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Mono-allelic mutations of SLC26A4 is over-presented in deaf patients with non-syndromic enlarged vestibular aqueduct
International Journal of Pediatric Otorhinolaryngology 79 (2015) 1351–1353
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Mono-allelic mutations of SLC26A4 is over-presented in deaf patients with non-syndromic enlarged vestibular aqueduct Xiuhong Pang a,b,c,d,1, Yongchuan Chai a,b,c,1, Penghui Chen a,b,c, Longxia He a,b,c, Xiaowen Wang a,b,c, Hao Wu a,b,c,**, Tao Yang a,b,c,* a
Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, China d Department of Otorhinolaryngology-Head and Neck Surgery, Taizhou People’s Hospital, Jiangsu Province, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 March 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online 11 June 2015
Objectives: Recessive mutations of SLC26A4 are the major cause of hearing impairment associated with enlarged vestibular aqueduct (EVA). In a significant percentage of non-syndromic EVA patients, however, only mono-allelic mutations of SLC26A4 can be identified. In this study, we aimed to evaluate whether presence of mono-allelic mutations of SLC26A4 in those patients was coincidental or etiologically associated with the disorder. Methods: The exons and flanking splicing sites of SLC26A4 were sequenced in 150 Chinese Han deaf probands with non-syndromic EVA. c.919-2A >G and p.H723R, two frequent mutations of SLC26A4 in Chinese Hans, were screened by an allele-specific PCR-based array in 3056 ethnically-matched normal hearing controls. The frequency of mono-allelic c.919-2A >G and p.H723R mutations was determined in each group. The statistical significance of the difference was analyzed by Fisher0 s exact test. Results: Bi-allelic, mono-allelic and no mutation of SLC26A4 were detected in 98 (65.3%), 18 (12%) and 34 (22.67%) deaf probands with non-syndromic EVA, respectively. The frequency of mono-allelic c.9192A >G and p.H723R mutations were significantly higher in the 150 deaf probands with non-syndromic EVA (8.67%) than in the 3056 normal hearing controls (1.4%, P = 1.8 106). Conclusion: Presence of mono-allelic mutations of SLC26A4 in non-syndromic EVA patients is etiologically associated with this disorder. Additional genetic or environmental causes may be present in those patients and demand further investigation and consideration during the genetic diagnosis and counseling. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Enlarged vestibular aqueduct SLC26A4 Mono-allelic mutation Genetic diagnosis
1. Introduction Enlarged vestibular aqueduct (EVA) is the most common inner ear malformation seen in the deaf children, accounting for approximately 12% of the permanent hearing impairment at age 4 [1]. The associated hearing impairment can be non-syndromic (DFNB4, OMIM#600791) or with additional thyroid dysfunction (Pendred Syndrome, OMIM#274600). In either case, autosomal
* Corresponding author at: Ear Institute, Shanghai Jiaot Tong University School of Medicine, Shanghai, China. Tel.: +86 21 25078893; fax: +86 21 65152394. ** Corresponding author at: Department of Otolaryngology-Head & Neck Surgery, Xinhua Hospital, Shanghai Jiaot Tong University School of Medicine, Shanghai, China. Tel.: +86 21 55570010; fax: +86 21 65152394. E-mail addresses: [email protected] (H. Wu), [email protected] (T. Yang). 1 Those two authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijporl.2015.06.009 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
recessive mutations of SLC26A4 are the major cause for the disorder [2,3]. Two large-scale mutation screenings of SLC26A4, for example, showed that bi-allelic mutations of SLC26A4 can be detected in 88.4% of non-syndromic EVA patients in Chinese Hans [4] and 24% in Caucasians [5]. SLC26A4 encodes pendrin, an anion transporter that plays a key role in maintaining the endocochlear potential [6]. Mutant mouse homozygous for the targeted deletion of SLC26A4 recapitulate the EVA phenotype and is profoundly deaf, while the heterozygous mouse is phenotypically normal [7]. Both animal models and human familial cases of EVA support the recessive inheritance of this disorder. In the aforementioned mutation screenings, however, sequencing of the exons and flanking splicing sites of SLC26A4 detected mono-allelic mutations in 9.5% of non-syndromic EVA patients in Chinese Hans [4] and 16% in Caucasians [5].
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In this study, we aimed to investigate whether the presence of mono-allelic mutations of SLC26A4 in non-syndromic EVA patients is coincidental or etiologically associated with the disorder. The frequencies of mono-allelic mutations of SLC26A4 were determined and compared between 150 Chinese Han deaf probands with non-syndromic EVA and 3056 ethnically-matched normal hearing controls.
Table 1 SLC26A4 mutations identified in the 150 probands with nonsyndromic EVA and the 3056 controls*. Subjects
Allele 1
Allele 2
No. of subjects (%)
EVA patients
c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G p.H723R p.H723R p.H723R p.H723R p.H723R p.H723R p.H723R p.A360V p.N392Y p.N392Y p.N392Y p.N392Y c.916-917insG p.T410 M p.V650D c.919-2A>G p.H723R c.916_917insG p.G197R p.V163I p.S532R Wild type c.919-2A>G p.H723R Wild type
c.919-2A > G p.H723R p.R409H p.G197R c.1520delT p.T410 M p.E682X c.916-917insG p.N392Y c.349delC c.1685_1686insA p.Q413R p.R409L p.T721 M p.G139V p.L676Q p.S252P c.416-1G > A p.M147V c.162 + 3delG p.S532R c.2085 + 1C > T p.W83X p.G497S c.1707 + 5G > A p.V650D c.1264-12T > A p.T94I p.E37X c.1001 + 2T > C p.L215S p.L703V c.1547_1548insC c.1133_1135delCCA p.S447L p.T132I p.K440X p.H723R p.Q446X p.R409 C p.R409H p.M147V p.T410 M p.S252P p.L676Q c.1547_1548insC p.I529S p.V659L p.S532R p.M147V p.D661Y c.1707 + 5G > A Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type
20 (13.33) 9 (6) 7 (4.67) 3 (2) 3 (2) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 2 (1.33) 2 (1.33) 1(0.67) 1(0.67) 1(0.67) 1(0.67) 1(0.67) 1(0.67) 10 (6.67) 3 (2) 2 (1.33) 1(0.67) 1(0.67) 1(0.67) 34 (22.67) 33 (1.07) 9 (0.30) 3014 (98.63)
2. Materials and methods 2.1. Subjects and clinical evaluation One hundred and fifty Chinese Han deaf probands with nonsyndromic EVA and 3056 Chinese Han normal hearing controls were recruited from Xinhua Hospital, Shanghai, China. A comprehensive family history and physical examinations (with special attention to the thyroid, skeletal, ophthalmologic and mental abnormalities) were performed in all patients. Auditory evaluations included pure-tone audiometry, tympanometry and otoscope examination. Enlargement of the vestibular aqueduct were confirmed by high-resolution computed tomography (CT) scan of the temporal bone. The diagnostic criterion was defined as diameter of >1.5 mm at the midpoint between the common crus and the external aperture. All subjects gave written, informed consent to participate in this study. This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. 2.2. Mutational analysis Genomic DNA of all subjects were extracted from the whole blood using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China). All exons and flanking introns of SLC26A4 were PCR amplified and sequenced in the 150 deaf probands with nonsyndromic EVA as previously described [4,5]. Two frequent mutations of SLC26A4 in Chinese Hans, c.919-2A>G and p.H723R, were screened in the 3056 normal hearing controls by an allelespecific PCR-based array (Catalog# 300065, CapitalBio Corp., Beijing, China) as previously described [8]. Briefly, single-strand DNA containing the targeted mutation sites were amplified by asymmetric PCR, hybridized to the array and imaged with a LuxScan 10 K-B Microarray Scanner (CapitalBio Corp., Beijing, China) following the manufacture’s protocol. All SLC26A4 mutations identified by the array were verified by Sanger sequencing. The frequencies of mono-allelic c.919-2A>G and p.H723R mutations were determined in the patients and controls. The statistical significance of the difference was analyzed by Fisher0 s exact test using SPSS software version-19 (SPSS Inc., Chicago, USA). 3. Results In the 150 Chinese Han deaf probands with non-syndromic EVA, bi-allelic, mono-allelic and no mutation of SLC26A4 were detected in 98 (65.3%), 18 (12.0%) and 34 (22.7%) probands, respectively (Table 1). Among them, c.919-2A>G and p.H723R were most frequent, accounting for 51.9% (111/214) and 9.4% (20/214) of all mutant alleles, respectively. We therefore selected c.919-2A>G and p.H723R as the representing mutations to test whether the presence of monoallelic mutations of SLC26A4 was etiologically associated with nonsyndromic EVA. Mono-allelic mutations of c.919-2A>G and p.H723R were detected in 10 and 3 non-syndromic EVA probands, respectively, accounting for a combined 8.7% of the probands (Table 2). In contrast, in the 3056 ethnically-matched normal hearing controls, mono-allelic c.919-2A>G and p.H723R were
Controls
* Only the c.919-2A >G and p.H723R mutations of SLC26A4 were genotyped in the 3056 controls.
identified in 33 and 9 controls, respectively, accounting for a combined 1.4% in the control group. The frequency of monoallelic c.919-2A>G and p.H723R mutations was significantly higher in the non-syndromic EVA probands than in the controls (P = 1.8 106).
X. Pang et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 1351–1353 Table 2 Mono-allelic c.919-2A>G and p.H723R mutations of SLC26A4 in 150 deaf probands with non-syndromic EVA and 3056 normal hearing controls. Mono-allelic mutations
c.919-2A>G/+ p.H723R/+ Total
EVA (n = 150)
Control (n = 3056)
No. of subjects
Percentage (%)
No. of subjects
Percentage (%)
10 3 13
6.7 2.0 8.7
33 9 42
1.1 0.3 1.4
P-value
1.8 106
1353
In summary, our study demonstrated that mono-allelic mutations of SLC26A4 in non-syndromic EVA probands are etiologically associated with this disorder, which warrants important consideration for patients with such genetic diagnosis. Further investigations should be performed to elucidate the additional causes. Conflict of interest No competing financial interests exist.
4. Discussion The present study aimed to compare the frequencies of monoallelic mutations of SLC26A4 between the deaf probands with nonsyndromic EVA and the normal hearing controls. For that purpose, sequencing of all exons and flanking introns of SLC26A4 were performed in the 150 probands. This approach, however, was not chosen for the large-scale screening of the 3056 controls for costefficiency reasons. Alternatively, we selected c.919-2A>G and p.H723R as the representative mutations of SLC26A4 for genotyping and further analysis, as these two mutations accounted for a combined 61.2% of all mutant alleles of SLC26A4 in our study. Since the heterozygous c.919-2A>G and p.H723R mutations were identified in controls with normal hearing, they are expected to be mono-allelic without further screening for other mutations of SLC26A4. Our study showed that mono-allelic mutations of SLC26A4 accounted for 12.0% of the 150 deaf probands with non-syndromic EVA, among which 8.7% were mono-allelic c.919-2A>G and p.H723R. This percentage was significantly higher than that (1.4%) of the 3056 normal hearing controls (P = 1.75 106, Table 2), suggesting that mono-allelic mutations of SLC26A4 in non-syndromic EVA probands are etiologically associated with this disorder. Considering the recessive inheritance of the non-syndromic EVA, we postulate that additional genetic variants may contribute to the etiology of the non-syndromic EVA patients with monoallelic mutations of SLC26A4. In some patients, for example, a hidden mutation may be present in the non-coding regions of SLC26A4. One such example is the c.103T>C mutation in the SLC26A4 promoter, which has been demonstrated to interfere with the transcriptional activation of SLC26A4 [9]. Genomic deletions of SLC26A4, as shown in a recent study [10], may be another type of SLC26A4 mutations that are undetectable by the regular mutation screening methods. Alternatively, genetic variants in other genes or even the environmental factors may also be involved. Examples of the former included mutations in FOXI1 and KCNJ10, two genes implicated in the pathogenesis of the non-syndromic EVA in patients with mono-allelic mutations of SLC26A4 [9,11].
Acknowledgments This research was supported by grants from National Natural Science Foundation of China (81222010 to TY), National Basic Research Program of China (2011CB504501 to HW), Minister of Science and Technology (2012BAI09B02 to HW), Shanghai Municipal Science and Technology Commission (14DZ1940102 to TY, 14DZ2260300 and 14DJ1400201 to HW, 15YF1407600 to YC), and Program for New Century Excellent Talents in University (NCET-130376 to TY). References [1] C.C. Morton, W.E. Nance, Newborn hearing screening—a silent revolution, N. Engl. J. Med. 354 (20) (2006) 2151–2164. [2] L.A. Everett, B. Glaser, J.C. Beck, J.R. Idol, A. Buchs, M. Heyman, et al., Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS), Nat. Genet. 17 (4) (1997) 411–422. [3] X.C. Li, L.A. Everett, A.K. Lalwani, D. Desmukh, T.B. Friedman, E.D. Green, et al., A mutation in PDS causes non-syndromic recessive deafness, Nat. Genet. 18 (3) (1998) 215–217. [4] Q.J. Wang, Y.L. Zhao, S.Q. Rao, Y.F. Guo, H. Yuan, L. Zong, et al., A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China, Clin. Genet. 72 (3) (2007) 245–254. [5] S. Albert, H. Blons, L. Jonard, D. Feldmann, P. Chauvin, N. Loundon, et al., SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations, Eur. J. Hum. Genet. 14 (6) (2006) 773–779. [6] L.A. Everett, H. Morsli, D.K. Wu, E.D. Green, Expression pattern of the mouse ortholog of the Pendred’s syndrome gene (Pds) suggests a key role for pendrin in the inner ear, Proc. Natl. Acad. Sci. U S A 96 (17) (1999) 9727–9732. [7] L.A. Everett, I.A. Belyantseva, K. Noben-Trauth, R. Cantos, A. Chen, S.I. Thakkar, et al., Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome, Hum. Mol. Genet. 10 (2) (2001) 153–161. [8] Qu C, Sun X, Shi Y, Gong A, Liang S, Zhao M, et al.;1; Microarray-based mutation detection of pediatric sporadic nonsyndromic hearing loss in China. Int J Pediatr Otorhinolaryngol 76 (2) 235–239. [9] T. Yang, H. Vidarsson, S. Rodrigo-Blomqvist, S.S. Rosengren, S. Enerback, R.J. Smith, Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4), Am. J. Hum. Genet. 80 (6) (2007) 1055–1063. [10] L.M. Pique, M.L. Brennan, C.J. Davidson, F. Schaefer, J. Greinwald Jr., I. Schrijver, Mutation analysis of the SLC26A4, FOXI1 and KCNJ10 genes in individuals with congenital hearing loss, Peer J. 2 (2014) e384. [11] T. Yang, J.G. Gurrola 2nd, H. Wu, S.M. Chiu, P. Wangemann, P.M. Snyder, et al., Mutations of KCNJ10 together with mutations of SLC26A4 cause digenic nonsyndromic hearing loss associated with enlarged vestibular aqueduct syndrome, Am. J. Hum. Genet. 84 (5) (2009) 651–657.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Mono-allelic mutations of SLC26A4 is over-presented in deaf patients with non-syndromic enlarged vestibular aqueduct Xiuhong Pang a,b,c,d,1, Yongchuan Chai a,b,c,1, Penghui Chen a,b,c, Longxia He a,b,c, Xiaowen Wang a,b,c, Hao Wu a,b,c,**, Tao Yang a,b,c,* a
Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, China d Department of Otorhinolaryngology-Head and Neck Surgery, Taizhou People’s Hospital, Jiangsu Province, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 March 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online 11 June 2015
Objectives: Recessive mutations of SLC26A4 are the major cause of hearing impairment associated with enlarged vestibular aqueduct (EVA). In a significant percentage of non-syndromic EVA patients, however, only mono-allelic mutations of SLC26A4 can be identified. In this study, we aimed to evaluate whether presence of mono-allelic mutations of SLC26A4 in those patients was coincidental or etiologically associated with the disorder. Methods: The exons and flanking splicing sites of SLC26A4 were sequenced in 150 Chinese Han deaf probands with non-syndromic EVA. c.919-2A >G and p.H723R, two frequent mutations of SLC26A4 in Chinese Hans, were screened by an allele-specific PCR-based array in 3056 ethnically-matched normal hearing controls. The frequency of mono-allelic c.919-2A >G and p.H723R mutations was determined in each group. The statistical significance of the difference was analyzed by Fisher0 s exact test. Results: Bi-allelic, mono-allelic and no mutation of SLC26A4 were detected in 98 (65.3%), 18 (12%) and 34 (22.67%) deaf probands with non-syndromic EVA, respectively. The frequency of mono-allelic c.9192A >G and p.H723R mutations were significantly higher in the 150 deaf probands with non-syndromic EVA (8.67%) than in the 3056 normal hearing controls (1.4%, P = 1.8 106). Conclusion: Presence of mono-allelic mutations of SLC26A4 in non-syndromic EVA patients is etiologically associated with this disorder. Additional genetic or environmental causes may be present in those patients and demand further investigation and consideration during the genetic diagnosis and counseling. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Enlarged vestibular aqueduct SLC26A4 Mono-allelic mutation Genetic diagnosis
1. Introduction Enlarged vestibular aqueduct (EVA) is the most common inner ear malformation seen in the deaf children, accounting for approximately 12% of the permanent hearing impairment at age 4 [1]. The associated hearing impairment can be non-syndromic (DFNB4, OMIM#600791) or with additional thyroid dysfunction (Pendred Syndrome, OMIM#274600). In either case, autosomal
* Corresponding author at: Ear Institute, Shanghai Jiaot Tong University School of Medicine, Shanghai, China. Tel.: +86 21 25078893; fax: +86 21 65152394. ** Corresponding author at: Department of Otolaryngology-Head & Neck Surgery, Xinhua Hospital, Shanghai Jiaot Tong University School of Medicine, Shanghai, China. Tel.: +86 21 55570010; fax: +86 21 65152394. E-mail addresses: [email protected] (H. Wu), [email protected] (T. Yang). 1 Those two authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijporl.2015.06.009 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
recessive mutations of SLC26A4 are the major cause for the disorder [2,3]. Two large-scale mutation screenings of SLC26A4, for example, showed that bi-allelic mutations of SLC26A4 can be detected in 88.4% of non-syndromic EVA patients in Chinese Hans [4] and 24% in Caucasians [5]. SLC26A4 encodes pendrin, an anion transporter that plays a key role in maintaining the endocochlear potential [6]. Mutant mouse homozygous for the targeted deletion of SLC26A4 recapitulate the EVA phenotype and is profoundly deaf, while the heterozygous mouse is phenotypically normal [7]. Both animal models and human familial cases of EVA support the recessive inheritance of this disorder. In the aforementioned mutation screenings, however, sequencing of the exons and flanking splicing sites of SLC26A4 detected mono-allelic mutations in 9.5% of non-syndromic EVA patients in Chinese Hans [4] and 16% in Caucasians [5].
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X. Pang et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 1351–1353
In this study, we aimed to investigate whether the presence of mono-allelic mutations of SLC26A4 in non-syndromic EVA patients is coincidental or etiologically associated with the disorder. The frequencies of mono-allelic mutations of SLC26A4 were determined and compared between 150 Chinese Han deaf probands with non-syndromic EVA and 3056 ethnically-matched normal hearing controls.
Table 1 SLC26A4 mutations identified in the 150 probands with nonsyndromic EVA and the 3056 controls*. Subjects
Allele 1
Allele 2
No. of subjects (%)
EVA patients
c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G c.919-2A > G p.H723R p.H723R p.H723R p.H723R p.H723R p.H723R p.H723R p.A360V p.N392Y p.N392Y p.N392Y p.N392Y c.916-917insG p.T410 M p.V650D c.919-2A>G p.H723R c.916_917insG p.G197R p.V163I p.S532R Wild type c.919-2A>G p.H723R Wild type
c.919-2A > G p.H723R p.R409H p.G197R c.1520delT p.T410 M p.E682X c.916-917insG p.N392Y c.349delC c.1685_1686insA p.Q413R p.R409L p.T721 M p.G139V p.L676Q p.S252P c.416-1G > A p.M147V c.162 + 3delG p.S532R c.2085 + 1C > T p.W83X p.G497S c.1707 + 5G > A p.V650D c.1264-12T > A p.T94I p.E37X c.1001 + 2T > C p.L215S p.L703V c.1547_1548insC c.1133_1135delCCA p.S447L p.T132I p.K440X p.H723R p.Q446X p.R409 C p.R409H p.M147V p.T410 M p.S252P p.L676Q c.1547_1548insC p.I529S p.V659L p.S532R p.M147V p.D661Y c.1707 + 5G > A Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type
20 (13.33) 9 (6) 7 (4.67) 3 (2) 3 (2) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 2 (1.33) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 1 (0.67) 2 (1.33) 2 (1.33) 1(0.67) 1(0.67) 1(0.67) 1(0.67) 1(0.67) 1(0.67) 10 (6.67) 3 (2) 2 (1.33) 1(0.67) 1(0.67) 1(0.67) 34 (22.67) 33 (1.07) 9 (0.30) 3014 (98.63)
2. Materials and methods 2.1. Subjects and clinical evaluation One hundred and fifty Chinese Han deaf probands with nonsyndromic EVA and 3056 Chinese Han normal hearing controls were recruited from Xinhua Hospital, Shanghai, China. A comprehensive family history and physical examinations (with special attention to the thyroid, skeletal, ophthalmologic and mental abnormalities) were performed in all patients. Auditory evaluations included pure-tone audiometry, tympanometry and otoscope examination. Enlargement of the vestibular aqueduct were confirmed by high-resolution computed tomography (CT) scan of the temporal bone. The diagnostic criterion was defined as diameter of >1.5 mm at the midpoint between the common crus and the external aperture. All subjects gave written, informed consent to participate in this study. This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. 2.2. Mutational analysis Genomic DNA of all subjects were extracted from the whole blood using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China). All exons and flanking introns of SLC26A4 were PCR amplified and sequenced in the 150 deaf probands with nonsyndromic EVA as previously described [4,5]. Two frequent mutations of SLC26A4 in Chinese Hans, c.919-2A>G and p.H723R, were screened in the 3056 normal hearing controls by an allelespecific PCR-based array (Catalog# 300065, CapitalBio Corp., Beijing, China) as previously described [8]. Briefly, single-strand DNA containing the targeted mutation sites were amplified by asymmetric PCR, hybridized to the array and imaged with a LuxScan 10 K-B Microarray Scanner (CapitalBio Corp., Beijing, China) following the manufacture’s protocol. All SLC26A4 mutations identified by the array were verified by Sanger sequencing. The frequencies of mono-allelic c.919-2A>G and p.H723R mutations were determined in the patients and controls. The statistical significance of the difference was analyzed by Fisher0 s exact test using SPSS software version-19 (SPSS Inc., Chicago, USA). 3. Results In the 150 Chinese Han deaf probands with non-syndromic EVA, bi-allelic, mono-allelic and no mutation of SLC26A4 were detected in 98 (65.3%), 18 (12.0%) and 34 (22.7%) probands, respectively (Table 1). Among them, c.919-2A>G and p.H723R were most frequent, accounting for 51.9% (111/214) and 9.4% (20/214) of all mutant alleles, respectively. We therefore selected c.919-2A>G and p.H723R as the representing mutations to test whether the presence of monoallelic mutations of SLC26A4 was etiologically associated with nonsyndromic EVA. Mono-allelic mutations of c.919-2A>G and p.H723R were detected in 10 and 3 non-syndromic EVA probands, respectively, accounting for a combined 8.7% of the probands (Table 2). In contrast, in the 3056 ethnically-matched normal hearing controls, mono-allelic c.919-2A>G and p.H723R were
Controls
* Only the c.919-2A >G and p.H723R mutations of SLC26A4 were genotyped in the 3056 controls.
identified in 33 and 9 controls, respectively, accounting for a combined 1.4% in the control group. The frequency of monoallelic c.919-2A>G and p.H723R mutations was significantly higher in the non-syndromic EVA probands than in the controls (P = 1.8 106).
X. Pang et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 1351–1353 Table 2 Mono-allelic c.919-2A>G and p.H723R mutations of SLC26A4 in 150 deaf probands with non-syndromic EVA and 3056 normal hearing controls. Mono-allelic mutations
c.919-2A>G/+ p.H723R/+ Total
EVA (n = 150)
Control (n = 3056)
No. of subjects
Percentage (%)
No. of subjects
Percentage (%)
10 3 13
6.7 2.0 8.7
33 9 42
1.1 0.3 1.4
P-value
1.8 106
1353
In summary, our study demonstrated that mono-allelic mutations of SLC26A4 in non-syndromic EVA probands are etiologically associated with this disorder, which warrants important consideration for patients with such genetic diagnosis. Further investigations should be performed to elucidate the additional causes. Conflict of interest No competing financial interests exist.
4. Discussion The present study aimed to compare the frequencies of monoallelic mutations of SLC26A4 between the deaf probands with nonsyndromic EVA and the normal hearing controls. For that purpose, sequencing of all exons and flanking introns of SLC26A4 were performed in the 150 probands. This approach, however, was not chosen for the large-scale screening of the 3056 controls for costefficiency reasons. Alternatively, we selected c.919-2A>G and p.H723R as the representative mutations of SLC26A4 for genotyping and further analysis, as these two mutations accounted for a combined 61.2% of all mutant alleles of SLC26A4 in our study. Since the heterozygous c.919-2A>G and p.H723R mutations were identified in controls with normal hearing, they are expected to be mono-allelic without further screening for other mutations of SLC26A4. Our study showed that mono-allelic mutations of SLC26A4 accounted for 12.0% of the 150 deaf probands with non-syndromic EVA, among which 8.7% were mono-allelic c.919-2A>G and p.H723R. This percentage was significantly higher than that (1.4%) of the 3056 normal hearing controls (P = 1.75 106, Table 2), suggesting that mono-allelic mutations of SLC26A4 in non-syndromic EVA probands are etiologically associated with this disorder. Considering the recessive inheritance of the non-syndromic EVA, we postulate that additional genetic variants may contribute to the etiology of the non-syndromic EVA patients with monoallelic mutations of SLC26A4. In some patients, for example, a hidden mutation may be present in the non-coding regions of SLC26A4. One such example is the c.103T>C mutation in the SLC26A4 promoter, which has been demonstrated to interfere with the transcriptional activation of SLC26A4 [9]. Genomic deletions of SLC26A4, as shown in a recent study [10], may be another type of SLC26A4 mutations that are undetectable by the regular mutation screening methods. Alternatively, genetic variants in other genes or even the environmental factors may also be involved. Examples of the former included mutations in FOXI1 and KCNJ10, two genes implicated in the pathogenesis of the non-syndromic EVA in patients with mono-allelic mutations of SLC26A4 [9,11].
Acknowledgments This research was supported by grants from National Natural Science Foundation of China (81222010 to TY), National Basic Research Program of China (2011CB504501 to HW), Minister of Science and Technology (2012BAI09B02 to HW), Shanghai Municipal Science and Technology Commission (14DZ1940102 to TY, 14DZ2260300 and 14DJ1400201 to HW, 15YF1407600 to YC), and Program for New Century Excellent Talents in University (NCET-130376 to TY). References [1] C.C. Morton, W.E. Nance, Newborn hearing screening—a silent revolution, N. Engl. J. Med. 354 (20) (2006) 2151–2164. [2] L.A. Everett, B. Glaser, J.C. Beck, J.R. Idol, A. Buchs, M. Heyman, et al., Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS), Nat. Genet. 17 (4) (1997) 411–422. [3] X.C. Li, L.A. Everett, A.K. Lalwani, D. Desmukh, T.B. Friedman, E.D. Green, et al., A mutation in PDS causes non-syndromic recessive deafness, Nat. Genet. 18 (3) (1998) 215–217. [4] Q.J. Wang, Y.L. Zhao, S.Q. Rao, Y.F. Guo, H. Yuan, L. Zong, et al., A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China, Clin. Genet. 72 (3) (2007) 245–254. [5] S. Albert, H. Blons, L. Jonard, D. Feldmann, P. Chauvin, N. Loundon, et al., SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations, Eur. J. Hum. Genet. 14 (6) (2006) 773–779. [6] L.A. Everett, H. Morsli, D.K. Wu, E.D. Green, Expression pattern of the mouse ortholog of the Pendred’s syndrome gene (Pds) suggests a key role for pendrin in the inner ear, Proc. Natl. Acad. Sci. U S A 96 (17) (1999) 9727–9732. [7] L.A. Everett, I.A. Belyantseva, K. Noben-Trauth, R. Cantos, A. Chen, S.I. Thakkar, et al., Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome, Hum. Mol. Genet. 10 (2) (2001) 153–161. [8] Qu C, Sun X, Shi Y, Gong A, Liang S, Zhao M, et al.;1; Microarray-based mutation detection of pediatric sporadic nonsyndromic hearing loss in China. Int J Pediatr Otorhinolaryngol 76 (2) 235–239. [9] T. Yang, H. Vidarsson, S. Rodrigo-Blomqvist, S.S. Rosengren, S. Enerback, R.J. Smith, Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4), Am. J. Hum. Genet. 80 (6) (2007) 1055–1063. [10] L.M. Pique, M.L. Brennan, C.J. Davidson, F. Schaefer, J. Greinwald Jr., I. Schrijver, Mutation analysis of the SLC26A4, FOXI1 and KCNJ10 genes in individuals with congenital hearing loss, Peer J. 2 (2014) e384. [11] T. Yang, J.G. Gurrola 2nd, H. Wu, S.M. Chiu, P. Wangemann, P.M. Snyder, et al., Mutations of KCNJ10 together with mutations of SLC26A4 cause digenic nonsyndromic hearing loss associated with enlarged vestibular aqueduct syndrome, Am. J. Hum. Genet. 84 (5) (2009) 651–657.