Mutations of KCNJ10 Together with Mutations of SLC26A4 Cause Digenic Nonsyndromic Hearing Loss Associated with Enlarged Vestibular Aqueduct Syndrome
2009; Elsevier BV; Volume: 84; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2009.04.014
ISSN1537-6605
AutoresTao Yang, José Gurrola, Hao Wu, Sui Mei Chiu, Philine Wangemann, Peter M. Snyder, Richard J. Smith,
Tópico(s)Hearing Loss and Rehabilitation
ResumoMutations in SLC26A4 cause nonsyndromic hearing loss associated with an enlarged vestibular aqueduct (EVA, also known as DFNB4) and Pendred syndrome (PS), the most common type of autosomal-recessive syndromic deafness. In many patients with an EVA/PS phenotype, mutation screening of SLC26A4 fails to identify two disease-causing allele variants. That a sizable fraction of patients carry only one SLC26A4 mutation suggests that EVA/PS is a complex disease involving other genetic factors. Here, we show that mutations in the inwardly rectifying K+ channel gene KCNJ10 are associated with nonsyndromic hearing loss in carriers of SLC26A4 mutations with an EVA/PS phenotype. In probands from two families, we identified double heterozygosity in affected individuals. These persons carried single mutations in both SLC26A4 and KCNJ10. The identified SLC26A4 mutations have been previously implicated in EVA/PS, and the KCNJ10 mutations reduce K+ conductance activity, which is critical for generating and maintaining the endocochlear potential. In addition, we show that haploinsufficiency of Slc26a4 in the Slc26a4+/− mouse mutant results in reduced protein expression of Kcnj10 in the stria vascularis of the inner ear. Our results link KCNJ10 mutations with EVA/PS and provide further support for the model of EVA/PS as a multigenic complex disease. Mutations in SLC26A4 cause nonsyndromic hearing loss associated with an enlarged vestibular aqueduct (EVA, also known as DFNB4) and Pendred syndrome (PS), the most common type of autosomal-recessive syndromic deafness. In many patients with an EVA/PS phenotype, mutation screening of SLC26A4 fails to identify two disease-causing allele variants. That a sizable fraction of patients carry only one SLC26A4 mutation suggests that EVA/PS is a complex disease involving other genetic factors. Here, we show that mutations in the inwardly rectifying K+ channel gene KCNJ10 are associated with nonsyndromic hearing loss in carriers of SLC26A4 mutations with an EVA/PS phenotype. In probands from two families, we identified double heterozygosity in affected individuals. These persons carried single mutations in both SLC26A4 and KCNJ10. The identified SLC26A4 mutations have been previously implicated in EVA/PS, and the KCNJ10 mutations reduce K+ conductance activity, which is critical for generating and maintaining the endocochlear potential. In addition, we show that haploinsufficiency of Slc26a4 in the Slc26a4+/− mouse mutant results in reduced protein expression of Kcnj10 in the stria vascularis of the inner ear. Our results link KCNJ10 mutations with EVA/PS and provide further support for the model of EVA/PS as a multigenic complex disease. Enlargement of the vestibular aqueduct (EVA) is the most common bony inner-ear malformation resolved by computed tomography. It is associated with nonsyndromic hearing loss (DFNB4 [MIM 600791]) and with Pendred syndrome (PS [MIM 274600]), a common type of syndromic hearing loss that includes thyroid dysfunction as part of the disease phenotype. EVA/PS has been causally linked to mutations in the anion transporter gene SLC26A4, which encodes the protein pendrin (MIM 605646).1Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. et al.Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (948) Google Scholar, 2Li X.C. Everett L.A. Lalwani A.K. Desmukh D. Friedman T.B. Green E.D. Wilcox E.R. A mutation in PDS causes non-syndromic recessive deafness.Nat. Genet. 1998; 18: 215-217Crossref PubMed Scopus (317) Google Scholar In the inner ear, pendrin is expressed in cells of the external sulcus, epithelium cells of the endolymphatic duct and sac, and nonsensory cells at the margin of the maculae of the utricle and saccule.3Everett L.A. Morsli H. Wu D.K. Green E.D. 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. USA. 1999; 96: 9727-9732Crossref PubMed Scopus (254) Google Scholar, 4Yoshino T. Sato E. Nakashima T. Nagashima W. Teranishi M.A. Nakayama A. Mori N. Murakami H. Funahashi H. Imai T. The immunohistochemical analysis of pendrin in the mouse inner ear.Hear. Res. 2004; 195: 9-16Crossref PubMed Scopus (26) Google Scholar In all of these cell types, the apical surface is exposed to endolymph, consistent with pendrin's role as an anion transporter. The mouse mutant homozygous for the targeted deletion of Slc26a4 recapitulates the human phenotype—it is profoundly deaf and has EVA.5Everett L.A. Belyantseva I.A. Noben-Trauth K. Cantos R. Chen A. Thakkar S.I. Hoogstraten-Miller S.L. Kachar B. Wu D.K. Green E.D. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.Hum. Mol. Genet. 2001; 10: 153-161Crossref PubMed Scopus (328) Google Scholar Although pathogenic mutations in SLC26A4 have been shown to abolish membrane targeting or abrogate ion transport,6Pera A. Dossena S. Rodighiero S. Gandia M. Botta G. Meyer G. Moreno F. Nofziger C. Hernandez-Chico C. Paulmichl M. Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred syndrome and nonsyndromic EVA.Proc. Natl. Acad. Sci. USA. 2008; 105: 18608-18613Crossref PubMed Scopus (84) Google Scholar, 7Rotman-Pikielny P. Hirschberg K. Maruvada P. Suzuki K. Royaux I.E. Green E.D. Kohn L.D. Lippincott-Schwartz J. Yen P.M. Retention of pendrin in the endoplasmic reticulum is a major mechanism for Pendred syndrome.Hum. Mol. Genet. 2002; 11: 2625-2633Crossref PubMed Scopus (79) Google Scholar the exact link between the loss of functional pendrin and hearing impairment is not well understood. There is evidence to support a role for pendrin in mediating secretion of HCO3− from epithelial cells of the spiral prominence into the cochlear endolymph, where loss of pendrin in Slc26a4−/− mice causes endolymphatic acidification and Ca2+ overloading. These ionic changes could potentially inhibit mechanosensory transduction and lead to hair cell degeneration.8Wangemann P. Nakaya K. Wu T. Maganti R.J. Itza E.M. Sanneman J.D. Harbidge D.G. Billings S. Marcus D.C. Loss of cochlear HCO3- secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model.Am. J. Physiol. Renal Physiol. 2007; 292: F1345-F1353Crossref PubMed Scopus (176) Google Scholar However, it has also been postulated that cochlear development in Slc26a4−/− mice is compromised by local hypothyroidism (S. Billings et al., 2008, Assoc. Res. Otolaryngol., abstract). Wangemann and colleagues have raised another intriguing possibility based on protein expression studies in the Slc26a4−/− mouse, which show that loss of pendrin leads to reduced protein levels of the K+ channel Kcnj10.9Singh R. Wangemann P. Free radical stress-mediated loss of Kcnj10 protein expression in stria vascularis contributes to deafness in Pendred syndrome mouse model.Am. J. Physiol. Renal Physiol. 2008; 294: F139-F148Crossref PubMed Scopus (53) Google Scholar, 10Wangemann P. Itza E.M. Albrecht B. Wu T. Jabba S.V. Maganti R.J. Lee J.H. Everett L.A. Wall S.M. Royaux I.E. et al.Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.BMC Med. 2004; 2: 30Crossref PubMed Scopus (186) Google Scholar KCNJ10 (MIM 602208) is an inwardly rectifying K+ channel subunit abundantly expressed in the plasma membrane of intermediate cells of the stria vascularis. Temporal expression of murine Kcnj10 correlates with the onset of the endocochlear potential, a potential essential for auditory function. This potential is absent in Kcnj10−/− mice and is abolished when K+ channel blockers are used to inhibit Kcnj10 channel activity, suggesting that deafness in the Slc26a4−/− mutant mouse is secondary to loss of Kcnj10 function.11Marcus D.C. Characterization of potassium permeability of cochlear duct by perilymphatic perfusion of barium.Am. J. Physiol. 1984; 247: C240-C246PubMed Google Scholar, 12Marcus D.C. Wu T. Wangemann P. Kofuji P. KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential.Am. J. Physiol. Cell Physiol. 2002; 282: C403-C407Crossref PubMed Scopus (242) Google Scholar, 13Takeuchi S. Ando M. Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils.Cell Tissue Res. 1998; 293: 271-275Crossref PubMed Scopus (45) Google Scholar, 14Takeuchi S. Kakigi A. Takeda T. Saito H. Irimajiri A. Intravascularly applied K(+)-channel blockers suppress differently the positive endocochlear potential maintained by vascular perfusion.Hear. Res. 1996; 101: 181-185Crossref PubMed Scopus (16) Google Scholar The onset of hearing loss varies considerably in EVA/PS. Although many patients are congenitally deaf, in others the onset of hearing loss occurs during childhood, suggesting that EVA/PS is compatible with hearing and that environmental or other genetic factors contribute to the loss of hearing. On the basis of the close link between protein expression levels of KCNJ10 and pendrin and the functional importance of KCNJ10 in the generation of the endocochlear potential, we hypothesized that mutations in KCNJ10 might play a role in the pathogenesis of EVA/PS. Persons with EVA/PS were ascertained through hearing loss referrals based on results of temporal bone computed tomography or magnetic resonance imaging. Their evaluation also included a complete history and physical examination. For classification of EVA, enlargement of the vestibular aqueduct had to be greater than 1.5 mm at a point midway between the endolymphatic sac and vestibule. For classification of Mondini dysplasia, the cochlea also had to be abnormal, with incomplete partition and a scala communis. All procedures were approved by the institution review board at the University of Iowa, and all subjects or the parents of minors gave written informed consent for genetic testing. DNA was extracted from whole blood via standard procedures.15Grimberg J. Nawoschik S. Belluscio L. McKee R. Turck A. Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood.Nucleic Acids Res. 1989; 17: 8390Crossref PubMed Scopus (434) Google Scholar Mutation screening of SLC26A4 was completed by denaturing high-performance liquid chromatography (DHPLC) and bidirectional sequencing, as previously described.15Grimberg J. Nawoschik S. Belluscio L. McKee R. Turck A. Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood.Nucleic Acids Res. 1989; 17: 8390Crossref PubMed Scopus (434) Google Scholar, 16Prasad S. Kolln K.A. Cucci R.A. Trembath R.C. Van Camp G. Smith R.J. Pendred syndrome and DFNB4-mutation screening of SLC26A4 by denaturing high-performance liquid chromatography and the identification of eleven novel mutations.Am. J. Med. Genet. 2004; 124A: 1-9Crossref PubMed Scopus (56) Google Scholar For EVA/PS patients who carry only one SLC26A4 coding-sequence mutation, KCNJ10 was screened by bidirectional sequencing of amplicons generated by PCR amplification of all exons and splice sites. Primer sequences are listed in Table S1 (available online). The KCNJ10 coding region was PCR amplified from cDNA and cloned into the pSP64 Poly(A) vector (Promega, Madison, WI). The p.P194H and p.R348C mutations were introduced into KCNJ10 expression constructs by site-directed mutagenesis with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). In vitro transcription of KCNJ10 was performed with the mMESSAGE mMACHINE kit (Applied Biosystems/Ambion, Austin, TX), and the generated transcript (50 nl of a 1 ug/ul solution) was injected into defolliculated Xenopus oocytes. After injection, oocytes were incubated in MES-buffered saline (MBS) at 18°C. Electrophysiological studies were performed 24 hr later. Whole-cell electrophysiological studies were performed via two-electrode voltage clamping. In brief, currents were amplified with an Oocyte Clamp OC-725C (Warner Instruments, Hamden, CT), digitized with a MacLab/200 interface (ADInstruments, Colorado Springs, CO), and recorded and analyzed with Chart software (ADInstruments). The microelectrodes were filled with 3 M KCl, and during experiments, oocytes were bathed in a solution containing 90 mM KCl, 3 mM MgCl2, 5 mM HEPES, and 150 μM niflumic acid at pH 7.4. Oocytes were held at a potential of 0 mV by voltage clamping, and voltage steps of 2 s were applied in 20 mV increments. Differences between currents of wild-type (WT) and mutant KCNJ10 (n = 6) were compared with the Student's unpaired t test. Slopes of the current-voltage relationship in the linear range (−120 mV to 0 mV) were calculated by linear regression analysis. Cochleae were removed from P18 Slc26a4+/+, Slc26a4+/−, and Slc26a4−/− mice (n > 4 for each group) via procedures approved by the Institutional Animal Care and Use Committee of the University of Iowa. Stria vascularis fractions were obtained by microdissection under the surgical microscope and were then immediately transferred to 40 μl Tris-Triton buffer (50 mM Tris, 150 mM NaCl, 1% Triton-X) and homogenized on ice for 30 s by ultrasonic homogenization (Model 150VT, BioLogics, Manassas, VA). Extracted protein was stored at −80°C until use. Immunoblotting was performed as previously described,9Singh R. Wangemann P. Free radical stress-mediated loss of Kcnj10 protein expression in stria vascularis contributes to deafness in Pendred syndrome mouse model.Am. J. Physiol. Renal Physiol. 2008; 294: F139-F148Crossref PubMed Scopus (53) Google Scholar with slight modification, and all procedures were performed at room temperature unless otherwise noted. In brief, equal volumes of protein extract and Laemelli buffer containing 5% β-mercaptoethanol were mixed and heated at 70°C for 5 min, followed by SDS-PAGE gel electrophoresis (10% Ready-gel, BioRad Laboratories, Hercules, CA). Separated proteins were electrophoretically transferred to a nitrocellulose membrane (ProTran BA85, 0.45 μm pore size, Whatman, Florham Park, NJ), blocked at 4°C overnight in 5% nonfat dry milk in TBS with 0.1% Tween-20, then incubated with primary antibodies (rabbit anti-Kcnj10, 1:1000, Cat# APC-035, Alomone Labs, Jerusalem, Israel; rabbit anti-tubulin, 1:500, Cat# ab6046, Abcam, Cambridge, MA) in blocking buffer for 1 hr. Unbound primary antibody was removed by three 10 min washes with Tween TBS, and the membrane was then incubated with HRP-conjugated secondary antibody (Peroxidase AffiniPure Goat anti-Rabbit IgG, 1:4000, Cat# 111-035-045, Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 min. After three washes with Tween TBS, the membrane was developed by enhanced chemiluminescence (Amersham ECL Western Blotting Detection Reagents, GE Healthcare, Florham Park, NJ), read on a low-light digital camera (LAS-1000, Fujifilm Medical Systems, Stamford, CT), and quantified with Image Gauge software (Fujifilm Medical Systems, Stamford, CT). All experiments were repeated in triplicate. Kcnj10 protein expression differences in Slc26a4+/+, Slc26a4+/−, and Slc26a4−/− mice were compared by one-way analysis of variance (ANOVA) and the Dunn's post hoc test. We completed KCNJ10 mutation screening in 89 patients who had a clinical diagnosis of EVA/PS and were known carriers of only one SLC26A4 coding sequence mutation; promoter mutations and deletions of SCL26A4 were also excluded in this patient cohort. In two patients, we identified missense mutations in KCNJ10—a p.P194H (c.581C→A) mutation in patient 7740-1 and a p.R348C (c.1042C→T) mutation in patient 82120-1. Patient 7740-1 also carries a p.F335L (c.1003T→C) mutation in SLC26A4, and in Patient 82120-1, we identified a c.919-2A→G mutation (Table 1). In Family 82120, we were able to reconstruct haplotypes, which showed that the mother carries KCNJ10 p.R348C and the father carries SLC26A4 c.919-2A→G. An unaffected sibling carries only the KCNJ10 p.R348C variant (Figure 1). Both amino acid changes in KCNJ10 are conserved across most mammalian species (Figure S1), and neither change was found in ethnically matched normal-hearing controls of European (n = 200, 400 chromosomes) and Chinese (n = 200, 400 chromosomes) descent.Table 1KCNJ10 and SLC26A4 Mutations Identified in Two Probands with EVA/PSPatient No.Inner-Ear MalformationKCNJ10 MutationSLC26A4 Mutation82120-1EVAp.R348C / +c.919-2A→G / +7740-1EVA, Mondini dysplasiap.P194H / +p.F335L / + Open table in a new tab To determine whether the identified KCNJ10 mutations change K+ channel currents, we expressed WT and mutant KCNJ10 in Xenopus oocytes by mRNA injection and recorded evoked K+ currents in a two-electrode voltage clamp experiment. Figure 2A shows a representative current recording in an oocyte expressing WT KCNJ10. Control oocytes injected with equal volumes of water showed little current under evoking voltage commands (data not shown). So that the specificity of the measured currents was ensured, 150 μM niflumic acid was added in the bathing solution, blocking endogenous Cl− currents. In addition, the evoked current could be abolished when 1 mM Ba2+ was applied. Expression of WT KCNJ10 in Xenopus oocytes resulted in a weak inwardly rectifying K+ current similar to previously reported data.17Pessia M. Tucker S.J. Lee K. Bond C.T. Adelman J.P. Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.EMBO J. 1996; 15: 2980-2987Crossref PubMed Scopus (161) Google Scholar In oocytes expressing either H194 or C348 mutant KCNJ10, K+ channel conductance markedly decreased (Figure 2B). Compared to WT, the K+ currents of the mutant KCNJ10 were significantly reduced (p < 0.05) in the linear range (−120 mV to −20 mV), and the mean slopes (ΔI/ΔV) of the mutant KCNJ10 current-voltage relationships were reduced by 44% and 51% for C348 and H194, respectively (R2 > 0.99). These results suggest that K+ conductance is severely impaired by the mutations that we identified in these two patients. In family 82120, the c.919-2A→G splice-site mutation in SLC26A4 is predicted to cause skipping of exon 8, leading to premature protein truncation and haploinsufficiency. In the mouse mutant with a targeted deletion of Slc26a4, exon 8 is replaced by a neoR-containing cassette that similarly results in loss of functional protein.5Everett L.A. Belyantseva I.A. Noben-Trauth K. Cantos R. Chen A. Thakkar S.I. Hoogstraten-Miller S.L. Kachar B. Wu D.K. Green E.D. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.Hum. Mol. Genet. 2001; 10: 153-161Crossref PubMed Scopus (328) Google Scholar Given that deficiency of pendrin in Slc26a4−/− mice leads to loss of Kcnj10 protein in stria vascularis, we investigated whether haploinsufficiency would affect the Kcnj10 protein level in the stria vascularis by studying Slc26a4+/− animals. The stria vascularis of P18 mouse cochleae from Slc26a4−/−, Slc26a4+/−, and Slc26a4+/+ mice was isolated by microdissection, and total protein extracted from these tissue fractions was used for quantitative immunoblot analysis (Figure 3). In Slc26a4+/− mice, we observed reduced protein expression of Kcnj10 as compared to Slc26a4+/+ WT controls (75%, p < 0.05). In Slc26a4−/− mice, Kcnj10 protein expression was further reduced (49%, p < 0.05). These results indicate that haploinsufficiency of pendrin leads to reduced KCNJ10 protein expression. Many studies have suggested that EVA/PS is a complex disease.18Park H.J. Lee S.J. Jin H.S. Lee J.O. Go S.H. Jang H.S. Moon S.K. Lee S.C. Chun Y.M. Lee H.K. et al.Genetic basis of hearing loss associated with enlarged vestibular aqueducts in Koreans.Clin. Genet. 2005; 67: 160-165Crossref PubMed Scopus (108) Google Scholar, 19Pryor S.P. Madeo A.C. Reynolds J.C. Sarlis N.J. Arnos K.S. Nance W.E. Yang Y. Zalewski C.K. Brewer C.C. Butman J.A. et al.SLC26A4/PDS genotype-phenotype correlation in hearing loss with enlargement of the vestibular aqueduct (EVA): evidence that Pendred syndrome and non-syndromic EVA are distinct clinical and genetic entities.J. Med. Genet. 2005; 42: 159-165Crossref PubMed Scopus (200) Google Scholar, 20Tsukamoto K. Suzuki H. Harada D. Namba A. Abe S. Usami S. Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese.Eur. J. Hum. Genet. 2003; 11: 916-922Crossref PubMed Scopus (199) Google Scholar However, in the ten-year span following discovery of SLC26A4 as the primary gene responsible for EVA/PS, identification of factors that promote the onset of hearing loss in patients with EVA/PS has been limited to infections and head injuries and to genetic factors such as the expression of FOXI1, which controls the expression of SLC26A4.21Azaiez H. Yang T. Prasad S. Sorensen J.L. Nishimura C.J. Kimberling W.J. Smith R.J. Genotype-phenotype correlations for SLC26A4-related deafness.Hum. Genet. 2007; 122: 451-457Crossref PubMed Scopus (82) Google Scholar, 22Yang T. Vidarsson H. Rodrigo-Blomqvist S. Rosengren S.S. Enerback S. Smith R.J. Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4).Am. J. Hum. Genet. 2007; 80: 1055-1063Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar The goal of identifying genetic factors has been limited by the availablity of a sufficient number of families showing clear digenic inheritance that would make conventional positional cloning approaches feasible. As an alternative, we have adopted a candidate gene approach, selecting for in-depth study genes that have a strong functional link to SLC26A4. In addition, we have identified a cohort of patients in which to screen these candidates. Using this strategy in our earlier work, we selected FOXI1 for study on the basis of the observation that endolymphatic sac expression of Slc26a4 is absent in the Foxi1−/− mouse. We were able to show that heterozygosity for mutations in this transcription factor and SLC26A4 are causally related to the EVA/PS phenotype in humans.22Yang T. Vidarsson H. Rodrigo-Blomqvist S. Rosengren S.S. Enerback S. Smith R.J. Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4).Am. J. Hum. Genet. 2007; 80: 1055-1063Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar In this study, on the basis of work demonstrating that loss of Kcnj10 protein expression is a key event in the etiology of deafness in Slc26a4−/− mice, we hypothesized that genetic factors affecting expression of functional KCNJ10 channels might also contribute to penetrance of hearing loss in EVA/PS patients. We identified two patients who have been diagnosed with EVA/PS and who segregate mutations in both SLC26A4 and KCNJ10. The p.P194H and p.R348C mutations in KCNJ10 change two well-conserved aminio acids and were not observed in 800 control chromosomes. Our electrophysiologic study showed that these mutations are detrimental to channel activity and reduce K+ conductance by 44%–51% (Figure 2). Notably, given that KCNJ10 may form homomeric tetramers,17Pessia M. Tucker S.J. Lee K. Bond C.T. Adelman J.P. Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.EMBO J. 1996; 15: 2980-2987Crossref PubMed Scopus (161) Google Scholar heterozygosity for these mutations is predicted to affect over 90% of multimeric KCNJ10 channels. The p.F335L SLC26A4 mutation carried by patient 7740-1 has been reported in 14 of 668 EVA/PS patients but in none of 358 normal-hearing controls (p < 0.01, Fisher's exact test).19Pryor S.P. Madeo A.C. Reynolds J.C. Sarlis N.J. Arnos K.S. Nance W.E. Yang Y. Zalewski C.K. Brewer C.C. Butman J.A. et al.SLC26A4/PDS genotype-phenotype correlation in hearing loss with enlargement of the vestibular aqueduct (EVA): evidence that Pendred syndrome and non-syndromic EVA are distinct clinical and genetic entities.J. Med. Genet. 2005; 42: 159-165Crossref PubMed Scopus (200) Google Scholar, 22Yang T. Vidarsson H. Rodrigo-Blomqvist S. Rosengren S.S. Enerback S. Smith R.J. Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4).Am. J. Hum. Genet. 2007; 80: 1055-1063Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 23Albert S. Blons H. Jonard L. Feldmann D. Chauvin P. Loundon N. Sergent-Allaoui A. Houang M. Joannard A. Schmerber S. et al.SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations.Eur. J. Hum. Genet. 2006; 14: 773-779Crossref PubMed Scopus (170) Google Scholar, 24Choi B.Y. Stewart A.K. Madeo A.C. Pryor S.P. Lenhard S. Kittles R. Eisenman D. Kim H.J. Niparko J. Thomsen J. et al.Hypo-functional SLC26A4 variants associated with nonsyndromic hearing loss and enlargement of the vestibular aqueduct: genotype-phenotype correlation or coincidental polymorphisms?.Hum. Mutat. 2009; 30: 599-608Crossref PubMed Scopus (115) Google Scholar, 25Pera A. Villamar M. Vinuela A. Gandia M. Meda C. Moreno F. Hernandez-Chico C. A mutational analysis of the SLC26A4 gene in Spanish hearing-impaired families provides new insights into the genetic causes of Pendred syndrome and DFNB4 hearing loss.Eur. J. Hum. Genet. 2008; 16: 888-896Crossref PubMed Scopus (50) Google Scholar Functional studies of this variant have shown decreased Cl−/HCO3− exchange, suggesting that leucine at this position results in a hypofunctional protein.24Choi B.Y. Stewart A.K. Madeo A.C. Pryor S.P. Lenhard S. Kittles R. Eisenman D. Kim H.J. Niparko J. Thomsen J. et al.Hypo-functional SLC26A4 variants associated with nonsyndromic hearing loss and enlargement of the vestibular aqueduct: genotype-phenotype correlation or coincidental polymorphisms?.Hum. Mutat. 2009; 30: 599-608Crossref PubMed Scopus (115) Google Scholar Family 82120 is of Chinese origin, and the affected child carries the SLC26A4 c.919-2A→G mutation, which is highly prevalent in the Chinese population, where it accounts for more than half of all reported SLC26A4 mutant alleles.26Wang Q.J. Zhao Y.L. Rao S.Q. Guo Y.F. Yuan H. Zong L. Guan J. Xu B.C. Wang D.Y. Han M.K. et al.A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China.Clin. Genet. 2007; 72: 245-254Crossref PubMed Scopus (127) Google Scholar This mutation is within the 3′ splicing site of exon 8 and is predicted to lead to premature protein truncation and haploinsufficiency. Our quantitative immunoblot analysis in Slc26a4−/−, Slc26a4+/−, and Slc26a4+/+ mice indicates that haploinsufficiency for Slc26a4 leads to decreased expression of Kcnj10 in the stria vascularis, providing a pathogenic link between mutations in these two genes. Haploinsufficiency of only Slc26a4 in Slc26a4+/− mice or Kcnj10 in Kcnj10+/− mice, however, does not affect the magnitude of the endocochlear potential; in contrast, homozygous loss of Slc26a4 in Slc26a4−/− mice or of Kcnj10 in Kcnj10−/− mice leads to a complete loss of this potential.8Wangemann P. Nakaya K. Wu T. Maganti R.J. Itza E.M. Sanneman J.D. Harbidge D.G. Billings S. Marcus D.C. Loss of cochlear HCO3- secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model.Am. J. Physiol. Renal Physiol. 2007; 292: F1345-F1353Crossref PubMed Scopus (176) Google Scholar, 12Marcus D.C. Wu T. Wangemann P. Kofuji P. KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential.Am. J. Physiol. Cell Physiol. 2002; 282: C403-C407Crossref PubMed Scopus (242) Google Scholar Confirmation of a digenic disease mechanism in the patients that we have described will require generation of a mouse model carrying orthologous mutations in Slc26a4 and Kcnj10. The mechanism for the reduction of Kcnj10 protein expression in Slc26a4+/− mice is not understood. Previous studies have suggested that maintenance of normal endolymphatic K+ concentration in Slc26a4−/− mice leads to free-radical stress in the stria vascularis and that this free-radical stress causes loss of strial Kcnj10 and thereby loss of the endocochlear potential.8Wangemann P. Nakaya K. Wu T. Maganti R.J. Itza E.M. Sanneman J.D. Harbidge D.G. Billings S. Marcus D.C. Loss of cochlear HCO3- secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model.Am. J. Physiol. Renal Physiol. 2007; 292: F1345-F1353Crossref PubMed Scopus (176) Google Scholar, 9Singh R. 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Peters B.A. van der Heijden M.S. et al.Mutational analysis of the tyrosine phosphatome in colorectal cancers.Science. 2004; 304: 1164-1166Crossref PubMed Scopus (410) Google Scholar The effectiveness of this strategy is clearly demonstrated by our finding of single KCNJ10 mutations in two patients who also carry single mutations in SLC26A4 and have an EVA/PS phenotype. While this article was being written, Scholl and colleagues reported homozygous or compound heterozygous mutations in KCNJ10 in members of four kindreds with a recessive complex syndrome that includes seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance.31Scholl U.I. Choi M. Liu T. Ramaekers V.T. Hausler M.G. Grimmer J. Tobe S.W. Farhi A. Nelson-Williams C. Lifton R.P. Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10.Proc. Natl. Acad. Sci. USA. 2009; 106: 5842-5847Crossref PubMed Scopus (316) Google Scholar This complex phenotype is similar to that seen in Kcnj10−/− mouse mutants that exhibit severe ataxia, stress-induced seizures, spongiform vacuolation, axonal swellings, and degeneration, in addition to hearing loss.32Djukic B. Casper K.B. Philpot B.D. Chin L.S. McCarthy K.D. Conditional knock-out of Kir4.1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation.J. Neurosci. 2007; 27: 11354-11365Crossref PubMed Scopus (385) Google Scholar, 33Neusch C. Rozengurt N. Jacobs R.E. Lester H.A. Kofuji P. Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination.J. Neurosci. 2001; 21: 5429-5438Crossref PubMed Google Scholar In contrast, the EVA/PS phenotype associated with double heterozygosity for mutations in KCNJ10 and SLC26A4 represents a distinct disease entity in which only hearing loss is observed. Our functional characterization of the pathogenic interaction between SLC26A4 and KCNJ10 mutations suggests that this digenic combination of mutations is specifically associated with inner-ear dysfunction. On the basis of our data, we propose that KCNJ10 is an important genetic factor in the pathogenesis of EVA/PS. The mutations in SLC26A4 lead to hypofunction or haploinsufficiency. In association with EVA/PS, haploinsufficiency of SLC26A4 and oxidative stress converge to reduce strial expression of KCNJ10 protein. The consequence of decreased KCNJ10 expression is a reduced supply of K+ to marginal cells in the stria vascularis. In turn, these cells reduce their rate of K+ secretion. This self-limiting mechanism might account for the intermittent hearing-threshold recovery that is well documented in EVA/PS patients and leads to fluctuating and progressive hearing loss.21Azaiez H. Yang T. Prasad S. Sorensen J.L. Nishimura C.J. Kimberling W.J. Smith R.J. Genotype-phenotype correlations for SLC26A4-related deafness.Hum. Genet. 2007; 122: 451-457Crossref PubMed Scopus (82) Google Scholar, 34Abe S. Usami S. Hoover D.M. Cohn E. Shinkawa H. Kimberling W.J. Fluctuating sensorineural hearing loss associated with enlarged vestibular aqueduct maps to 7q31, the region containing the Pendred gene.Am. J. Med. Genet. 1999; 82: 322-328Crossref PubMed Scopus (68) Google Scholar, 35Stinckens C. Huygen P.L. Van Camp G. Cremers C.W. Pendred syndrome redefined. Report of a new family with fluctuating and progressive hearing loss.Adv. Otorhinolaryngol. 2002; 61: 131-141PubMed Google Scholar This paradigm provides new insight into the pathogenesis of EVA/PS and, as a corollary, suggests that if strial expression of KCNJ10 can be maintained, perhaps through controlling endolymph pH or limiting oxidative stress through medical therapy, hearing loss might be preventable in some persons with an EVA/PS phenotype. This study was supported in part by grants from the National Institute on Deafness and Other Communication Disorders (NIDCD) to R.J.H.S. (R01-DC02842) and P.W. (R01-DC1098), from the National Heart, Lung, and Blood Institute (NHLBI) to P.M.S. (HL072256), and from the Science and Technology Commission of Shanghai Municipality, China (STCSM) to H.W. (08411954500). The authors would like to thank the families who made this research possible. Download .pdf (.07 MB) Help with pdf files Document S1. One Figure and One Table The URLs for data presented herein are as follows:GenBank, http://ncbi.nlm.nih.gov/Genbank/Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.gov/Omim/
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