
Chinesec1007delC
Japanese
Exon1 Exon2
pArg326LysKorean
POU-specific domain POU homeodomain
pPro164ArgChinese
pPro246ThrJapanese
c602delTChinese
lowast
c120+1GgtClowast c603_604delGGlowast
(a)
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
Leu311
Xenopus laevis
Danio rerio
Mus musculus
Pan troglodytes
Homo sapiens
(b)
Figure 2 Summary and conservation of the POU4F3 mutations (a) Schematic illustration of the thirteen reported POU4F3 mutationsassociated with DFNA15 Asterisks indicated the mutations reported in the present study (b) Multispecies sequence alignment showing theevolutionary conserved amino acid Leu311
ranged from congenital to 40 years In Family P59 withthe c603 604delGG mutation the proband III-8 had onlymoderate hearing loss at 54 years of age (Figure 1(c)) andall affected individuals in this family had a rather late ageof onset around 40s For other auditory symptoms tinnitushas been complained by proband P1748-III-1 No vestibulardysfunction was shown in any affected individuals
4 Discussion
Combined with our present and previous studies we iden-tified mutations in POU4F3 as the pathogenic cause ofdeafness in 3 of the 16 (18) Chinese Han ADNSHL familiessuggesting that it is a relatively common cause for ADNSHLin Chinese Hans Consistently seven of the ten previously
Neural Plasticity 5
reportedPOU4F3mutations fromother research groupswerealso from the East Asians (three in Korean two in Japaneseand two in Chinese Figure 2(a)) suggesting that this geneshould be routinely screened in ADNSHL cases of East Asiandescent In contrast only three (one in Israeli Jewish and twoin Dutch Figure 2(a)) POU4F3 mutations were previouslyreported from regions other thanEastAsiaThedistinguishedmutation spectrum among different ethnical groups has alsobeen reported for other deafness genes such as SLC26A4 inwhich case biallelic SLC26A4 mutations can be identified in884 of deaf patients with nonsyndromic EVA in Chinesebut only 15 in Caucasians [19 20]
Our study also expanded the mutation spectrum ofPOU4F3 Figure 2(a) summarized the type position andassociated ethnicity of the thirteen POU4F3 mutationsreported to date Four of them including the c603 604delGG(pLeu201fslowast12) mutation reported in our previous studywere truncating mutations that were predicted to lead toprematurely stopped protein product or nonsense-mediateddecay of themRNA while another seven POU4F3mutationsincluding the pLeu311Pro mutation reported in the presentstudy were missense mutations leading to single amino acidsubstitutionsNotably these twelvemutationswere all locatedwithin or close to the POU-specific domain or the POUhomeodomain the two conserved DNA-binding domains ofPOU4F3 encoded in exon 2 On the contrary the c120+1GgtCmutation identified in the present study is the only reportedmutation outside exon 2 of POU4F3
Consistent with previous reports [5 11 13ndash16] thePOU4F3mutations identified in the present study were asso-ciated with progressive hearing loss with considerable vari-ability in the ages of onset and the degrees of severity andthis variable hearing phenotype can be seen both interfa-milially and intrafamilially Apparently no simple genotype-phenotype correlation can be drawn based on the positionor the truncatingnontruncating nature of the POU4F3muta-tions In a previous study of the Pou4f3 mutant deaf micedeficiency of Pou4f3 has been found to result in reducedexpression of its hair cell specific downstream target Gfi1which was suggested as the direct cause of the outer hair celldegeneration in the Pou4f3mutant mice [10] In future stud-ies therefore it will be interesting to correlate the presumablyreduced levels of theGfi1 transcriptionwith differentPOU4F3mutations and the severity of the associated hearing loss
5 Conclusions
Mutations in POU4F3 are a relatively common cause forADNSHL in Chinese Hans The hearing loss associated withPOU4F3mutations has considerable variability in the ages ofonset and the degrees of severity
Competing Interests
The authors declare no competing financial interests
Authorsrsquo Contributions
Longxia He Xiuhong Pang and Penghui Chen contributedequally to this work
Acknowledgments
This research was supported by grants from National Sci-ence Foundation of China (81570930 and 81371101 to TaoYang 81330023 to Hao Wu) Shanghai Municipal Scienceand Technology Commission (14DZ1940102 to Tao Yang14DZ2260300 and 14DJ1400201 to Hao Wu) and ShanghaiMunicipal Education Commission-Gaofeng Clinical Medi-cine Grant (20152519 to Tao Yang)
References
[1] R J H Smith A E Shearer M S Hildebrand and G VanCamp ldquoDeafness and hereditary hearing loss overviewrdquo inGeneReviews R A Pagon M P Adam H H Ardinger et alEds University of Washington Seattle Wash USA 1993
[2] A E Shearer A P DeLuca M S Hildebrand et al ldquoCompre-hensive genetic testing for hereditary hearing loss using massi-vely parallel sequencingrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 49 pp21104ndash21109 2010
[3] A E Shearer and R J H Smith ldquoGenetics advances in genetictesting for deafnessrdquo Current Opinion in Pediatrics vol 24 no6 pp 679ndash686 2012
[4] Z Brownstein Y Bhonker and K B Avraham ldquoHigh-through-put sequencing to decipher the genetic heterogeneity of deaf-nessrdquo Genome Biology vol 13 article 245 2012
[5] O Vahava R Morell E D Lynch et al ldquoMutation in transcrip-tion factor POU4F3 associated with inherited progressive hear-ing loss in humansrdquo Science vol 279 no 5358 pp 1950ndash19541998
[6] L Erkman R J McEvilly L Luo et al ldquoRole of transcriptionfactors Brn-31 and Brn-32 in auditory and visual systemdevelopmentrdquo Nature vol 381 no 6583 pp 603ndash606 1996
[7] S W Wang X Mu W J Bowers et al ldquoBrn3bBrn3c doubleknockout mice reveal an unsuspected role for Brn3c in retinalganglion cell axon outgrowthrdquo Development vol 129 no 2 pp467ndash477 2002
[8] M Xiang L Gan D Li et al ldquoEssential role of POU-domainfactor Brn-3c in auditory and vestibular hair cell developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 17 pp 9445ndash9450 1997
[9] MXiangW-QGao THasson and J J Shin ldquoRequirement forBrn-3c inmaturation and survival but not in fate determinationof inner ear hair cellsrdquo Development vol 125 no 20 pp 3935ndash3946 1998
[10] R Hertzano M Montcouquiol S Rashi-Elkeles et al ldquoTran-scription profiling of inner ears from Pou4f3119889119889119897119889119889119897 identifiesGfi1 as a target of the Pou4f3 deafness generdquo Human MolecularGenetics vol 13 no 18 pp 2143ndash2153 2004
[11] R W J Collin R Chellappa R-J Pauw et al ldquoMissense muta-tions in POU4F3 cause autosomal dominant hearing impair-ment DFNA15 and affect subcellular localization and DNAbindingrdquo Human Mutation vol 29 no 4 pp 545ndash554 2008
6 Neural Plasticity
[12] HMutai N Suzuki A Shimizu et al ldquoDiverse spectrumof raredeafness genes underlies early-childhood hearing loss in Japa-nese patients a cross-sectional multi-center next-generationsequencing studyrdquo Orphanet Journal of Rare Diseases vol 8article 172 2013
[13] H-J Kim H-H Won K-J Park et al ldquoSNP linkage analysisand whole exome sequencing identify a novel POU4F3 muta-tion in autosomal dominant late-onset nonsyndromic hearingloss (DFNA15)rdquo PLoS ONE vol 8 no 11 Article ID e790632013
[14] H K Lee H J Park K Y Lee R Park and U K Kim ldquoA novelframeshiftmutation of POU4F3 gene associatedwith autosomaldominant non-syndromic hearing lossrdquo Biochemical and Bio-physical Research Communications vol 396 no 3 pp 626ndash6302010
[15] X Z Cai Y Li L Xia et al ldquoExome sequencing identifiesPOU4F3 as the causative gene for a large Chinese family withnon-syndromic hearing lossrdquo Journal of Human Genetics 2016
[16] Q Wei H Zhu X Qian et al ldquoTargeted genomic capture andmassively parallel sequencing to identify novel variants caus-ing Chinese hereditary hearing lossrdquo Journal of TranslationalMedicine vol 12 article 311 2014
[17] M Miyagawa T Naito S-Y Nishio N Kamatani and S-IUsami ldquoTargeted exon sequencing successfully discovers rarecausative genes and clarifies the molecular epidemiology ofJapanese deafness patientsrdquo PLoS ONE vol 8 no 8 Article IDe71381 2013
[18] T Yang X Wei Y Chai L Li and H Wu ldquoGenetic etiologystudy of the non-syndromic deafness in Chinese Hans by tar-geted next-generation sequencingrdquo Orphanet Journal of RareDiseases vol 8 no 1 article 85 2013
[19] Q-J Wang Y-L Zhao A Q Rao et al ldquoA distinct spectrumof SLC26A4 mutations in patients with enlarged vestibularaqueduct in ChinardquoClinical Genetics vol 72 no 3 pp 245ndash2542007
[20] T Yang H Vidarsson S Rodrigo-Blomqvist et al ldquoTranscrip-tional control of SLC26A4 is involved in Pendred syndrome andnonsyndromic enlargement of vestibular aqueduct (DFNB4)rdquoAmerican Journal of Human Genetics vol 80 no 6 pp 1055ndash1063 2007
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
4 Neural Plasticity
Table 1 Candidate pathogenic mutations identified in probands of Families P1748 and PD6 by targeted NGS
Proband Gene (referencesequence) Mutation MAF
(ExAC)
MAF(NHLBIESP)
MutationTaster
PROVEAN(score) SIFT (score)
PolyPhen-2(HumVarscore)
Intrafamilialphenotype
cosegregation
P1748-III-1
POU4F3(NM 002700)
pLeu311Pro(c932TgtC) 0 0 Disease
causingDeleterious(minus363)
Damaging(0)
Probablydamaging (1) Yes
TECTA(NM 005422)
pVal1830Met(c5488GgtA) 00003871 0 Disease
causingNeutral(minus083)
Damaging(0008)
Probablydamaging(0969)
No
TMC1(NM 138691)
pSer697X(c2090CgtG) 0 0 Disease
causingDeleterious(minus1026) mdash mdash No
PD6-IV-1 POU4F3(NM 002700) c120+1GgtC 0 0 Disease
causing mdash mdash mdash Yes
pLeu223ProDutch
c662_675del14Korean
pGlu232LysKorean
c880_887del8Israeli Jewish
pLeu289PheDutch
Chinese
pLeu311ProChinese
Chinesec1007delC
Japanese
Exon1 Exon2
pArg326LysKorean
POU-specific domain POU homeodomain
pPro164ArgChinese
pPro246ThrJapanese
c602delTChinese
lowast
c120+1GgtClowast c603_604delGGlowast
(a)
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
QPRPSSEKIAAIAEKLDLKKNVVRVWFCNQR
Leu311
Xenopus laevis
Danio rerio
Mus musculus
Pan troglodytes
Homo sapiens
(b)
Figure 2 Summary and conservation of the POU4F3 mutations (a) Schematic illustration of the thirteen reported POU4F3 mutationsassociated with DFNA15 Asterisks indicated the mutations reported in the present study (b) Multispecies sequence alignment showing theevolutionary conserved amino acid Leu311
ranged from congenital to 40 years In Family P59 withthe c603 604delGG mutation the proband III-8 had onlymoderate hearing loss at 54 years of age (Figure 1(c)) andall affected individuals in this family had a rather late ageof onset around 40s For other auditory symptoms tinnitushas been complained by proband P1748-III-1 No vestibulardysfunction was shown in any affected individuals
4 Discussion
Combined with our present and previous studies we iden-tified mutations in POU4F3 as the pathogenic cause ofdeafness in 3 of the 16 (18) Chinese Han ADNSHL familiessuggesting that it is a relatively common cause for ADNSHLin Chinese Hans Consistently seven of the ten previously
Neural Plasticity 5
reportedPOU4F3mutations fromother research groupswerealso from the East Asians (three in Korean two in Japaneseand two in Chinese Figure 2(a)) suggesting that this geneshould be routinely screened in ADNSHL cases of East Asiandescent In contrast only three (one in Israeli Jewish and twoin Dutch Figure 2(a)) POU4F3 mutations were previouslyreported from regions other thanEastAsiaThedistinguishedmutation spectrum among different ethnical groups has alsobeen reported for other deafness genes such as SLC26A4 inwhich case biallelic SLC26A4 mutations can be identified in884 of deaf patients with nonsyndromic EVA in Chinesebut only 15 in Caucasians [19 20]
Our study also expanded the mutation spectrum ofPOU4F3 Figure 2(a) summarized the type position andassociated ethnicity of the thirteen POU4F3 mutationsreported to date Four of them including the c603 604delGG(pLeu201fslowast12) mutation reported in our previous studywere truncating mutations that were predicted to lead toprematurely stopped protein product or nonsense-mediateddecay of themRNA while another seven POU4F3mutationsincluding the pLeu311Pro mutation reported in the presentstudy were missense mutations leading to single amino acidsubstitutionsNotably these twelvemutationswere all locatedwithin or close to the POU-specific domain or the POUhomeodomain the two conserved DNA-binding domains ofPOU4F3 encoded in exon 2 On the contrary the c120+1GgtCmutation identified in the present study is the only reportedmutation outside exon 2 of POU4F3
Consistent with previous reports [5 11 13ndash16] thePOU4F3mutations identified in the present study were asso-ciated with progressive hearing loss with considerable vari-ability in the ages of onset and the degrees of severity andthis variable hearing phenotype can be seen both interfa-milially and intrafamilially Apparently no simple genotype-phenotype correlation can be drawn based on the positionor the truncatingnontruncating nature of the POU4F3muta-tions In a previous study of the Pou4f3 mutant deaf micedeficiency of Pou4f3 has been found to result in reducedexpression of its hair cell specific downstream target Gfi1which was suggested as the direct cause of the outer hair celldegeneration in the Pou4f3mutant mice [10] In future stud-ies therefore it will be interesting to correlate the presumablyreduced levels of theGfi1 transcriptionwith differentPOU4F3mutations and the severity of the associated hearing loss
5 Conclusions
Mutations in POU4F3 are a relatively common cause forADNSHL in Chinese Hans The hearing loss associated withPOU4F3mutations has considerable variability in the ages ofonset and the degrees of severity
Competing Interests
The authors declare no competing financial interests
Authorsrsquo Contributions
Longxia He Xiuhong Pang and Penghui Chen contributedequally to this work
Acknowledgments
This research was supported by grants from National Sci-ence Foundation of China (81570930 and 81371101 to TaoYang 81330023 to Hao Wu) Shanghai Municipal Scienceand Technology Commission (14DZ1940102 to Tao Yang14DZ2260300 and 14DJ1400201 to Hao Wu) and ShanghaiMunicipal Education Commission-Gaofeng Clinical Medi-cine Grant (20152519 to Tao Yang)
References
[1] R J H Smith A E Shearer M S Hildebrand and G VanCamp ldquoDeafness and hereditary hearing loss overviewrdquo inGeneReviews R A Pagon M P Adam H H Ardinger et alEds University of Washington Seattle Wash USA 1993
[2] A E Shearer A P DeLuca M S Hildebrand et al ldquoCompre-hensive genetic testing for hereditary hearing loss using massi-vely parallel sequencingrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 49 pp21104ndash21109 2010
[3] A E Shearer and R J H Smith ldquoGenetics advances in genetictesting for deafnessrdquo Current Opinion in Pediatrics vol 24 no6 pp 679ndash686 2012
[4] Z Brownstein Y Bhonker and K B Avraham ldquoHigh-through-put sequencing to decipher the genetic heterogeneity of deaf-nessrdquo Genome Biology vol 13 article 245 2012
[5] O Vahava R Morell E D Lynch et al ldquoMutation in transcrip-tion factor POU4F3 associated with inherited progressive hear-ing loss in humansrdquo Science vol 279 no 5358 pp 1950ndash19541998
[6] L Erkman R J McEvilly L Luo et al ldquoRole of transcriptionfactors Brn-31 and Brn-32 in auditory and visual systemdevelopmentrdquo Nature vol 381 no 6583 pp 603ndash606 1996
[7] S W Wang X Mu W J Bowers et al ldquoBrn3bBrn3c doubleknockout mice reveal an unsuspected role for Brn3c in retinalganglion cell axon outgrowthrdquo Development vol 129 no 2 pp467ndash477 2002
[8] M Xiang L Gan D Li et al ldquoEssential role of POU-domainfactor Brn-3c in auditory and vestibular hair cell developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 17 pp 9445ndash9450 1997
[9] MXiangW-QGao THasson and J J Shin ldquoRequirement forBrn-3c inmaturation and survival but not in fate determinationof inner ear hair cellsrdquo Development vol 125 no 20 pp 3935ndash3946 1998
[10] R Hertzano M Montcouquiol S Rashi-Elkeles et al ldquoTran-scription profiling of inner ears from Pou4f3119889119889119897119889119889119897 identifiesGfi1 as a target of the Pou4f3 deafness generdquo Human MolecularGenetics vol 13 no 18 pp 2143ndash2153 2004
[11] R W J Collin R Chellappa R-J Pauw et al ldquoMissense muta-tions in POU4F3 cause autosomal dominant hearing impair-ment DFNA15 and affect subcellular localization and DNAbindingrdquo Human Mutation vol 29 no 4 pp 545ndash554 2008
6 Neural Plasticity
[12] HMutai N Suzuki A Shimizu et al ldquoDiverse spectrumof raredeafness genes underlies early-childhood hearing loss in Japa-nese patients a cross-sectional multi-center next-generationsequencing studyrdquo Orphanet Journal of Rare Diseases vol 8article 172 2013
[13] H-J Kim H-H Won K-J Park et al ldquoSNP linkage analysisand whole exome sequencing identify a novel POU4F3 muta-tion in autosomal dominant late-onset nonsyndromic hearingloss (DFNA15)rdquo PLoS ONE vol 8 no 11 Article ID e790632013
[14] H K Lee H J Park K Y Lee R Park and U K Kim ldquoA novelframeshiftmutation of POU4F3 gene associatedwith autosomaldominant non-syndromic hearing lossrdquo Biochemical and Bio-physical Research Communications vol 396 no 3 pp 626ndash6302010
[15] X Z Cai Y Li L Xia et al ldquoExome sequencing identifiesPOU4F3 as the causative gene for a large Chinese family withnon-syndromic hearing lossrdquo Journal of Human Genetics 2016
[16] Q Wei H Zhu X Qian et al ldquoTargeted genomic capture andmassively parallel sequencing to identify novel variants caus-ing Chinese hereditary hearing lossrdquo Journal of TranslationalMedicine vol 12 article 311 2014
[17] M Miyagawa T Naito S-Y Nishio N Kamatani and S-IUsami ldquoTargeted exon sequencing successfully discovers rarecausative genes and clarifies the molecular epidemiology ofJapanese deafness patientsrdquo PLoS ONE vol 8 no 8 Article IDe71381 2013
[18] T Yang X Wei Y Chai L Li and H Wu ldquoGenetic etiologystudy of the non-syndromic deafness in Chinese Hans by tar-geted next-generation sequencingrdquo Orphanet Journal of RareDiseases vol 8 no 1 article 85 2013
[19] Q-J Wang Y-L Zhao A Q Rao et al ldquoA distinct spectrumof SLC26A4 mutations in patients with enlarged vestibularaqueduct in ChinardquoClinical Genetics vol 72 no 3 pp 245ndash2542007
[20] T Yang H Vidarsson S Rodrigo-Blomqvist et al ldquoTranscrip-tional control of SLC26A4 is involved in Pendred syndrome andnonsyndromic enlargement of vestibular aqueduct (DFNB4)rdquoAmerican Journal of Human Genetics vol 80 no 6 pp 1055ndash1063 2007
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Neural Plasticity 5
reportedPOU4F3mutations fromother research groupswerealso from the East Asians (three in Korean two in Japaneseand two in Chinese Figure 2(a)) suggesting that this geneshould be routinely screened in ADNSHL cases of East Asiandescent In contrast only three (one in Israeli Jewish and twoin Dutch Figure 2(a)) POU4F3 mutations were previouslyreported from regions other thanEastAsiaThedistinguishedmutation spectrum among different ethnical groups has alsobeen reported for other deafness genes such as SLC26A4 inwhich case biallelic SLC26A4 mutations can be identified in884 of deaf patients with nonsyndromic EVA in Chinesebut only 15 in Caucasians [19 20]
Our study also expanded the mutation spectrum ofPOU4F3 Figure 2(a) summarized the type position andassociated ethnicity of the thirteen POU4F3 mutationsreported to date Four of them including the c603 604delGG(pLeu201fslowast12) mutation reported in our previous studywere truncating mutations that were predicted to lead toprematurely stopped protein product or nonsense-mediateddecay of themRNA while another seven POU4F3mutationsincluding the pLeu311Pro mutation reported in the presentstudy were missense mutations leading to single amino acidsubstitutionsNotably these twelvemutationswere all locatedwithin or close to the POU-specific domain or the POUhomeodomain the two conserved DNA-binding domains ofPOU4F3 encoded in exon 2 On the contrary the c120+1GgtCmutation identified in the present study is the only reportedmutation outside exon 2 of POU4F3
Consistent with previous reports [5 11 13ndash16] thePOU4F3mutations identified in the present study were asso-ciated with progressive hearing loss with considerable vari-ability in the ages of onset and the degrees of severity andthis variable hearing phenotype can be seen both interfa-milially and intrafamilially Apparently no simple genotype-phenotype correlation can be drawn based on the positionor the truncatingnontruncating nature of the POU4F3muta-tions In a previous study of the Pou4f3 mutant deaf micedeficiency of Pou4f3 has been found to result in reducedexpression of its hair cell specific downstream target Gfi1which was suggested as the direct cause of the outer hair celldegeneration in the Pou4f3mutant mice [10] In future stud-ies therefore it will be interesting to correlate the presumablyreduced levels of theGfi1 transcriptionwith differentPOU4F3mutations and the severity of the associated hearing loss
5 Conclusions
Mutations in POU4F3 are a relatively common cause forADNSHL in Chinese Hans The hearing loss associated withPOU4F3mutations has considerable variability in the ages ofonset and the degrees of severity
Competing Interests
The authors declare no competing financial interests
Authorsrsquo Contributions
Longxia He Xiuhong Pang and Penghui Chen contributedequally to this work
Acknowledgments
This research was supported by grants from National Sci-ence Foundation of China (81570930 and 81371101 to TaoYang 81330023 to Hao Wu) Shanghai Municipal Scienceand Technology Commission (14DZ1940102 to Tao Yang14DZ2260300 and 14DJ1400201 to Hao Wu) and ShanghaiMunicipal Education Commission-Gaofeng Clinical Medi-cine Grant (20152519 to Tao Yang)
References
[1] R J H Smith A E Shearer M S Hildebrand and G VanCamp ldquoDeafness and hereditary hearing loss overviewrdquo inGeneReviews R A Pagon M P Adam H H Ardinger et alEds University of Washington Seattle Wash USA 1993
[2] A E Shearer A P DeLuca M S Hildebrand et al ldquoCompre-hensive genetic testing for hereditary hearing loss using massi-vely parallel sequencingrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 49 pp21104ndash21109 2010
[3] A E Shearer and R J H Smith ldquoGenetics advances in genetictesting for deafnessrdquo Current Opinion in Pediatrics vol 24 no6 pp 679ndash686 2012
[4] Z Brownstein Y Bhonker and K B Avraham ldquoHigh-through-put sequencing to decipher the genetic heterogeneity of deaf-nessrdquo Genome Biology vol 13 article 245 2012
[5] O Vahava R Morell E D Lynch et al ldquoMutation in transcrip-tion factor POU4F3 associated with inherited progressive hear-ing loss in humansrdquo Science vol 279 no 5358 pp 1950ndash19541998
[6] L Erkman R J McEvilly L Luo et al ldquoRole of transcriptionfactors Brn-31 and Brn-32 in auditory and visual systemdevelopmentrdquo Nature vol 381 no 6583 pp 603ndash606 1996
[7] S W Wang X Mu W J Bowers et al ldquoBrn3bBrn3c doubleknockout mice reveal an unsuspected role for Brn3c in retinalganglion cell axon outgrowthrdquo Development vol 129 no 2 pp467ndash477 2002
[8] M Xiang L Gan D Li et al ldquoEssential role of POU-domainfactor Brn-3c in auditory and vestibular hair cell developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 17 pp 9445ndash9450 1997
[9] MXiangW-QGao THasson and J J Shin ldquoRequirement forBrn-3c inmaturation and survival but not in fate determinationof inner ear hair cellsrdquo Development vol 125 no 20 pp 3935ndash3946 1998
[10] R Hertzano M Montcouquiol S Rashi-Elkeles et al ldquoTran-scription profiling of inner ears from Pou4f3119889119889119897119889119889119897 identifiesGfi1 as a target of the Pou4f3 deafness generdquo Human MolecularGenetics vol 13 no 18 pp 2143ndash2153 2004
[11] R W J Collin R Chellappa R-J Pauw et al ldquoMissense muta-tions in POU4F3 cause autosomal dominant hearing impair-ment DFNA15 and affect subcellular localization and DNAbindingrdquo Human Mutation vol 29 no 4 pp 545ndash554 2008
6 Neural Plasticity
[12] HMutai N Suzuki A Shimizu et al ldquoDiverse spectrumof raredeafness genes underlies early-childhood hearing loss in Japa-nese patients a cross-sectional multi-center next-generationsequencing studyrdquo Orphanet Journal of Rare Diseases vol 8article 172 2013
[13] H-J Kim H-H Won K-J Park et al ldquoSNP linkage analysisand whole exome sequencing identify a novel POU4F3 muta-tion in autosomal dominant late-onset nonsyndromic hearingloss (DFNA15)rdquo PLoS ONE vol 8 no 11 Article ID e790632013
[14] H K Lee H J Park K Y Lee R Park and U K Kim ldquoA novelframeshiftmutation of POU4F3 gene associatedwith autosomaldominant non-syndromic hearing lossrdquo Biochemical and Bio-physical Research Communications vol 396 no 3 pp 626ndash6302010
[15] X Z Cai Y Li L Xia et al ldquoExome sequencing identifiesPOU4F3 as the causative gene for a large Chinese family withnon-syndromic hearing lossrdquo Journal of Human Genetics 2016
[16] Q Wei H Zhu X Qian et al ldquoTargeted genomic capture andmassively parallel sequencing to identify novel variants caus-ing Chinese hereditary hearing lossrdquo Journal of TranslationalMedicine vol 12 article 311 2014
[17] M Miyagawa T Naito S-Y Nishio N Kamatani and S-IUsami ldquoTargeted exon sequencing successfully discovers rarecausative genes and clarifies the molecular epidemiology ofJapanese deafness patientsrdquo PLoS ONE vol 8 no 8 Article IDe71381 2013
[18] T Yang X Wei Y Chai L Li and H Wu ldquoGenetic etiologystudy of the non-syndromic deafness in Chinese Hans by tar-geted next-generation sequencingrdquo Orphanet Journal of RareDiseases vol 8 no 1 article 85 2013
[19] Q-J Wang Y-L Zhao A Q Rao et al ldquoA distinct spectrumof SLC26A4 mutations in patients with enlarged vestibularaqueduct in ChinardquoClinical Genetics vol 72 no 3 pp 245ndash2542007
[20] T Yang H Vidarsson S Rodrigo-Blomqvist et al ldquoTranscrip-tional control of SLC26A4 is involved in Pendred syndrome andnonsyndromic enlargement of vestibular aqueduct (DFNB4)rdquoAmerican Journal of Human Genetics vol 80 no 6 pp 1055ndash1063 2007
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
6 Neural Plasticity
[12] HMutai N Suzuki A Shimizu et al ldquoDiverse spectrumof raredeafness genes underlies early-childhood hearing loss in Japa-nese patients a cross-sectional multi-center next-generationsequencing studyrdquo Orphanet Journal of Rare Diseases vol 8article 172 2013
[13] H-J Kim H-H Won K-J Park et al ldquoSNP linkage analysisand whole exome sequencing identify a novel POU4F3 muta-tion in autosomal dominant late-onset nonsyndromic hearingloss (DFNA15)rdquo PLoS ONE vol 8 no 11 Article ID e790632013
[14] H K Lee H J Park K Y Lee R Park and U K Kim ldquoA novelframeshiftmutation of POU4F3 gene associatedwith autosomaldominant non-syndromic hearing lossrdquo Biochemical and Bio-physical Research Communications vol 396 no 3 pp 626ndash6302010
[15] X Z Cai Y Li L Xia et al ldquoExome sequencing identifiesPOU4F3 as the causative gene for a large Chinese family withnon-syndromic hearing lossrdquo Journal of Human Genetics 2016
[16] Q Wei H Zhu X Qian et al ldquoTargeted genomic capture andmassively parallel sequencing to identify novel variants caus-ing Chinese hereditary hearing lossrdquo Journal of TranslationalMedicine vol 12 article 311 2014
[17] M Miyagawa T Naito S-Y Nishio N Kamatani and S-IUsami ldquoTargeted exon sequencing successfully discovers rarecausative genes and clarifies the molecular epidemiology ofJapanese deafness patientsrdquo PLoS ONE vol 8 no 8 Article IDe71381 2013
[18] T Yang X Wei Y Chai L Li and H Wu ldquoGenetic etiologystudy of the non-syndromic deafness in Chinese Hans by tar-geted next-generation sequencingrdquo Orphanet Journal of RareDiseases vol 8 no 1 article 85 2013
[19] Q-J Wang Y-L Zhao A Q Rao et al ldquoA distinct spectrumof SLC26A4 mutations in patients with enlarged vestibularaqueduct in ChinardquoClinical Genetics vol 72 no 3 pp 245ndash2542007
[20] T Yang H Vidarsson S Rodrigo-Blomqvist et al ldquoTranscrip-tional control of SLC26A4 is involved in Pendred syndrome andnonsyndromic enlargement of vestibular aqueduct (DFNB4)rdquoAmerican Journal of Human Genetics vol 80 no 6 pp 1055ndash1063 2007
