Original Paper Audiology Neurotology
Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
Received: March 17, 2014 Accepted after revision: July 28, 2014 Published online: October 24, 2014
Identification of Novel Functional Null Allele of SLC26A4 Associated with Enlarged Vestibular Aqueduct and Its Possible Implication Jeong Hun Jang a Jinsei Jung b, c Ah Reum Kim d Young Mi Cho e Min Young Kim e Sang Yeon Lee d Jae Young Choi c Jun Ho Lee d Byung Yoon Choi e a
Department of Otorhinolaryngology, Kyungpook National University College of Medicine, Daegu, Department of Pharmacology, Brain Korea 21 Project for Medical Science, c Department of Otorhinolaryngology, College of Medicine, Yonsei University, and d Department of Otorhinolaryngology, Seoul National University College of Medicine, Seoul, and e Department of Otorhinolaryngology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea
Key Words SLC26A4 · Vestibular aqueduct · Sensorineural hearing loss
Abstract Mutations in the SLC26A4 gene, which encodes pendrin, cause congenital hearing loss as a manifestation of Pendred syndrome (PS) with an iodide organification defect or nonsyndromic enlarged vestibular aqueduct (NSEVA, DFNB4). There have been reports of differences between PS and NSEVA, including their auditory phenotypes and molecular genetic bases. For appropriate genetic diagnosis and counseling, it is important to functionally characterize SLC26A4 variants. In this study, we identified and evaluated a novel null mutation of SLC26A4 and report our method of assessing the pathogenic potential of mutations in SLC26A4, one of the most frequent causative genes of deafness in humans. A 3-year-old female with progressive sensorineural hearing loss and her parents were recruited. They underwent clinical, audiological, radiological and genetic evaluations, which revealed that the female patient had an enlarged vestibular
© 2014 S. Karger AG, Basel 1420–3030/14/0195–0319$39.50/0 E-Mail [email protected] www.karger.com/aud
aqueduct and an incomplete partition type II anomaly in the cochlea bilaterally. Sanger sequencing of the SLC26A4 gene was also performed. For a confirmatory genetic diagnosis, we first characterized the anion/base exchange ability of mutant pendrin products in HEK 293 cells and, if necessary, evaluated whether the mutant pendrin traffics to the plasma membrane in COS-7 cells. We also expressed a null function mutant, p.H723R, and a previously documented polymorphism, p.P542R, as controls. The pure tone average was 66 dB HL in the right ear and 75 dB HL in the left ear. Sequencing of SLC26A4 revealed a known pathogenic mutation (p.H723R) and a novel missense variant (p.V510D) as a compound heterozygote. When we expressed the p.V510D mutant pendrin in mammalian cells, the rate constants for Cl–/ HCO3– exchange were 10.96 ± 4.79% compared with those of wild-type pendrin. This figure was comparable to that of p.H723R, indicating p.V510D to be another pathogenic mutation with a null function. The p.V510D pendrin product was shown to be entrapped in the endoplasmic reticulum (ER) at 24–30 h after transfection, and not trafficked to the plasma membrane in COS-7 cells, suggesting retention in
Byung Yoon Choi, MD, PhD Department of Otorhinolaryngology, Seoul National University Bundang Hospital Seoul National University College of Medicine 300 Gumi-dong, Bundang-gu, Seongnam 463-707 (Republic of Korea) E-Mail choiby @ snubh.org
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© 2014 S. Karger AG, Basel
Introduction
The SLC26A4 gene encodes pendrin, a transmembrane protein mediating the exchange of anions, such as Cl–, HCO3– and I– [Mount and Romero, 2004]. Mutations in this gene cause congenital sensorineural hearing loss associated with enlarged vestibular aqueduct (EVA; NIM 600709), either as part of the Pendred syndrome (PS; NIM 274600) or as a nonsyndromic form (NSEVA, DFNB4; NIM 600791) [Everett et al., 1997; Li et al., 1998; Usami et al., 1999]. EVA is the most common radiological malformation of the inner ear associated with hereditary sensorineural hearing loss [Valvassori and Clemis, 1978]. EVA is diagnosed based on the criterion of a vestibular aqueduct diameter greater than 1.5 mm at the midpoint between the common crus and the external aperture on temporal bone computed tomography. PS is characterized by an ‘iodide organification defect’ that may lead to goiter later, at the time of puberty, in addition to bilateral sensorineural hearing loss related to the EVA [Everett et al., 1997]. There has been controversy over the molecular genetic factors distinguishing PS from NSEVA. Recent studies have suggested that the phenotype of EVA (PS vs. NSEVA) tends to correlate more with the number of mutant alleles of SLC26A4, rather than with the type of mutation in Western populations [Azaiez et al., 2007; Choi et al., 2009b; Pryor et al., 2005]. In these studies, PS with iodide organification defect was shown to be strongly associated with 2 mutant alleles of SLC26A4 (M2), while NSEVA was frequently correlated with only 1 (M1) or no mutation (M0). Some researchers have also reported that the degree of hearing loss was correlated with the number of mutant alleles of SLC26A4 in a Caucasian population [Albert et al., 2006; Ishihara et al., 2010]. Furthermore, a correlation between the degree of residual hearing and the type of SLC26A4 variant within the M2 EVA group was reported recently in a Korean population with a higher 320
Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
proportion of M2 EVA subjects [Lee et al., 2013]. Thus, it is important to evaluate the pathogenic potential of SLC26A4 variants and correctly categorize EVA subjects into M1 or M2 for a correct diagnosis, prediction of the prognosis and counseling of these families. Pendrin, a 73-kDa polytopic transmembrane protein containing 12 putative domains, exchanges Cl– and I– across the apical membrane of thyroid follicular cells [Gillam et al., 2004] and exchanges Cl– and HCO–3 in the nonsensory epithelia of the inner ear [Wangemann et al., 2007]. Previous studies have suggested that the mechanism of pathogenesis involves disruption of trafficking and alterations in the anion exchange activity of mutant pendrins [Rotman-Pikielny et al., 2002; Taylor et al., 2002]. Incorrect N-glycan processing has been associated with the intracellular retention and endoplasmic-reticulum (ER)-associated degradation of mutant pendrins [Yoon et al., 2008]. To date, more than 150 mutations of the SLC26A4 gene have been found to be associated with human deafness. However, only a subset of these mutations has been characterized functionally. Recently, several studies have reported that the abnormal transport function and abolished anion exchange activity of some missense pendrin mutants, such as p.H723R, could be restored substantially simply by inducing plasma membrane targeting of the mutant using a chemical chaperone, low temperature incubation or drugs such as salicylate [Ishihara et al., 2010; Yoon et al., 2008]. Missense pendrin mutants produce abnormal proteins which are retained in the ER as a result of misfolding, which is the main pathogenic mechanism of PS [Rotman-Pikielny et al., 2002]. Thus, it is vital to identify the pathogenic mechanism (mislocalization vs. intrinsic defect in transporter activity) of the missense variants of SLC26A4 to find the best candidates for future pharmacotherapy. The aims of this study were to define the pathogenic potential of a novel missense variant of SLC26A4 and to determine the pathogenic mechanism.
Material and Methods Subjects Written informed consent was obtained from parents of SH123-253 according to a protocol approved by the Seoul National University Hospital Institutional Review Board prior to the study (IRB Y-H-0905-041-281). Among the patients at the genetic hearing loss clinic at the Department of Otorhinolaryngology at Seoul National University
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the ER and abnormal trafficking as the pathogenic mechanism. This was similar to p.H723R, which is a null function founder mutant in this population but is a candidate variant for future drug therapy to rescue the abnormal cell trafficking. Impaired cellular trafficking due to ER retention and abolished exchange activity of the newly detected p.V510D indicates the pathogenic potential of this variant. These missense variants may be good candidate variants for drug therapy if the intrinsic exchange activity is not damaged by the change.
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Fig. 1. a Pedigree of the family, showing the probability of an autosomal recessive inheritance pattern. Squares = Males; circles = females; arrow = proband; black circle = individual with hearing loss. b Pure-tone audiogram of the patient with the novel mutation. c Axial view of temporal bone computed tomography shows the EVAs (white arrows) and cochlear hypoplasia (black arrowheads).
SLC26A4 Genotype Analysis Figure 1a shows the phenotype and family history of the patient. Genomic DNA of the patient and parents was isolated from peripheral blood leukocytes using GentraPureGene (Qiagen, Valencia, Calif., USA). Polymerase chain reaction amplification of the 21 coding exons and intron-exon boundaries of the SLC26A4 gene was performed using specific primers. DNA samples were evaluated to determine segregation and the meiotic phase configuration of the mutant alleles of SLC26A4 by sequence analyses of affected exons in the parents and the proband. Generation of Constructs Wild-type SCL26A4 cDNA was cloned into the KpnI and XhoI sites of pEGFP (enhanced green fluorescent protein)-N1 [Taylor et al., 2002] and into the KpnI and NotI sites of pcDNA3.1(+) for site-directed mutagenesis to generate missense expression constructs. The mutagenesis primer set designed was 510F: 5′-TATATTTGGACTGTTGACTGTGGACCTGAGAGTTCAGTT-3′ and 510R: 5′-AACTGAACTCTCAGGTCCACAGTCAACAGTCCAAATATA-3′. The entire cDNA inserts were sequenced to confirm the absence of any unintended mutation. For a control, we also subcloned p.P542R into the same site of pcDNA3.1(+) as a polymorphism control and p.H723R as a null allele control for anion exchange (using primers 542F: 5′-TTA-
Null Mutant Allele of SLC26A4
CAAAAACATTGAAGAACGTCAAGGAGTGAAGATTCTT-3′ and 542R: 5′-AAGAATCTTCACTCCTTGACGTTCTTCAATGTTTTTGTAA-3′). Again, the entire cDNA inserts were sequenced to confirm the absence of any unintended mutation. Intracellular pH and Cl–/HCO3– Exchange Measurements of pHi in human embryonic kidney 293 cells, which have minimal intrinsic anion exchange activity, were performed using a pH-sensitive fluorescent probe, biscarboxyethylcarboxyfluorescein (BCECF) [Yoon et al., 2008]. Briefly, cells were transiently transfected with wild-type or mutant pendrin, and pHi was monitored. Control cells were transfected with mock vector. After adding BCECF, cells were perfused with a HCO–3-buffered solution (in mmol/l): 116 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Dglucose, 5 HEPES and 25 NaHCO3, pH 7.4. BCECF fluorescence was recorded at excitation wavelengths of 490 and 440 nm at a resolution of 2/s on a recording setup (Delta Ram; PTI Inc., Birmingham, N.J., USA). Cl–/HCO–3 exchange activities were estimated from the initial slope of the 490/440 ratio increase as a result of replacing chloride with equimolar gluconate in the HCO–3-containing buffer (25 mM HCO–3 with 5% CO2). The anion exchange activities of the p.H723R and p.P542R mutant pendrins, known as a functional null allele mutant and a benign polymorphic mutant, respectively [Choi et al., 2009c; Van Hauwe et al., 1998], were measured for comparison. The intrinsic buffering capacity (βi) was measured as described previously [Yoon et al., 2008]. This value was not significantly different for cells transfected with wild-type, H723R, V510D and P542R mutant pendrins. Expression and Localization in COS-7 Cells COS-7 cells were cultured and transfected with SLC26A4EGFP cDNA expression constructs [Choi et al., 2009b]. After in-
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Hospital, a 3-year-old female (SH123-253) who visited for an evaluation of progressive bilateral hearing loss was assessed. The patient was born without any history of relevant disease. Birth occurred at term without any known pre-, peri- or postnatal risk factors for hearing loss. No other family member showed hearing impairment or thyroid diseases. The patient had used hearing aids bilaterally since 1 year of age. Physical and otoscopic examinations and medical history were unremarkable.
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Fig. 2. a DNA sequences of the SLC26A4 genes of the family show the c.1529T>A change (left) and c.2168A>G change (right). b Multiple sequence alignment of pendrin from a human, monkey, cow, pig, mouse, rat, frog and zebrafish. Amino acid residue V510 is conserved among orthologs. c Amino acid residue V510 is conserved in
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Fig. 3. a–d Measurements of Cl–/HCO–3 exchange activity. Representative traces of cells transfected with wild-type (a), p.V510D (b), p.P542R (c) and p.H723R (d) pendrins are shown. e A summary of Cl–/HCO–3 exchange activ-
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ity. All experiments were performed a minimum of 4 times using transfected cells. WT = Wild type. * p ≤ 0.001.
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Fig. 4. Localization of SLC26A4/pendrin variants
in COS-7 cells. Representative merged images show green fluorescent protein-tagged wild-type (WT) or missense allele products (green) and concanavalin A staining (red) of the plasma membrane. Colocalization (yellow) demonstrates targeting of pendrin to the plasma membrane. Scale bar = 20 μm.
Results
Phenotype Analysis The pure tone average, calculated as the mean threshold measured at 0.5/1/2/4 kHz, was 66 dB HL in the right ear and 75 dB HL in the left ear (fig. 1b). Temporal bone computed tomography showed bilaterally EVA as well as bilateral incomplete cochlear turns, known as the Mondini malformation (fig. 1c). Genotype Analysis Sanger sequencing of the SLC26A4 gene revealed 2 missense variants of SLC26A4 that were in a trans configuration (fig. 2a). One was the p.H723R variant reported previously as a null function variant [Yoon et al., 2008] and the other was a novel missense variant (p.V510D). Null Mutant Allele of SLC26A4
The p.H723R allele was inherited from the mother and p.V510D from the father (fig. 2a). The novel missense variant (p.V510D), which is located between the sulfate transporter and the ‘sulfate transporter and anti-sigma’ domain, caused a thymine-to-adenine transition at nucleotide position 1529 (c.1529T>A), resulting in the substitution of aspartate for valine. This residue was highly conserved among SLC26A4 orthologs (fig. 2b) and conserved among SLC26A paralogs (http://genome.ucsc. edu/) (fig. 2c). Anion Exchange Activity of the Novel Missense Variant The Cl–/HCO–3 exchange activity of the p.V510D mutant pendrin was evaluated to address the pathogenic potential, compared with those of the wild type, a known polymorphism, p.P542R, and a known null function control, p.H723R. Representative traces and summarized results are shown in figure 3a–d and figure 3e, respectively. The Cl–/HCO–3 exchange activities for the novel p.V510D and p.H723R variants were found to be 11.0 ± 4.8 and 12.6 ± 3.7%, respectively, of the wild-type Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
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cubation for 24–30 h, cells were stained with concanavalin A [Choi et al., 2009b]. We analyzed mutant allele products fused at their C termini to EGFP. Images were captured using a confocal microscope (LSM510, Zeiss, Thornwood, N.Y., USA).
Trafficking of the Missense Pendrin Variant Because the p.V510D mutant protein showed almost null exchange activity in our assay, we sought to identify whether the loss of exchange activity was due to an abnormal subcellular localization of the mutant protein. The variant pendrin product (p.V510D) was expressed in COS-7 cells, fused at its C terminus to EGFP, and the subcellular localization of the mutant protein was evaluated after incubation for 24–30 h. The p.V510D pendrin product showed a reticular pattern in the cytoplasm consistent with ER retention, and was not trafficked to the plasma membrane (fig. 4) in COS-7 cells. In contrast, the wildtype product showed a pattern of colocalization with concanavalin A at the cell periphery.
Discussion
Understanding the pathogenic potential of novel variants of SLC26A4 detected in EVA patients, especially those with indeterminate thyroid organification ability, is important in the correct clinical diagnosis and the appropriate genetic counseling. Because PS and NSEVA are known to be variable manifestations of the same underlying genetic alteration [Azaiez et al., 2007; Tsukamoto et al., 2003], there have been various studies of the role of missense pendrin variants on the EVA phenotype, hearing loss severity, and phenotypic variability between PS and NSEVA [Albert et al., 2006; Choi et al., 2009a; LopezBigas et al., 2002; Pryor et al., 2005; Scott et al., 2000; Taylor et al., 2002]. Despite several exceptions, it has been reported that there is a tendency that PS is associated with bi-allelic SLC26A4 mutations while NSEVA is correlated with one or no mutations in SLC26A4 in the Caucasian populations [Azaiez et al., 2007; Choi et al., 2009a; Pryor et al., 2005]. Perchlorate discharge testing could not be performed in SH123-253 due to her young age (3 years), precluding a definite diagnosis. SH123-253, carrying one known functionally null allele, p.H723R, could be classified as either M1 or M2, depending on the pathogenic potential of p.V510D in trans with p.H723R. In this study, the abnormal cellular localization and minimal anion exchange activity showed that p.V510D was func324
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tionally a null allele, like p.H723R, suggesting that SH123253 belongs to the M2 group. If p.V510D had turned out to be a simple polymorphism with normal anion exchange activity, then SH123-253 with only 1 detectable pathogenic mutant allele of SLC26A4 would have been predicted likely to be a case of NSEVA rather than PS. Previously, it was suggested that a second undetected mutant allele of SLC26A4 in Caucasian M1 EVA subjects would encode some residual pendrin, leading to a less severe phenotype than the M2 EVA group [Choi et al., 2009a–c]. However, it is possible that this genotype-phenotype correlation does not apply to East Asian populations since some studies in these populations failed to delineate the same genotype-phenotype correlation [Miyagawa et al., 2014; Wu et al., 2010]. These studies showed a similar severity of clinical features between the M1 and M2 groups. Classification of PS mainly based upon the presence of goiter instead of perchlorate discharge test results in these studies with East Asian populations may account for the failure of observation of the correlation. Alternatively, the pathogenic potential of the second undetected occult mutant alleles of SLC26A4 in East Asian M1 EVA subjects might be as severe as those of the known pathogenic SLC26A4 mutations, eliminating the phenotypic difference between the M1 and M2 groups. Rigorous thyroid phenotyping including perchlorate discharge tests should be performed in a larger cohort to draw any conclusive genotype-phenotype correlation in this population. In addition, we should also take into account a possibility that our result might have been affected by the protein degradation or ER stress caused by transient overexpression of the mutant protein or by utilization of nonpolarized COS-7 cells or human embryonic kidney 293 cells. Genetic counseling should be cautiously provided. Understanding the pathogenic mechanism of SLC26A4 missense mutations may also be a basis of appropriate selection of a candidate mutation for future drug therapy [Gong et al., 2004]. The p.V510D mutant protein did not exhibit anion exchange activity and was retained in the ER of the cytoplasm, not targeted to the plasma membrane, by 24 h after transfection. It is as yet unclear whether the abolished anion exchange activity of p.V510D is attributable to protein mislocalization, an intrinsic defect in exchange activity or both. Complete loss of anion base exchange activity despite significant plasma membrane trafficking of a mutant pendrin likely indicates an intrinsic defect in exchange activity, as described previously for p.E303Q [Dai et al., 2009] (online suppl. table 1; for all online suppl. material, see www. Jang /Jung /Kim /Cho /Kim /Lee /Choi /Lee / Choi
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value. In contrast, the exchange activity of the polymorphism control, p.P542R, was 82.4 ± 10.1% of the wildtype value. The anion exchange activities of the p.V510D and p.H723R pendrin mutants were similar, but both showed a significant difference from that of wild-type pendrin (p < 0.001).
karger.com/doi/10.1159/000366190). However, the null or significantly reduced exchange activity of some mutant pendrin products, including p.H723R and perhaps p.V510D in this study, together with their altered subcellular localization is likely due to various processing defects [Yoon et al., 2008]. Aberrant cellular localization caused by processing defects of a mutant pendrin is evident in the ER retention pattern – perinuclear, centrosomal or other – depending on the mutation [Yoon et al., 2008]. Some pendrin mutants showing persistent ER retention, such as p. H723R, exhibited membrane expression and complex glycosylation in response to a chemical chaperone or low temperature incubation, leading to restoration of significant anion exchange activity [Yoon et al., 2008]. Recently, Ishihara et al. [2010] reported that salicylate restored the transport function and anion exchanger activity of missense pendrin mutants. In that study, among 8 pendrin mutants (p.P123S, p.M147V, p.A372V, p.N392Y, p.S657N, p.S666F, p.T721M and p.H723R) that remained in the cytoplasm, 4 (p.P123S, p.M147V, p.S657S and p.H723R) were transported from the cytoplasm to the plasma membrane with 10 mM salicylate, leading to restoration of anion exchange activity [Ishihara et al., 2010]. They proposed that the difference in responses to salicylate among the 8 mutants might be due to different processing or localization in the cytoplasm. In another study, restoration of anion exchange activity, by a chemical chaperone or low temperature incubation, for the p.H723R mutant was not observed in another null function mutant, p.L236P [Yoon et al., 2008]. In detail, the p.L236P mutant was initially retained in the ER in the cytoplasm but later censored by the ER-associated degradation system and transported to the centrosome to be degraded [Yoon et al., 2008]. In contrast, the intracellular reticular pattern indicating ER retention of p.V501D at 24 h after transfection suggested that p.V510D is likely to be processed in a similar fashion to p.H723R. Thus, clarification of the processing of each pendrin mutant would be a fundamental step to judge the possibility of restoration of anion exchanger activity. Recently, molecular mechanisms associated with ER stress-induced cell surface trafficking of the ER core-glycosylated wildtype and ∆F508 cystic fibrosis transmembrane conductance regulators via a GRASP-dependent pathway [Gee et al., 2011]. Such selective retrafficking from the ER to the plasma membrane by an unconventional pathway could be a potential therapeutic strategy for the treatment of misfolded-protein-related diseases.
Here, we reviewed all 42 pendrin mutants (including p.V510D in this study) for which anion exchange activity has been reported in the literature to be either a loss of function or significant reduction (online suppl. table 1). The mutations were first grouped according to the subcellular localization pattern: I, plasma membrane (n = 6), II, intracellular (n = 24), or III, indeterminate (n = 12). Mutations in group II are more likely to be better candidates for future drug therapy than those in group I, because their activity may be restored by induction of plasma membrane retrafficking if their intrinsic exchanger activity is unimpaired. This grouping may facilitate the selection of candidates.
Null Mutant Allele of SLC26A4
Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
Conclusions
In summary, abolished exchange activity and impaired cellular trafficking of the newly detected p.V510D variant of SLC26A4 indicate it to be a pathogenic mutation. Clarification of the pathogenic mechanism of mutations is important for the selection of candidate mutations for future drug therapy.
Acknowledgements
References
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This study was supported by the Seoul National University Bundang Hospital Research Fund (No. 02-2011-024 to B.Y. Choi) and the Korean Health Technology R & D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea [No. A111377 (HI11C13310000) to B.Y. Choi]. The Industrial Strategic Technology Development Program, 10047943, ‘Development of microsurgical apparatus based on 3D tomographic operating microscope’ was funded by the Ministry of Trade, Industry and Energy (MI, Korea; No. 10047943).
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Taylor JP, Metcalfe RA, Watson PF, Weetman AP, Trembath RC: Mutations of the PDS gene, encoding pendrin, are associated with protein mislocalization and loss of iodide efflux: implications for thyroid dysfunction in Pendred syndrome. J Clin Endocrinol Metab 2002;87:1778–1784. Tsukamoto 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;11:916–922. Usami S, Abe S, Weston MD, Shinkawa H, Van Camp G, Kimberling WJ: Non-syndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations. Hum Genet 1999;104:188–192. Valvassori GE, Clemis JD: The large vestibular aqueduct syndrome. Laryngoscope 1978; 88: 723–728. Van Hauwe P, Everett LA, Coucke P, Scott DA, Kraft ML, Ris-Stalpers C, Bolder C, Otten B, de Vijlder JJ, Dietrich NL, Ramesh A, Srisailapathy SC, Parving A, Cremers CW, Willems PJ, Smith RJ, Green ED, Van Camp G: Two frequent missense mutations in Pendred syndrome. Hum Mol Genet 1998; 7: 1099– 1104. Wangemann P, Nakaya K, Wu T, Maganti RJ, Itza EM, Sanneman JD, Harbidge DG, Billings S, Marcus DC: 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– F1353. Wu CC, Lu YC, Chen PJ, Yeh PL, Su YN, Hwu WL, Hsu CJ: Phenotypic analyses and mutation screening of the SLC26A4 and FOXI1 genes in 101 Taiwanese families with bilateral nonsyndromic enlarged vestibular aqueduct (DFNB4) or Pendred syndrome. Audiol Neurotol 2010;15:57–66. Yoon JS, Park HJ, Yoo SY, Namkung W, Jo MJ, Koo SK, Park HY, Lee WS, Kim KH, Lee MG: Heterogeneity in the processing defect of SLC26A4 mutants. J Med Genet 2008;45:411–419.
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Choi BY, Madeo AC, King KA, Zalewski CK, Pryor SP, Muskett JA, Nance WE, Butman JA, Brewer CC, Griffith AJ: Segregation of enlarged vestibular aqueducts in families with non-diagnostic SLC26A4 genotypes. J Med Genet 2009a;46:856–861. Choi BY, Stewart AK, Madeo AC, Pryor SP, Lenhard S, Kittles R, Eisenman D, Kim HJ, Niparko J, Thomsen J, Arnos KS, Nance WE, King KA, Zalewski CK, Brewer CC, Shawker T, Reynolds JC, Butman JA, Karniski LP, Alper SL, Griffith AJ: Hypo-functional SLC26A4 variants associated with nonsyndromic hearing loss and enlargement of the vestibular aqueduct: genotype-phenotype correlation or coincidental polymorphisms? Hum Mutat 2009b;30:599–608. Choi BY, Stewart AK, Nishimura KK, Cha WJ, Seong MW, Park SS, Kim SW, Chun YS, Chung JW, Park SN, Chang SO, Kim CS, Alper SL, Griffith AJ, Oh SH: Efficient molecular genetic diagnosis of enlarged vestibular aqueducts in East Asians. Genet Test Mol Biomarkers 2009c;13:679–687. Dai P, Stewart AK, Chebib F, Hsu A, Rozenfeld J, Huang D, Kang D, Lip V, Fang H, Shao H, Liu X, Yu F, Yuan H, Kenna M, Miller DT, Shen Y, Yang W, Zelikovic I, Platt OS, Han D, Alper SL, Wu BL: Distinct and novel SLC26A4/ pendrin mutations in Chinese and US patients with nonsyndromic hearing loss. Physiol Genomics 2009;38:281–290. Everett LA, Glaser B, Beck JC, Idol JR, Buchs A, Heyman M, Adawi F, Hazani E, Nassir E, Baxevanis AD, Sheffield VC, Green ED: Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet 1997;17:411–422. Gee HY, Noh SH, Tang BL, Kim KH, Lee MG: Rescue of deltaf508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway. Cell 2011;146:746–760. Gillam MP, Sidhaye AR, Lee EJ, Rutishauser J, Stephan CW, Kopp P: Functional characterization of pendrin in a polarized cell system. Evidence for pendrin-mediated apical iodide efflux. J Biol Chem 2004; 279: 13004– 13010. Gong Q, Anderson CL, January CT, Zhou Z: Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long qt mutant N470D subunits. Am J Physiol Heart Circ Physiol 2004; 287:H652–H658.
Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
Received: March 17, 2014 Accepted after revision: July 28, 2014 Published online: October 24, 2014
Identification of Novel Functional Null Allele of SLC26A4 Associated with Enlarged Vestibular Aqueduct and Its Possible Implication Jeong Hun Jang a Jinsei Jung b, c Ah Reum Kim d Young Mi Cho e Min Young Kim e Sang Yeon Lee d Jae Young Choi c Jun Ho Lee d Byung Yoon Choi e a
Department of Otorhinolaryngology, Kyungpook National University College of Medicine, Daegu, Department of Pharmacology, Brain Korea 21 Project for Medical Science, c Department of Otorhinolaryngology, College of Medicine, Yonsei University, and d Department of Otorhinolaryngology, Seoul National University College of Medicine, Seoul, and e Department of Otorhinolaryngology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea
Key Words SLC26A4 · Vestibular aqueduct · Sensorineural hearing loss
Abstract Mutations in the SLC26A4 gene, which encodes pendrin, cause congenital hearing loss as a manifestation of Pendred syndrome (PS) with an iodide organification defect or nonsyndromic enlarged vestibular aqueduct (NSEVA, DFNB4). There have been reports of differences between PS and NSEVA, including their auditory phenotypes and molecular genetic bases. For appropriate genetic diagnosis and counseling, it is important to functionally characterize SLC26A4 variants. In this study, we identified and evaluated a novel null mutation of SLC26A4 and report our method of assessing the pathogenic potential of mutations in SLC26A4, one of the most frequent causative genes of deafness in humans. A 3-year-old female with progressive sensorineural hearing loss and her parents were recruited. They underwent clinical, audiological, radiological and genetic evaluations, which revealed that the female patient had an enlarged vestibular
© 2014 S. Karger AG, Basel 1420–3030/14/0195–0319$39.50/0 E-Mail [email protected] www.karger.com/aud
aqueduct and an incomplete partition type II anomaly in the cochlea bilaterally. Sanger sequencing of the SLC26A4 gene was also performed. For a confirmatory genetic diagnosis, we first characterized the anion/base exchange ability of mutant pendrin products in HEK 293 cells and, if necessary, evaluated whether the mutant pendrin traffics to the plasma membrane in COS-7 cells. We also expressed a null function mutant, p.H723R, and a previously documented polymorphism, p.P542R, as controls. The pure tone average was 66 dB HL in the right ear and 75 dB HL in the left ear. Sequencing of SLC26A4 revealed a known pathogenic mutation (p.H723R) and a novel missense variant (p.V510D) as a compound heterozygote. When we expressed the p.V510D mutant pendrin in mammalian cells, the rate constants for Cl–/ HCO3– exchange were 10.96 ± 4.79% compared with those of wild-type pendrin. This figure was comparable to that of p.H723R, indicating p.V510D to be another pathogenic mutation with a null function. The p.V510D pendrin product was shown to be entrapped in the endoplasmic reticulum (ER) at 24–30 h after transfection, and not trafficked to the plasma membrane in COS-7 cells, suggesting retention in
Byung Yoon Choi, MD, PhD Department of Otorhinolaryngology, Seoul National University Bundang Hospital Seoul National University College of Medicine 300 Gumi-dong, Bundang-gu, Seongnam 463-707 (Republic of Korea) E-Mail choiby @ snubh.org
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© 2014 S. Karger AG, Basel
Introduction
The SLC26A4 gene encodes pendrin, a transmembrane protein mediating the exchange of anions, such as Cl–, HCO3– and I– [Mount and Romero, 2004]. Mutations in this gene cause congenital sensorineural hearing loss associated with enlarged vestibular aqueduct (EVA; NIM 600709), either as part of the Pendred syndrome (PS; NIM 274600) or as a nonsyndromic form (NSEVA, DFNB4; NIM 600791) [Everett et al., 1997; Li et al., 1998; Usami et al., 1999]. EVA is the most common radiological malformation of the inner ear associated with hereditary sensorineural hearing loss [Valvassori and Clemis, 1978]. EVA is diagnosed based on the criterion of a vestibular aqueduct diameter greater than 1.5 mm at the midpoint between the common crus and the external aperture on temporal bone computed tomography. PS is characterized by an ‘iodide organification defect’ that may lead to goiter later, at the time of puberty, in addition to bilateral sensorineural hearing loss related to the EVA [Everett et al., 1997]. There has been controversy over the molecular genetic factors distinguishing PS from NSEVA. Recent studies have suggested that the phenotype of EVA (PS vs. NSEVA) tends to correlate more with the number of mutant alleles of SLC26A4, rather than with the type of mutation in Western populations [Azaiez et al., 2007; Choi et al., 2009b; Pryor et al., 2005]. In these studies, PS with iodide organification defect was shown to be strongly associated with 2 mutant alleles of SLC26A4 (M2), while NSEVA was frequently correlated with only 1 (M1) or no mutation (M0). Some researchers have also reported that the degree of hearing loss was correlated with the number of mutant alleles of SLC26A4 in a Caucasian population [Albert et al., 2006; Ishihara et al., 2010]. Furthermore, a correlation between the degree of residual hearing and the type of SLC26A4 variant within the M2 EVA group was reported recently in a Korean population with a higher 320
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proportion of M2 EVA subjects [Lee et al., 2013]. Thus, it is important to evaluate the pathogenic potential of SLC26A4 variants and correctly categorize EVA subjects into M1 or M2 for a correct diagnosis, prediction of the prognosis and counseling of these families. Pendrin, a 73-kDa polytopic transmembrane protein containing 12 putative domains, exchanges Cl– and I– across the apical membrane of thyroid follicular cells [Gillam et al., 2004] and exchanges Cl– and HCO–3 in the nonsensory epithelia of the inner ear [Wangemann et al., 2007]. Previous studies have suggested that the mechanism of pathogenesis involves disruption of trafficking and alterations in the anion exchange activity of mutant pendrins [Rotman-Pikielny et al., 2002; Taylor et al., 2002]. Incorrect N-glycan processing has been associated with the intracellular retention and endoplasmic-reticulum (ER)-associated degradation of mutant pendrins [Yoon et al., 2008]. To date, more than 150 mutations of the SLC26A4 gene have been found to be associated with human deafness. However, only a subset of these mutations has been characterized functionally. Recently, several studies have reported that the abnormal transport function and abolished anion exchange activity of some missense pendrin mutants, such as p.H723R, could be restored substantially simply by inducing plasma membrane targeting of the mutant using a chemical chaperone, low temperature incubation or drugs such as salicylate [Ishihara et al., 2010; Yoon et al., 2008]. Missense pendrin mutants produce abnormal proteins which are retained in the ER as a result of misfolding, which is the main pathogenic mechanism of PS [Rotman-Pikielny et al., 2002]. Thus, it is vital to identify the pathogenic mechanism (mislocalization vs. intrinsic defect in transporter activity) of the missense variants of SLC26A4 to find the best candidates for future pharmacotherapy. The aims of this study were to define the pathogenic potential of a novel missense variant of SLC26A4 and to determine the pathogenic mechanism.
Material and Methods Subjects Written informed consent was obtained from parents of SH123-253 according to a protocol approved by the Seoul National University Hospital Institutional Review Board prior to the study (IRB Y-H-0905-041-281). Among the patients at the genetic hearing loss clinic at the Department of Otorhinolaryngology at Seoul National University
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the ER and abnormal trafficking as the pathogenic mechanism. This was similar to p.H723R, which is a null function founder mutant in this population but is a candidate variant for future drug therapy to rescue the abnormal cell trafficking. Impaired cellular trafficking due to ER retention and abolished exchange activity of the newly detected p.V510D indicates the pathogenic potential of this variant. These missense variants may be good candidate variants for drug therapy if the intrinsic exchange activity is not damaged by the change.
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Fig. 1. a Pedigree of the family, showing the probability of an autosomal recessive inheritance pattern. Squares = Males; circles = females; arrow = proband; black circle = individual with hearing loss. b Pure-tone audiogram of the patient with the novel mutation. c Axial view of temporal bone computed tomography shows the EVAs (white arrows) and cochlear hypoplasia (black arrowheads).
SLC26A4 Genotype Analysis Figure 1a shows the phenotype and family history of the patient. Genomic DNA of the patient and parents was isolated from peripheral blood leukocytes using GentraPureGene (Qiagen, Valencia, Calif., USA). Polymerase chain reaction amplification of the 21 coding exons and intron-exon boundaries of the SLC26A4 gene was performed using specific primers. DNA samples were evaluated to determine segregation and the meiotic phase configuration of the mutant alleles of SLC26A4 by sequence analyses of affected exons in the parents and the proband. Generation of Constructs Wild-type SCL26A4 cDNA was cloned into the KpnI and XhoI sites of pEGFP (enhanced green fluorescent protein)-N1 [Taylor et al., 2002] and into the KpnI and NotI sites of pcDNA3.1(+) for site-directed mutagenesis to generate missense expression constructs. The mutagenesis primer set designed was 510F: 5′-TATATTTGGACTGTTGACTGTGGACCTGAGAGTTCAGTT-3′ and 510R: 5′-AACTGAACTCTCAGGTCCACAGTCAACAGTCCAAATATA-3′. The entire cDNA inserts were sequenced to confirm the absence of any unintended mutation. For a control, we also subcloned p.P542R into the same site of pcDNA3.1(+) as a polymorphism control and p.H723R as a null allele control for anion exchange (using primers 542F: 5′-TTA-
Null Mutant Allele of SLC26A4
CAAAAACATTGAAGAACGTCAAGGAGTGAAGATTCTT-3′ and 542R: 5′-AAGAATCTTCACTCCTTGACGTTCTTCAATGTTTTTGTAA-3′). Again, the entire cDNA inserts were sequenced to confirm the absence of any unintended mutation. Intracellular pH and Cl–/HCO3– Exchange Measurements of pHi in human embryonic kidney 293 cells, which have minimal intrinsic anion exchange activity, were performed using a pH-sensitive fluorescent probe, biscarboxyethylcarboxyfluorescein (BCECF) [Yoon et al., 2008]. Briefly, cells were transiently transfected with wild-type or mutant pendrin, and pHi was monitored. Control cells were transfected with mock vector. After adding BCECF, cells were perfused with a HCO–3-buffered solution (in mmol/l): 116 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Dglucose, 5 HEPES and 25 NaHCO3, pH 7.4. BCECF fluorescence was recorded at excitation wavelengths of 490 and 440 nm at a resolution of 2/s on a recording setup (Delta Ram; PTI Inc., Birmingham, N.J., USA). Cl–/HCO–3 exchange activities were estimated from the initial slope of the 490/440 ratio increase as a result of replacing chloride with equimolar gluconate in the HCO–3-containing buffer (25 mM HCO–3 with 5% CO2). The anion exchange activities of the p.H723R and p.P542R mutant pendrins, known as a functional null allele mutant and a benign polymorphic mutant, respectively [Choi et al., 2009c; Van Hauwe et al., 1998], were measured for comparison. The intrinsic buffering capacity (βi) was measured as described previously [Yoon et al., 2008]. This value was not significantly different for cells transfected with wild-type, H723R, V510D and P542R mutant pendrins. Expression and Localization in COS-7 Cells COS-7 cells were cultured and transfected with SLC26A4EGFP cDNA expression constructs [Choi et al., 2009b]. After in-
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Hospital, a 3-year-old female (SH123-253) who visited for an evaluation of progressive bilateral hearing loss was assessed. The patient was born without any history of relevant disease. Birth occurred at term without any known pre-, peri- or postnatal risk factors for hearing loss. No other family member showed hearing impairment or thyroid diseases. The patient had used hearing aids bilaterally since 1 year of age. Physical and otoscopic examinations and medical history were unremarkable.
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Fig. 2. a DNA sequences of the SLC26A4 genes of the family show the c.1529T>A change (left) and c.2168A>G change (right). b Multiple sequence alignment of pendrin from a human, monkey, cow, pig, mouse, rat, frog and zebrafish. Amino acid residue V510 is conserved among orthologs. c Amino acid residue V510 is conserved in
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ity. All experiments were performed a minimum of 4 times using transfected cells. WT = Wild type. * p ≤ 0.001.
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Fig. 4. Localization of SLC26A4/pendrin variants
in COS-7 cells. Representative merged images show green fluorescent protein-tagged wild-type (WT) or missense allele products (green) and concanavalin A staining (red) of the plasma membrane. Colocalization (yellow) demonstrates targeting of pendrin to the plasma membrane. Scale bar = 20 μm.
Results
Phenotype Analysis The pure tone average, calculated as the mean threshold measured at 0.5/1/2/4 kHz, was 66 dB HL in the right ear and 75 dB HL in the left ear (fig. 1b). Temporal bone computed tomography showed bilaterally EVA as well as bilateral incomplete cochlear turns, known as the Mondini malformation (fig. 1c). Genotype Analysis Sanger sequencing of the SLC26A4 gene revealed 2 missense variants of SLC26A4 that were in a trans configuration (fig. 2a). One was the p.H723R variant reported previously as a null function variant [Yoon et al., 2008] and the other was a novel missense variant (p.V510D). Null Mutant Allele of SLC26A4
The p.H723R allele was inherited from the mother and p.V510D from the father (fig. 2a). The novel missense variant (p.V510D), which is located between the sulfate transporter and the ‘sulfate transporter and anti-sigma’ domain, caused a thymine-to-adenine transition at nucleotide position 1529 (c.1529T>A), resulting in the substitution of aspartate for valine. This residue was highly conserved among SLC26A4 orthologs (fig. 2b) and conserved among SLC26A paralogs (http://genome.ucsc. edu/) (fig. 2c). Anion Exchange Activity of the Novel Missense Variant The Cl–/HCO–3 exchange activity of the p.V510D mutant pendrin was evaluated to address the pathogenic potential, compared with those of the wild type, a known polymorphism, p.P542R, and a known null function control, p.H723R. Representative traces and summarized results are shown in figure 3a–d and figure 3e, respectively. The Cl–/HCO–3 exchange activities for the novel p.V510D and p.H723R variants were found to be 11.0 ± 4.8 and 12.6 ± 3.7%, respectively, of the wild-type Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
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cubation for 24–30 h, cells were stained with concanavalin A [Choi et al., 2009b]. We analyzed mutant allele products fused at their C termini to EGFP. Images were captured using a confocal microscope (LSM510, Zeiss, Thornwood, N.Y., USA).
Trafficking of the Missense Pendrin Variant Because the p.V510D mutant protein showed almost null exchange activity in our assay, we sought to identify whether the loss of exchange activity was due to an abnormal subcellular localization of the mutant protein. The variant pendrin product (p.V510D) was expressed in COS-7 cells, fused at its C terminus to EGFP, and the subcellular localization of the mutant protein was evaluated after incubation for 24–30 h. The p.V510D pendrin product showed a reticular pattern in the cytoplasm consistent with ER retention, and was not trafficked to the plasma membrane (fig. 4) in COS-7 cells. In contrast, the wildtype product showed a pattern of colocalization with concanavalin A at the cell periphery.
Discussion
Understanding the pathogenic potential of novel variants of SLC26A4 detected in EVA patients, especially those with indeterminate thyroid organification ability, is important in the correct clinical diagnosis and the appropriate genetic counseling. Because PS and NSEVA are known to be variable manifestations of the same underlying genetic alteration [Azaiez et al., 2007; Tsukamoto et al., 2003], there have been various studies of the role of missense pendrin variants on the EVA phenotype, hearing loss severity, and phenotypic variability between PS and NSEVA [Albert et al., 2006; Choi et al., 2009a; LopezBigas et al., 2002; Pryor et al., 2005; Scott et al., 2000; Taylor et al., 2002]. Despite several exceptions, it has been reported that there is a tendency that PS is associated with bi-allelic SLC26A4 mutations while NSEVA is correlated with one or no mutations in SLC26A4 in the Caucasian populations [Azaiez et al., 2007; Choi et al., 2009a; Pryor et al., 2005]. Perchlorate discharge testing could not be performed in SH123-253 due to her young age (3 years), precluding a definite diagnosis. SH123-253, carrying one known functionally null allele, p.H723R, could be classified as either M1 or M2, depending on the pathogenic potential of p.V510D in trans with p.H723R. In this study, the abnormal cellular localization and minimal anion exchange activity showed that p.V510D was func324
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tionally a null allele, like p.H723R, suggesting that SH123253 belongs to the M2 group. If p.V510D had turned out to be a simple polymorphism with normal anion exchange activity, then SH123-253 with only 1 detectable pathogenic mutant allele of SLC26A4 would have been predicted likely to be a case of NSEVA rather than PS. Previously, it was suggested that a second undetected mutant allele of SLC26A4 in Caucasian M1 EVA subjects would encode some residual pendrin, leading to a less severe phenotype than the M2 EVA group [Choi et al., 2009a–c]. However, it is possible that this genotype-phenotype correlation does not apply to East Asian populations since some studies in these populations failed to delineate the same genotype-phenotype correlation [Miyagawa et al., 2014; Wu et al., 2010]. These studies showed a similar severity of clinical features between the M1 and M2 groups. Classification of PS mainly based upon the presence of goiter instead of perchlorate discharge test results in these studies with East Asian populations may account for the failure of observation of the correlation. Alternatively, the pathogenic potential of the second undetected occult mutant alleles of SLC26A4 in East Asian M1 EVA subjects might be as severe as those of the known pathogenic SLC26A4 mutations, eliminating the phenotypic difference between the M1 and M2 groups. Rigorous thyroid phenotyping including perchlorate discharge tests should be performed in a larger cohort to draw any conclusive genotype-phenotype correlation in this population. In addition, we should also take into account a possibility that our result might have been affected by the protein degradation or ER stress caused by transient overexpression of the mutant protein or by utilization of nonpolarized COS-7 cells or human embryonic kidney 293 cells. Genetic counseling should be cautiously provided. Understanding the pathogenic mechanism of SLC26A4 missense mutations may also be a basis of appropriate selection of a candidate mutation for future drug therapy [Gong et al., 2004]. The p.V510D mutant protein did not exhibit anion exchange activity and was retained in the ER of the cytoplasm, not targeted to the plasma membrane, by 24 h after transfection. It is as yet unclear whether the abolished anion exchange activity of p.V510D is attributable to protein mislocalization, an intrinsic defect in exchange activity or both. Complete loss of anion base exchange activity despite significant plasma membrane trafficking of a mutant pendrin likely indicates an intrinsic defect in exchange activity, as described previously for p.E303Q [Dai et al., 2009] (online suppl. table 1; for all online suppl. material, see www. Jang /Jung /Kim /Cho /Kim /Lee /Choi /Lee / Choi
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value. In contrast, the exchange activity of the polymorphism control, p.P542R, was 82.4 ± 10.1% of the wildtype value. The anion exchange activities of the p.V510D and p.H723R pendrin mutants were similar, but both showed a significant difference from that of wild-type pendrin (p < 0.001).
karger.com/doi/10.1159/000366190). However, the null or significantly reduced exchange activity of some mutant pendrin products, including p.H723R and perhaps p.V510D in this study, together with their altered subcellular localization is likely due to various processing defects [Yoon et al., 2008]. Aberrant cellular localization caused by processing defects of a mutant pendrin is evident in the ER retention pattern – perinuclear, centrosomal or other – depending on the mutation [Yoon et al., 2008]. Some pendrin mutants showing persistent ER retention, such as p. H723R, exhibited membrane expression and complex glycosylation in response to a chemical chaperone or low temperature incubation, leading to restoration of significant anion exchange activity [Yoon et al., 2008]. Recently, Ishihara et al. [2010] reported that salicylate restored the transport function and anion exchanger activity of missense pendrin mutants. In that study, among 8 pendrin mutants (p.P123S, p.M147V, p.A372V, p.N392Y, p.S657N, p.S666F, p.T721M and p.H723R) that remained in the cytoplasm, 4 (p.P123S, p.M147V, p.S657S and p.H723R) were transported from the cytoplasm to the plasma membrane with 10 mM salicylate, leading to restoration of anion exchange activity [Ishihara et al., 2010]. They proposed that the difference in responses to salicylate among the 8 mutants might be due to different processing or localization in the cytoplasm. In another study, restoration of anion exchange activity, by a chemical chaperone or low temperature incubation, for the p.H723R mutant was not observed in another null function mutant, p.L236P [Yoon et al., 2008]. In detail, the p.L236P mutant was initially retained in the ER in the cytoplasm but later censored by the ER-associated degradation system and transported to the centrosome to be degraded [Yoon et al., 2008]. In contrast, the intracellular reticular pattern indicating ER retention of p.V501D at 24 h after transfection suggested that p.V510D is likely to be processed in a similar fashion to p.H723R. Thus, clarification of the processing of each pendrin mutant would be a fundamental step to judge the possibility of restoration of anion exchanger activity. Recently, molecular mechanisms associated with ER stress-induced cell surface trafficking of the ER core-glycosylated wildtype and ∆F508 cystic fibrosis transmembrane conductance regulators via a GRASP-dependent pathway [Gee et al., 2011]. Such selective retrafficking from the ER to the plasma membrane by an unconventional pathway could be a potential therapeutic strategy for the treatment of misfolded-protein-related diseases.
Here, we reviewed all 42 pendrin mutants (including p.V510D in this study) for which anion exchange activity has been reported in the literature to be either a loss of function or significant reduction (online suppl. table 1). The mutations were first grouped according to the subcellular localization pattern: I, plasma membrane (n = 6), II, intracellular (n = 24), or III, indeterminate (n = 12). Mutations in group II are more likely to be better candidates for future drug therapy than those in group I, because their activity may be restored by induction of plasma membrane retrafficking if their intrinsic exchanger activity is unimpaired. This grouping may facilitate the selection of candidates.
Null Mutant Allele of SLC26A4
Audiol Neurotol 2014;19:319–326 DOI: 10.1159/000366190
Conclusions
In summary, abolished exchange activity and impaired cellular trafficking of the newly detected p.V510D variant of SLC26A4 indicate it to be a pathogenic mutation. Clarification of the pathogenic mechanism of mutations is important for the selection of candidate mutations for future drug therapy.
Acknowledgements
References
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This study was supported by the Seoul National University Bundang Hospital Research Fund (No. 02-2011-024 to B.Y. Choi) and the Korean Health Technology R & D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea [No. A111377 (HI11C13310000) to B.Y. Choi]. The Industrial Strategic Technology Development Program, 10047943, ‘Development of microsurgical apparatus based on 3D tomographic operating microscope’ was funded by the Ministry of Trade, Industry and Energy (MI, Korea; No. 10047943).
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