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Article

Functional Studies of Deafness-Associated Pendrin and Prestin Variants

by
Satoe Takahashi
1,
Takashi Kojima
1,2,
Koichiro Wasano
1,3 and
Kazuaki Homma
1,4,*
1
Department of Otolaryngology—Head and Neck Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
2
Department of Otolaryngology, Head and Neck Surgery, National Hospital Organization Tochigi Medical Center, Tochigi 320-0057, Japan
3
Department of Otolaryngology, Head and Neck Surgery, Tokai University School of Medicine, Isehara 259-1193, Japan
4
The Hugh Knowles Center for Clinical and Basic Science in Hearing and Its Disorders, Northwestern University, Evanston, IL 60208, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(5), 2759; https://doi.org/10.3390/ijms25052759
Submission received: 30 January 2024 / Revised: 21 February 2024 / Accepted: 23 February 2024 / Published: 27 February 2024
(This article belongs to the Special Issue Exploring the Molecular, Cellular, and Physiological Basis of Hearing Disorders)
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Abstract

:
Pendrin and prestin are evolutionary-conserved membrane proteins that are essential for normal hearing. Dysfunction of these proteins results in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here, we report results from our ongoing efforts to experimentally characterize pendrin and prestin variants using in vitro functional assays. With previously established fluorometric anion transport assays, we determined that many of the pendrin variants identified on transmembrane (TM) 10, which contains the essential anion binding site, and on the neighboring TM9 within the core domain resulted in impaired anion transport activity. We also determined the range of functional impairment in three deafness-associated prestin variants by measuring nonlinear capacitance (NLC), a proxy for motor function. Using the results from our functional analyses, we also evaluated the performance of AlphaMissense (AM), a computational tool for predicting the pathogenicity of missense variants. AM prediction scores correlated well with our experimental results; however, some variants were misclassified, underscoring the necessity of experimentally assessing the effects of variants. Together, our experimental efforts provide invaluable information regarding the pathogenicity of deafness-associated pendrin and prestin variants.

1. Introduction

The SLC26 family of membrane proteins includes two members, pendrin (SLC26A4) and prestin (SLC26A5), that are essential for normal hearing. Pendrin is an anion transporter required for normal development and maintenance of ion homeostasis in the inner ear [1,2]. Prestin is a voltage-dependent motor and mediates voltage-dependent somatic motility (electromotility) of cochlear outer hair cells (OHCs), which is essential for the high sensitivity and frequency selectivity of mammalian hearing [3,4,5,6]. Dysfunction or loss of these SLC26 proteins results in hearing loss. Pendrin-coding SLC26A4 is a causative gene for Pendred syndrome (PDS, OMIM 274600), which is one of the most common types of syndromic hearing loss with enlarged vestibular aqueduct (EVA) and goiter, as well as nonsyndromic hearing loss (DNFB4, OMIM600791) [7,8]. Prestin-coding SLC26A5 is associated with nonsyndromic hearing loss (DNFB61, OMIM613865). Since the advent of next-generation sequencing, a large number of deafness-associated variants have been identified in patients. As of January 2024, 704 and 16 variants have been identified in SLC26A4 and SLC26A5, respectively (The Human Gene Mutation Database [9]). However, experimental assessments of the functional phenotypes of these genetic variants have been lagging behind in determining their pathogenicity with confidence. Previously, we quantitated the anion transport functions of disease-associated pendrin variants in order to understand the pathology underlying the various severity of PDS and DFNB4 [10]. Similarly, we characterized the functional phenotypes of prestin variants both in vitro and in vivo to determine their pathogenicity [11,12]. Those experimental efforts are indispensable to providing information regarding the effects of variants on protein functions, which in turn improves the consistency and accuracy in genetic diagnoses of patients. This is in line with the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines for the interpretation of sequence variants adapted for genetic hearing loss [13].
In this study, we continue to assess the pathogenicity of deafness-associated pendrin and prestin variants in vitro to determine their roles in hearing loss in patients. For pendrin, highly reproducible fluorometric antiport assays previously developed by our group were used to interrogate the functional impacts of pendrin variants on the anion transport function [10], with a major focus on transmembrane (TM) 9 and 10 located within the core domain that accommodates an anion substrate and undergoes elevator-like motions with respect to the gate domain during the transport cycle. Since the number of variants identified was clustered around this functionally important region, we reasoned that the likelihood of identifying the pathogenic variants would be high. For prestin, nonlinear capacitance (NLC) measurement was used to determine the functional impacts of a few prestin variants reported to date on the motile function [14,15]. In addition, we compared our experimental results with predictions from AlphaMissense (AM) [16], a recently developed in silico tool that predicts variant effects using artificial intelligence. We found a decent correlation between AM pathogenicity scores and the antiport activity of pendrin variants, indicating good overall performance of this prediction tool (r = −0.6497). However, some of the variants were miscategorized, indicating that prediction tools still have limitations and that experimental efforts remain important to assess the pathogenicity of deafness-associated variants.

2. Results

2.1. Functional Characterization of Pendrin Variants

A recent cryo-EM study on mouse pendrin [17] revealed a homodimeric architecture that is similar to prestin [18,19,20,21] and other SLC26 proteins [22,23,24]. The transmembrane domain consists of the gate and the core domains, and the ion translocation pathway is located in between them (Figure 1A). The positive helix dipoles of TM3 and TM10 (their N-termini), together with a conserved basic residue (Arg409 and Arg399 in pendrin and prestin, respectively) in TM10, constitute a positively charged pocket that holds a variety of anion substrates. The N-terminus of TM10 is connected to TM9, which is located on the opposite side of the core domain. The importance of the conserved residues in TM9, TM10, and the linker portion connecting these two helices (Figure 1B) is suggested by the presence of multiple clustered missense variants identified in these regions: p.D380N (c.1138G>A), p.N382K (c.1146C>G), p.Q383E (c.1147C>G), p.Q383P (c.1148A>C), p.E384Q (c.1150G>C), p.E384G (c.1151A>G), p.I386V (c.1156A>G), p.A387V (c.1160C>T), p.G389R (c.1165G>A), p.G389R (c.1165G>C), p.S391R (c.1173C>A), p.S391N (c.1172G>A), p.N392S (c.1175A>G), p.N392Y (c.1174A>T), p.G396E (c.1187G>A), p.S399P (c.1195T>C), p.V402L (c.1204G>T), p.V402M (c.1204G>A), p.T404N (c.1211C>A), p.T404I (c.1211C>T), p.A406T (c.1216G>A), p.R409C (c.1225C>T), p.R409H (c.1226G>A), p.R409L (c.1226G>T), p.R409P (c.1226G>C), p.T410K (c.1229C>A), p.T410M (c.1229C>T), p.A411P (c.1231G>C), p.A411T (c.1231G>A), p.V412I (c.1234G>A), p.Q413R (c.1238A>G), p.Q413P (c.1238A>C), p.E414K (c.1240G>A), p.S415R (c.1245C>A), p.S415G (c.1243A>G), p.T416P (c.1246A>C) [10,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54]. Among them, p.E384G, p.N392Y, p.S399P, p.R409H, p.T410K, p.T410M, p.Q413R, p.Q413P, and p.T416P were functionally characterized and found to significantly impair the anion transport function of pendrin in previous studies by us and others [10,42,55,56,57,58,59,60,61]. The present study extended our experimental efforts to define the functional consequences of missense variants identified in the TM9-TM10 region. To this end, we established HEK293T stable cell lines expressing pendrin variants in a doxycycline (Dox) dosage-dependent manner and conducted HCO3−/Cl− and I−/Cl− antiport assays alongside wild-type (WT) control as described in detail in our previous report [10]. Figure 2A,B show the results of 25 pendrin missense variants evaluated here for their Dox dosage-dependent HCO3−/Cl− and I−/Cl− antiport activities. Many of the variants exhibited vastly reduced or no activity, with only a few having WT-like activity (summarized in Table 1). These observations further affirm the importance of the conserved residues in TM9-TM10 in the core domain for the anion transport function of pendrin.

2.2. Functional Characterization of Prestin Variants

Mammalian prestin is unique among the SLC26 family of proteins as it functions as a voltage-dependent motor instead of a transporter. Prestin also shares the same overall similarities in structure as pendrin and other SLC26 proteins, implying a common molecular principle underlying transport and motor functions. Thus, it is conceivable that conserved residues between pendrin and prestin are of similar functional importance in these structurally similar proteins. Interestingly, however, the number of prestin missense variants reported to date is only 13, six of which are associated with autism with unknown pathophysiological relevance to hearing [54,62,63,64,65,66,67,68]. In this study, we characterized p.A100T (c.298G>A), p.P119S (c.355C>T), and p.S441L (c.1322C>T) prestin variants that are all associated with hearing loss. The locations of these three prestin missense variants are indicated in Figure 3A. We established HEK293T cell lines expressing these prestin variants and measured the NLC as a proxy for their motor function. Figure 4A shows the NLC of p.A100T and p.P119S prestin compared to WT. Although reduced, the NLC was detected for both p.A100T and p.P119S prestin, indicating some residual activity for these variants (Figure 4B). These Ala and Pro residues are relatively well conserved among the SLC26 proteins (Ala104 and Pro123, respectively, in pendrin, Figure 3B), and previous studies by us and others have shown that the p.P123S variant significantly impairs the anion transport function of pendrin [10,57]. In this study, we also measured the HCO3−/Cl− antiporter activity for p.A104V pendrin (deafness-associated) and p.A104T pendrin (equivalent to p.A100T prestin but not identified in patients as of January 2024) and found that these two pendrin missense variants also significantly impair the anion transport function (Figure 5, left), suggesting the common importance of these conserved residues for pendrin and prestin.
Unlike p.A100T and p.P119S, the NLC of p.S441L prestin was undetectable, indicating that p.S441L completely abrogates the motor function of prestin (Figure 4A,B). Ser441 is located at the interface between the gate and core domains (Figure 3A, right). Equivalent residues at this site in the other SLC26 family members are either Ala or Gly (Ala451 in pendrin). These small residues may be important not to hinder the relative elevator-like movements of the core domain with respect to the gate domain. If true, p.S441L may sterically interfere with these interdomain motions. In line with this speculation, p.A451L in pendrin (equivalent to p.S441L in prestin) abolished the anion transport activity, whereas p.A451S and p.A451G did not (Figure 5, right). We also found that p.S441A prestin has a WT-like NLC (Figure 4A,B). These observations suggest the common importance of having a small residue at the Ser441 site in prestin and equivalent sites in the other SLC26 proteins.
Table 1. Summaries of the antiport rates of pendrin variants determined in this study. Numerical data from HCO3−/Cl− and I−/Cl− antiport assays and for HCO3−/Cl− antiport assays are listed as indicated. P-values from one-way ANOVA with Fisher’s LSD tests compared to uninduced basal rates were listed below the mean ± SD values for each Dox concentrations. The numbers in the parentheses indicate the sample size. Comparison to WT was performed using the F-test on slope values as in our previous study [10]. Box with gray shades indicate statistically not significant (p ≥ 0.05). Asterisks (*) indicate variants not found in human patients (as of January 2024).
Table 1. Summaries of the antiport rates of pendrin variants determined in this study. Numerical data from HCO3−/Cl− and I−/Cl− antiport assays and for HCO3−/Cl− antiport assays are listed as indicated. P-values from one-way ANOVA with Fisher’s LSD tests compared to uninduced basal rates were listed below the mean ± SD values for each Dox concentrations. The numbers in the parentheses indicate the sample size. Comparison to WT was performed using the F-test on slope values as in our previous study [10]. Box with gray shades indicate statistically not significant (p ≥ 0.05). Asterisks (*) indicate variants not found in human patients (as of January 2024).
Transport Activity [Mean ± SD (n)]Dox-Dependence
Dox: 0.1 µg/mLDox: 0.3 µg/mLDox: 1 µg/mLDox: 3 µg/mLDox: 10 µg/mLTransport Activity/
log10 [Dox] (Slope ± SE)		Comparison to WT (F-Test)
WTHCO3–/Cl–0.597 ± 0.109 (10)0.795 ± 0.125 (10)1.102 ± 0.163 (10)1.485 ± 0.290 (10)1.731 ± 0.253 (10)0.591 ± 0.040not applicable
[nM/sec]p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001
I–/Cl–0.08 ± 0.04 (3)0.14 ± 0.01 (3)0.18 ± 0.02 (3)0.23 ± 0.04 (3)0.21 ± 0.02 (3)0.069 ± 0.012not applicable
[mM/sec]p = 0.049p = 0.0002p < 0.0001p < 0.0001p < 0.0001p < 0.0001
Figure 2p.Gln383Glu (c.1147C>G)HCO3–/Cl–0.061 ± 0.023 (4)0.072 ± 0.029 (4)0.084 ± 0.060 (4)0.125 ± 0.059 (3)0.073 ± 0.020 (4)0.015 ± 0.014Function impaired
[nM/sec]p = 0.4446p = 0.6253p = 0.8678p = 0.3807p = 0.6457p = 0.0034p < 0.0001
I–/Cl–0.01 ± 0.003 (3)0.012 ± 0.003 (3)0.011 ± 0.004 (3)0.015 ± 0.001 (3)0.017 ± 0.005 (3)0.003 ± 0.001Function impaired
[mM/sec]p = 0.2067p = 0.2589p = 0.2398p = 0.3427p = 0.4135p = 0.0132p < 0.0001
p.Glu384Gly (c.1151G>C)HCO3–/Cl–0.055 ± 0.018 (4)0.063 ± 0.028 (4)0.081 ± 0.025 (4)0.088 ± 0.018 (4)0.081 ± 0.038 (4)0.016 ± 0.008Function impaired
[nM/sec]p = 0.2875p = 0.407p = 0.7755p = 0.9276p = 0.7679p = 0.066p < 0.0001
I–/Cl–0.008 ± 0.002 (3)0.009 ± 0.002 (3)0.016 ± 0.009 (3)0.013 ± 0.004 (3)0.012 ± 0.002 (3)0.002 ± 0.002Function impaired
[mM/sec]p = 0.1742p = 0.1931p = 0.3773p = 0.3016p = 0.2592p = 0.1892p < 0.0001
p.Ala387Val (c.1160C>T)HCO3–/Cl–0.046 ± 0.027 (4)0.054 ± 0.14 (4)0.067 ± 0.032 (4)0.082 ± 0.053 (4)0.100 ± 0.051 (4)0.028 ± 0.011Function impaired
[nM/sec]p = 0.2327p = 0.3218p = 0.5252p = 0.8158p = 0.7995p = 0.023p < 0.0001
I–/Cl–0.008 ± 0.005 (3)0.016 ± 0.005 (3)0.011 ± 0.002 (3)0.017 ± 0.008 (3)0.015 ± 0.003 (3)0.003 ± 0.002Function impaired
[mM/sec]p = 0.1662p = 0.4038p = 0.2439p = 0.4111p = 0.3413p = 0.1819p < 0.0001
p.Gly389Arg (c.1165G>A)HCO3–/Cl–0.054 ± 0.049 (3)0.064 ± 0.023 (3)0.064 ± 0.015 (3)0.071 ± 0.013 (3)0.075 ± 0.023 (3)0.010 ± 0.009Function impaired
[nM/sec]p = 0.3825p = 0.5245p = 0.5265p = 0.6367p = 0.6956p = 0.3057p < 0.0001
I–/Cl–0.011 ± 0.007 (3)0.009 ± 0.005 (3)0.012 ± 0.006 (3)0.011 ± 0.001 (3)0.013 ± 0.004 (3)0.001 ± 0.002Function impaired
[mM/sec]p = 0.2336p = 0.1889p = 0.2642p = 0.2429p = 0.2878p = 0.4214p < 0.0001
p.Gly389Arg (c.1165G>C)HCO3–/Cl–0.057 ± 0.015 (3)0.110 ± 0.090 (3)0.076 ± 0.019 (3)0.077 ± 0.012 (3)0.113 ± 0.066 (3)0.015 ± 0.018Function impaired
[nM/sec]p = 0.4957p = 0.6993p = 0.7679p = 0.7755p = 0.6584p = 0.408p < 0.0001
I–/Cl–0.018 ± 0.008 (3)0.014 ± 0.009 (3)0.013 ± 0.002 (3)0.015 ± 0.002 (3)0.013 ± 0.002 (3)slope < 0Function impaired
[mM/sec]p = 0.4607p = 0.3203p = 0.3036p = 0.375p = 0.2997p = 0.4403p < 0.0001
p.Ser391Asn (c.1172G>A)HCO3–/Cl–0.076 ± 0.026 (4)0.084 ± 0.043 (4)0.075 ± 0.024 (4)0.085 ± 0.048 (4)0.066 ± 0.022 (4)slope < 0Function impaired
[nM/sec]p = 0.6844p = 0.8509p = 0.652p = 0.879p = 0.4908p = 0.6983p < 0.0001
I–/Cl–0.012 ± 0.004 (3)0.015 ± 0.005 (3)0.018 ± 0.001 (3)0.019 ± 0.007 (3)0.024 ± 0.006 (3)0.005 ± 0.002Function impaired
[mM/sec]p = 0.2746p = 0.3545p = 0.462p = 0.4873p = 0.7334p = 0.0053p < 0.0001
p.Ser391Arg (c.1173C>A)HCO3–/Cl–0.049 ± 0.009 (3)0.038 ± 0.015 (3)0.036 ± 0.039 (3)0.056 ± 0.022 (3)0.106 ± 0.074 (3)0.026 ± 0.014Function impaired
[nM/sec]p = 0.3588p = 0.2441p = 0.2289p = 0.4346p = 0.7391p = 0.0897p < 0.0001
I–/Cl–0.014 ± 0.002 (3)0.014 ± 0.003 (3)0.015 ± 0.001 (3)0.013 ± 0.002 (3)0.013 ± 0.001 (3)slope < 0Function impaired
[mM/sec]p = 0.2964p = 0.3102p = 0.3437p = 0.2906p = 0.2652p = 0.4322p < 0.0001
p.Asn392Ser (c.1175A>G)HCO3–/Cl–0.049 ± 0.018 (3)0.052 ± 0.046 (3)0.089 ± 0.041 (3)0.056 ± 0.032 (3)0.096 ± 0.027 (3)0.020 ± 0.012Function impaired
[nM/sec]p = 0.3348p = 0.3763p = 0.9589p = 0.4347p = 0.9091p = 0.1275p < 0.0001
I–/Cl–0.009 ± 0.001 (3)0.016 ± 0.003 (3)0.015 ± 0.002 (3)0.015 ± 0.001 (3)0.023 ± 0.005 (3)0.005 ± 0.001Function impaired
[mM/sec]p = 0.1892p = 0.3664p = 0.3464p = 0.340p = 0.6698p = 0.0012p < 0.0001
p.Gly396Glu (c.1187G>A)HCO3–/Cl–0.089 ± 0.038 (4)0.096 ± 0.060 (4)0.121 ± 0.054 (4)0.084 ± 0.031 (4)0.108 ± 0.013 (4)0.006 ± 0.013Function impaired
[nM/sec]p = 0.9512p = 0.9048p = 0.4323p = 0.852p = 0.6605p = 0.6755p < 0.0001
I–/Cl–0.009 ± 0.005 (3)0.009 ± 0.001 (3)0.012 ± 0.002 (3)0.011 ± 0.003 (3)0.010 ± 0.002 (3)0.001 ± 0.001Function impaired
[mM/sec]p = 0.1751p = 0.1868p = 0.2536p = 0.2304p = 0.2133p = 0.3095p < 0.0001
p.Val402Met (c.1204G>A)HCO3–/Cl–0.086 ± 0.026 (3)0.075 ± 0.010 (3)0.072 ± 0.029 (3)0.108 ± 0.071 (3)0.095 ± 0.020 (3)0.010 ± 0.013Function impaired
[nM/sec]p = 0.9029p = 0.7134p = 0.6663p = 0.6978p = 0.9317p = 0.4368p < 0.0001
I–/Cl–0.013 ± 0.001 (3)0.011 ± 0.003 (3)0.016 ± 0.007 (3)0.018 ± 0.004 (3)0.018 ± 0.003 (3)0.004 ± 0.001Function impaired
[mM/sec]p = 0.2741p = 0.2302p = 0.3718p = 0.4644p = 0.4446p = 0.0321p < 0.0001
p.Thr404Ile (c.1211C>T)HCO3–/Cl–0.091 ± 0.032 (5)0.062 ± 0.059 (5)0.085 ± 0.043 (5)0.094 ± 0.059 (5)0.100 ± 0.047 (5)0.010 ± 0.013Function impaired
[nM/sec]p = 0.9989p = 0.4374p = 0.870p = 0.940p = 0.8092p = 0.4618p < 0.0001
I–/Cl–0.011 ± 0.004 (3)0.011 ± 0.004 (3)0.014 ± 0.004 (3)0.015 ± 0.003 (3)0.012 ± 0.007 (3)0.001 ± 0.002Function impaired
[mM/sec]p = 0.2324p = 0.2375p = 0.3348p = 0.365p = 0.2664p = 0.4046p < 0.0001
p.Ala406Thr (c.1216G>A)HCO3–/Cl–0.233 ± 0.068 (3)0.345 ± 0.073 (3)0.473 ± 0.053 (3)0.479 ± 0.026 (3)0.461 ± 0.088 (3)0.118 ± 0.028Function impaired
[nM/sec]p = 0.0172p = 0.0002p < 0.0001p < 0.0001p < 0.0001p = 0.0011p < 0.0001
I–/Cl–0.056 ± 0.009 (3)0.094 ± 0.021 (3)0.120 ± 0.037 (3)0.111 ± 0.040 (3)0.122 ± 0.033 (3)0.030 ± 0.011Function impaired
[mM/sec]p = 0.3394p = 0.0308p = 0.005p = 0.0095p = 0.0046p = 0.017p = 0.0212
p.Ser408Asp (c.1222_3TC>GA) *HCO3–/Cl–0.040 ± 0.019 (4)0.057 ± 0.020 (4)0.053 ± 0.012 (4)0.070 ± 0.031 (4)0.088 ± 0.031 (4)0.022 ± 0.007Function impaired
[nM/sec]p = 0.1339p = 0.3078p = 0.2554p = 0.5357p = 0.919p = 0.0068p < 0.0001
I–/Cl–0.011 ± 0.002 (3)0.013 ± 0.003 (3)0.017 ± 0.004 (3)0.016 ± 0.007 (3)0.019 ± 0.002 (3)0.004 ± 0.001Function impaired
[mM/sec]p = 0.2394p = 0.276p = 0.4036p = 0.3921p = 0.4893p = 0.0165p < 0.0001
p.Ser408Glu (c.1222_4TCC>GAG) *HCO3–/Cl–0.066 ± 0.032 (5)0.075 ± 0.021 (5)0.063 ± 0.034 (5)0.084 ± 0.059 (5)0.091 ± 0.057 (5)0.012 ± 0.012Function impaired
[nM/sec]p = 0.477p = 0.6504p = 0.427p = 0.8493p = 0.9929p = 0.3294p < 0.0001
I–/Cl–0.008 ± 0.003 (3)0.009 ± 0.005 (3)0.010 ± 0.002 (3)0.015 ± 0.001 (3)0.010 ± 0.001 (3)0.002 ± 0.001Function impaired
[mM/sec]p = 0.1561p = 0.1915p = 0.2025p = 0.3308p = 0.2164p = 0.0695p < 0.0001
p.Arg409Cys (c.1225C>T)HCO3–/Cl–0.103 ± 0.066 (3)0.091 ± 0.081 (3)0.065 ± 0.036 (3)0.076 ± 0.040 (3)0.168 ± 0.085 (3)0.023 ± 0.024Function impaired
[nM/sec]p = 0.8259p = 0.9943p = 0.6379p = 0.782p = 0.1685p = 0.3529p < 0.0001
I–/Cl–0.022 ± 0.002 (3)0.024 ± 0.002 (3)0.028 ± 0.003 (3)0.028 ± 0.004 (3)0.026 ± 0.004 (3)0.002 ± 0.001Function impaired
[mM/sec]p = 0.6456p = 0.7489p = 0.9292p = 0.9227p = 0.8445p = 0.0791p < 0.0001
p.Arg409Leu (c.1226G>T)HCO3–/Cl–0.234 ± 0.042 (4)0.275 ± 0.037 (4)0.318 ± 0.043 (4)0.334 ± 0.082 (4)0.380 ± 0.044 (3)0.071 ± 0.016Function impaired
[nM/sec]p = 0.225p = 0.1221p = 0.0603p = 0.0453p = 0.0003p = 0.0004p < 0.0001
I–/Cl–0.061 ± 0.002 (3)0.078 ± 0.005 (3)0.086 ± 0.003 (3)0.086 ± 0.005 (3)0.095 ± 0.013 (3)0.015 ± 0.003Function impaired
[mM/sec]p = 0.0552p = 0.0072p = 0.0027p = 0.0024p = 0.009p < 0.0001p = 0.0001
p.Ala411Pro (c.1231G>C)HCO3–/Cl–0.064 ± 0.021 (3)0.084 ± 0.021 (3)0.071 ± 0.033 (3)0.066 ± 0.013 (3)0.065 ± 0.014 (3)slope < 0Function impaired
[nM/sec]p = 0.5137p = 0.8711p = 0.6193p = 0.5427p = 0.529p = 0.6639p < 0.0001
I–/Cl–0.016 ± 0.002 (3)0.024 ± 0.002 (3)0.027 ± 0.005 (3)0.028 ± 0.005 (3)0.029 ± 0.005 (3)0.006 ± 0.002Function impaired
[mM/sec]p = 0.3929p = 0.7402p = 0.8987p = 0.9163p = 0.9784p = 0.0033p < 0.0001
p.Ala411Thr (c.1231G>A)HCO3–/Cl–0.890 ± 0.010 (3)1.176 ± 0.035 (3)1.370 ± 0.120 (3)1.732 ± 0.237 (3)1.752 ± 0.047 (3)0.455 ± 0.049WT-like
[nM/sec]p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p = 0.0847
I–/Cl–0.064 ± 0.012 (3)0.079 ± 0.014 (3)0.094 ± 0.011 (3)0.113 ± 0.023 (3)0.123 ± 0.022 (3)0.030 ± 0.006Function impaired
[mM/sec]p = 0.0955p = 0.0224p = 0.0054p = 0.0009p = 0.0004p = 0.0001p = 0.0062
p.Val412Ile (c.1234G>A)HCO3–/Cl–0.868 ± 0.186 (3)1.132 ± 0.503 (3)1.430 ± 0.706 (3)1.530 ± 0.532 (3)2.081 ± 0.849 (3)0.566 ± 0.194WT-like
[nM/sec]p = 0.041p = 0.0091p = 0.0016p = 0.0009p < 0.0001p = 0.0121p = 0.8432
I–/Cl–0.085 ± 0.009 (3)0.117 ± 0.005 (3)0.122 ± 0.016 (3)0.141 ± 0.015 (3)0.146 ± 0.006 (3)0.029 ± 0.004Function impaired
[mM/sec]p = 0.005p = 0.0002p < 0.0001p < 0.0001p < 0.0001p < 0.0001p = 0.0036
p.Gln413Pro (c.1238A>C)HCO3–/Cl–0.095 ± 0.021 (3)0.081 ± 0.027 (3)0.090 ± 0.057 (3)0.144 ± 0.056 (3)0.121 ± 0.100 (3)0.023 ± 0.020Function impaired
[nM/sec]p = 0.9349p = 0.8519p = 0.984p = 0.3123p = 0.5596p = 0.2745p < 0.0001
I–/Cl–0.019 ± 0.003 (3)0.026 ± 0.007 (3)0.027 ± 0.002 (3)0.024 ± 0.001 (3)0.022 ± 0.004 (3)0.001 ± 0.002Function impaired
[mM/sec]p = 0.4857p = 0.8454p = 0.8783p = 0.7352p = 0.6318p = 0.6167p < 0.0001
p.Gln413Arg (c.1238A>G)HCO3–/Cl–0.313 ± 0.107 (3)0.349 ± 0.156 (3)0.440 ± 0.212 (3)0.459 ± 0.203 (3)0.424 ± 0.208 (3)0.066 ± 0.059Function impaired
[nM/sec]p = 0.0652p = 0.0349p = 0.0069p = 0.0048p = 0.0092p = 0.2848p < 0.0001
I–/Cl–0.039 ± 0.011 (3)0.050 ± 0.009 (3)0.058 ± 0.002 (3)0.064 ± 0.002 (3)0.072 ± 0.007 (3)0.016 ± 0.002Function impaired
[mM/sec]p = 0.5267p = 0.1857p = 0.0756p = 0.0391p = 0.0139p < 0.0001p = 0.0001
p.Glu414Lys (c.1240G>A)HCO3–/Cl–0.305 ± 0.075 (3)0.314 ± 0.166 (3)0.327 ± 0.079 (3)0.404 ± 0.106 (3)0.429 ± 0.262 (3)0.067 ± 0.050Function impaired
[nM/sec]p = 0.0431p = 0.0356p = 0.0279p = 0.0055p = 0.0033p = 0.1998p < 0.0001
I–/Cl–0.040 ± 0.008 (3)0.045 ± 0.007 (3)0.049 ± 0.002 (3)0.051 ± 0.005 (3)0.061 ± 0.008 (3)0.010 ± 0.002Function impaired
[mM/sec]p = 0.4681p = 0.3109p = 0.2121p = 0.1617p = 0.0545p = 0.0007p < 0.0001
p.Ser415Gly (c.1243A>G)HCO3–/Cl–0.384 ± 0.171 (3)0.484 ± 0.075 (3)0.727 ± 0.184 (3)0.824 ± 0.260 (3)0.774 ± 0.260 (3)0.224 ± 0.070Function impaired
[nM/sec]p = 0.0303p = 0.0059p = 0.0001p < 0.0001p < 0.0001p = 0.0072p < 0.0001
I–/Cl–0.068 ± 0.011 (3)0.089 ± 0.011 (3)0.097 ± 0.014 (3)0.107 ± 0.016 (3)0.112 ± 0.007 (3)0.021 ± 0.004Function impaired
[mM/sec]p = 0.0377p = 0.0039p = 0.0017p = 0.0006p = 0.0003p = 0.0002p = 0.0007
p.Ser415Arg (c.1245C>A)HCO3–/Cl–0.634 ± 0.135 (3)0.923 ± 0.072 (3)0.915 ± 0.013 (3)1.084 ± 0.041 (3)1.242 ± 0.057 (3)0.275 ± 0.033Function impaired
[nM/sec]p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001
I–/Cl–0.052 ± 0.009 (3)0.069 ± 0.018 (3)0.080 ± 0.011 (3)0.094 ± 0.010 (3)0.091 ± 0.013 (3)0.021 ± 0.004Function impaired
[mM/sec]p = 0.203p = 0.0371p = 0.0102p = 0.0023p = 0.0033p = 0.0005p = 0.0006
p.Ser532Ile (c.1595G>T)HCO3–/Cl–0.549 ± 0.091 (4)0.793 ± 0.254 (4)0.816 ± 0.107 (4)0.845 ± 0.267 (4)0.912 ± 0.245 (4)0.155 ± 0.063Function impaired
[nM/sec]p = 0.0011p < 0.0001p < 0.0001p < 0.0001p < 0.0001p = 0.0244p < 0.0001
I–/Cl–0.053 ± 0.011 (3)0.055 ± 0.011 (3)0.069 ± 0.018 (3)0.086 ± 0.028 (3)0.095 ± 0.009 (3)0.023 ± 0.006Function impaired
[mM/sec]p = 0.2373p = 0.198p = 0.0572p = 0.0108p = 0.0045p = 0.0011p = 0.0015
Figure 5p.Ala104Val (c.311C>T)HCO3–/Cl–0.079 ± 0.017 (4)0.152 ± 0.080 (4)0.110 ± 0.035 (4)0.132 ± 0.029 (4)0.105 ± 0.039 (4)0.006 ± 0.015Function impaired
[nM/sec]p = 0.7598p = 0.1325p = 0.6295p = 0.3075p = 0.718p = 0.7015p < 0.0001
p.Ala104Thr (c.310G>A) *HCO3–/Cl–0.363 ± 0.051 (6)0.402 ± 0.075 (6)0.468 ± 0.039 (6)0.528 ± 0.100 (6)0.443 ± 0.084 (6)0.057 ± 0.022Function impaired
[nM/sec]p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p = 0.0159p < 0.0001
p.Ala451Gly (c.1352C>G) *HCO3–/Cl–0.479 ± 0.092 (3)0.911 ± 0.063 (3)0.993 ± 0.129 (3)1.284 ± 0.089 (3)1.836 ± 0.184 (3)0.618 ± 0.056WT-like
[nM/sec]p = 0.0062p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p = 0.7423
p.Ala451Ser (c.1351G>T) *HCO3–/Cl–0.572 ± 0.140 (3)0.921 ± 0.162 (3)1.168 ± 0.127 (3)1.406 ± 0.167 (3)1.749 ± 0.251 (3)0.568 ± 0.057WT-like
[nM/sec]p = 0.0027p < 0.0001p < 0.0001p < 0.0001p < 0.0001p < 0.0001p = 0.7667
p.Ala451Leu (c.1351_2GC>CT) *HCO3–/Cl–0.097 ± 0.047 (3)0.144 ± 0.013 (3)0.100 ± 0.036 (3)0.093 ± 0.014 (3)0.143 ± 0.064 (3)0.008 ± 0.015Function impaired
[nM/sec]p = 0.3455p = 0.0283p = 0.3058p = 0.4034p = 0.0299p = 0.6021p < 0.0001
Bold fonts were used to emphasize the amino acid changes of the variants from nucleotide changes in the parentheses.
Ijms 25 02759 g003
Figure 3. Ala100, Pro119, and Ser441 sites in prestin. (A) The homodimeric structure of human prestin (PDB: 7LGU). Protomers are shown in green and gray. In right panel, the core domain of one of the protomers is shown in bright orange. The Ala100, Pro119, and Ser441 sites and bound chlorides are indicated by cyan, blue, purple, and red spheres, respectively. (B) Partial amino acid sequences of human and mouse pendrin (A4) and prestin (A5). Numbers in parentheses indicate the residue numbers at the N- and C-terminal ends. The residues with asterisks indicate Ala100, Pro119, and Ser441 in human prestin (hA5) and equivalents in others.
Figure 3. Ala100, Pro119, and Ser441 sites in prestin. (A) The homodimeric structure of human prestin (PDB: 7LGU). Protomers are shown in green and gray. In right panel, the core domain of one of the protomers is shown in bright orange. The Ala100, Pro119, and Ser441 sites and bound chlorides are indicated by cyan, blue, purple, and red spheres, respectively. (B) Partial amino acid sequences of human and mouse pendrin (A4) and prestin (A5). Numbers in parentheses indicate the residue numbers at the N- and C-terminal ends. The residues with asterisks indicate Ala100, Pro119, and Ser441 in human prestin (hA5) and equivalents in others.
Ijms 25 02759 g003
Ijms 25 02759 g004
Figure 4. NLC measurements. (A) Examples of NLC recorded in HEK293T cells expressing WT, p.A100T, p.P119S, p.S441L, or p.S441A prestin. Different colors indicate individual recordings. (B) Summaries of the NLC parameters (α, Vpk, and CD). Error bars indicate SD. ns, p ≥ 0.05; ** 0.001 < p ≤ 0.01; *** 0.0001 < p ≤ 0.001; **** p ≤ 0.0001.
Figure 4. NLC measurements. (A) Examples of NLC recorded in HEK293T cells expressing WT, p.A100T, p.P119S, p.S441L, or p.S441A prestin. Different colors indicate individual recordings. (B) Summaries of the NLC parameters (α, Vpk, and CD). Error bars indicate SD. ns, p ≥ 0.05; ** 0.001 < p ≤ 0.01; *** 0.0001 < p ≤ 0.001; **** p ≤ 0.0001.
Ijms 25 02759 g004
Ijms 25 02759 g005
Figure 5. The effects of missense changes at Ala104 and Ala451 on the anion transport function of pendrin. HCO3−/Cl− antiport assay conducted for p.A104V (left), p.A104T (left), p.A451G (right), p.A451S (right), and p.A451L (right) pendrin alongside WT control. Error bars indicate SD. Horizontal dotted lines indicate transport rates of uninduced cells. Solid lines indicate linear regressions (log10 [Dox] vs. transport rates). Sample size information and statistics are provided in Table 1.
Figure 5. The effects of missense changes at Ala104 and Ala451 on the anion transport function of pendrin. HCO3−/Cl− antiport assay conducted for p.A104V (left), p.A104T (left), p.A451G (right), p.A451S (right), and p.A451L (right) pendrin alongside WT control. Error bars indicate SD. Horizontal dotted lines indicate transport rates of uninduced cells. Solid lines indicate linear regressions (log10 [Dox] vs. transport rates). Sample size information and statistics are provided in Table 1.
Ijms 25 02759 g005

2.3. Comparison to AlphaMissense Variant Effect Predictor

The pathogenicity of disease-associated variants is best assessed by functional analyses such as those presented here; however, experimental characterization of variants is laborious and remains reactive to the rapid identification of novel variants. Recent advancements in computational methods using machine learning have prompted us to evaluate the performance of AlphaMissense (AM), a novel tool for predicting the pathogenicity of missense variants [16]. First, we visualized the provided AM prediction scores of pendrin and prestin at every amino acid position as heatmaps (Supplementary Figures S1 and S2). AM pathogenicity predictions for both pendrin and prestin show similarity, such as (i) variants in regions that lack structural information (C- and N-termini, IDR) tend to be benign (i.e., predominantly “blue”); and (ii) variants in transmembrane regions tend to be less tolerant to missense changes (i.e., predominantly “red”) in the heatmap (Supplementary Figures S1 and S2). These observations are in line with the fact that AM utilizes structural information, and both pendrin and prestin share quite similar overall molecular architecture. To evaluate the accuracy of AM, we plotted the HCO3−/Cl− antiporter activities of pendrin variants measured in our previous study [10] and this study, which are normalized to WT control, against the AM pathogenicity scores (Figure 6). We found an overall correlation between our functional assay data and AM prediction scores of r = −0.6497 (nonparametric Spearman correlation), which is in line with the example given in their report (GCK, r = −0.65, Figure 3G in [16]). Many variants tested in this study are clustered around highly conserved TM9-10 that are essential for the transport function, and thus many had little or no activities, skewing the distribution of variants (Figure 6, inset). In addition, we found several variants that were misclassified by AM as benign when there is vastly reduced activity (for example, p.N392S) or vice versa (ambiguous/pathogenic when their activity is WT-like, such as p.T307A, p.L117F, and p.F354S) (Table 2). For prestin variants, AM scores for p.A100T, p.P119S, and p.S441L were 0.9138, 0.7712, and 0.916, respectively, all predicted to be pathogenic.

3. Discussion

Precisely understanding the relationships between genotypes and disease phenotypes is the holy grail of the field of human genetics. Although sequencing information becomes more and more accessible, assigning pathogenicity to variations of sequences remains challenging due to the complexity of determining the causality of variants to disease states [69]. Although experimental interrogation is ideal, our collective experimental efforts have only scratched the surface of building an atlas of variant effects in the genome. Consequently, the vast majority of disease-associated variants, including those of pendrin and prestin, remain as variants of unknown significance (VUS) [70]. To bridge the gap, in silico tools have been developed to predict whether a variant plays a causative role in a disease state. Large numbers of variant effect predictors (VEPs) have been reported over the years using various approaches with various degrees of performance [71]. For the deafness genes, a measure for protein-folding stability was used to re-classify VUSs in patients due to protein misfolding [72]. Recently developed AlphaMissense (AM) also uses structural information, although it does not predict protein stability. Rather, it classifies missense variants as benign, ambiguous, or pathogenic based on their prediction scores [16]. As the study provided AM prediction across the entire human proteome, we compared the available AM prediction scores for pendrin to our experimental results on quantitated transport activities. We found a good correlation (r = −0.6497, Figure 6), demonstrating their utility in informing the variant effects. It must be noted, however, that the uncertainty still exists as several pendrin variants were misclassified. Thus, the usage of VEPs, even the one that demonstrated superior performance over many existing VEPs across multiple benchmarks as AM, must exercise the utmost caution in clinical settings.
Recent efforts in large-scale systemic studies (multiplexed assay of variant effects, MAVE) have reported functional consequences of hundreds of thousands of variants across genome regions to date [73,74]. For proteins such as pendrin and prestin, however, large-scale multiplexing assays will be challenging as their dysfunctions may not result in easily discernible cellular phenotypes. Nevertheless, our small-scale in vitro assays are robust in determining the degrees of functional impairment of the variants found in human patients. The large variation in PDS and DFNB4 phenotypes suggests large variations in the activities of pendrin variants, thus warranting an experimental approach that can detect a subtle difference in transport activities. One caveat will be that our approach to heterologous expression systems may not be able to address the effects of variants on protein expression levels in the host environment, as we forcibly induce protein expression by doxycycline from cDNAs for our transporter assays. Also not addressed here are the variant effects on protein–protein interactions. For example, the intrinsically disordered region (IDR) within the cytosolic domain of prestin mediates the binding of calmodulin, thereby allowing modulation of protein activity by calcium, which is likely conserved in other SLC26 proteins, including pendrin [75]. Additionally, IQ-motif containing GTPase-activating protein 1 (IQGAP1) was shown to interact with the C-terminal region of pendrin and enhance its transport activity in kidney cells [76]. Interestingly, co-expression of IQGAP1 and pendrin also enhanced the plasma membrane (PM) localization of pendrin, suggesting another layer of regulatory interaction. It is curious that although both pendrin and prestin are conserved and the handful of disease-associated variants in prestin also similarly affected pendrin in this study, prestin is targeted to the lateral membrane in the outer hair cells (OHCs), while pendrin is targeted to the apical membranes of epithelial cells in the thyroid and in the inner ear [77,78,79,80]. Thus, it will require additional considerations such as co-expression of interacting partners or using the host cell lines in order to interrogate the effects on variants found in regions where such regulatory interactions were reported in the future.
Although functional annotation of deafness-associated variations in the genome may not be complete until the hearing phenotype is assessed in vivo, our continuing efforts to characterize missense variants in vitro provide invaluable information regarding the variant effects, which can be used to prioritize future efforts at the organismal level. With ever-improving VEPs such as AM, future studies will provide clinically useful information of variant effects with more confidence.

4. Materials and Methods

4.1. Generation of Stable Cell Lines

cDNAs coding human pendrin (NM_000441.2) and gerbil prestin (AF230376.2), (WT and variants) with a mTurquoise (mTq2) tag on the C-terminus were cloned into a pSBtet-pur vector (Addgene, Watertown, NY, USA) using SfiI sites. Stable cell lines carrying these constructs were established in HEK293T cells as previously described [10]. Stable cells were maintained in DMEM supplemented with 10% FBS and 1 µg/mL puromycin (Themo Fisher Scientific, Waltham, MA, USA), and the expression of the pendrin and prestin constructs were induced by the addition of doxycycline (Dox) in the media. For I−/Cl− antiport assay, the pendrin-expressing pSBtet-Pur vectors were transfected in the HEK293T cell line that constitutively express mVenusp.H148Q/p.I152L as previously described [10].

4.2. Fluorometric Anion Transport Assays

Fluorometric HCO3−/Cl− and I−/Cl− antiport assays were established and described in detail in a previous study [10]. Briefly, for HCO3−/Cl− antiport assay, stable HEK293T cells expressing pendrin constructs were loaded with a pH indicator, SNARF-5F (S23923, Thermo Fisher Scientific, Waltham, MA, USA) in a high chloride buffer containing (mM): 140 NaCl, 4.5 KCl, 1 MgCl2, 2.5 CaCl2, 20 HEPES (pH 7.4). The antiport assay was initiated by an automated injection of a low chloride buffer containing (mM): 125 Na-gluconate, 5 K-gluconate, 1 MgCl2, 1 CaCl2, 20 HEPES, 25 NaHCO3 with 5% CO2 in Synergy Neo2 (Agilent/BioTek, Santa Clara, CA, USA). The fluorescence of SNARF-5F were measured in a time dependent manner using Synergy Neo2 (Agilent/BioTek) and the data analyzed offline as described previously [10]. For I−/Cl− antiport assay, cells expressing both pendrin variants and iodide sensitive fluorescent protein, mVenusp.H148Q/p.I152L, were resuspended in 200 μL of a high Cl− buffer containing (mM): 150 NaCl, 1 MgCl2, 1 CaCl2, 20 HEPES, (pH 7.5). The I−/Cl− antiport assay using 160 µL of the cell suspension was initiated by an automated injection of 80 μL of a high I− buffer containing (mM): 150 NaI, 1 MgCl2, 1 CaCl2, 20 HEPES (pH 7.5) in Synergy Neo2 (Agilent/BioTek). The fluorescence intensities of mVenusp.H148Q/p.I152L and mTq2 were simultaneously measured in a time-dependent manner using Synergy Neo2 (Agilent/BioTek) and the data analyzed offline as described previously [10].

4.3. Whole-Cell Recordings

Whole-cell recordings were performed at room temperature using the Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA) with a 10 kHz low-pass filter. Recording pipettes pulled from borosilicate glass were filled with an ionic blocking intracellular solution containing (mM): 140 CsCl, 2 MgCl2, 10 EGTA, and 10 HEPES (pH 7.4). Cells were bathed in an extracellular solution containing (mM): 120 NaCl, 20 TEA-Cl, 2 CoCl2, 2 MgCl2, 10 HEPES (pH 7.4). Osmolality was adjusted to 309 mOsmol/kg with glucose. Holding potentials were set to 0 mV. NLC was measured using sinusoidal voltage stimuli (2.5-Hz, 120 or 150 mV amplitude) superimposed with two higher frequency stimuli (390.6 and 781.2 Hz, 10 mV amplitude). Data were collected by jClamp (SciSoft Company, New Haven, CT, USA) [81].

4.4. NLC Data Analysis

Voltage-dependent Cm data were analyzed using the following two-state Boltzmann equation:
C m = α Q m a x e x p [ α V m − V p k ] { 1 + e x p α V m − V p k } 2 + C l i n
where α is the slope factor of the voltage-dependence of charge transfer, Qmax is the maximum charge transfer, Vm is the membrane potential, Vpk is the voltage at which the maximum charge movement is attained, and Clin is the linear capacitance. The specific capacitance, Csp, was calculated as (Cm − Clin)/Clin.

4.5. Statistical Analyses

Statistical analyses for fluorometric antiport assays were performed as previously described [10]. Briefly, five different Dox dosage conditions were compared to the rates of uninduced cells by one-way ANOVA followed by an uncorrected Fisher’s Least Significant Difference (LSD). To assess the dependency of the transport rates on Dox dosage, linear regressions (log10 [Dox] vs. transport rates) were performed. F tests were performed to find the difference in the Dox-dependence between WT versus pendrin variants. To obtain normalized activities for each pendrin variant, the slope value from the linear regressions described above was divided by that of WT. The uncertainties (σ) associated with division computations were calculated as previously described [82] using the following equation:
σ A / B = A B ( σ A A ) 2 + ( σ B B ) 2
where A and B are the mean values with associated errors in linear regressions, σA and σB, respectively. These values are reported as errors in Table 2 under “%WT Activity”. The Spearman Correlation coefficient (r) between pendrin HCO3−/Cl− transport activities and the AM scores was calculated using Prism 10.2.0. For NLC data analyses, a one-way ANOVA combined with Tukey’s post hoc test was used for multiple comparisons. In all statistical tests, p < 0.05 was considered statistically significant.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25052759/s1.

Author Contributions

Conceptualization, K.H.; investigation, S.T., T.K., K.W. and K.H.; formal analysis, S.T. and K.H.; visualization, S.T. and K.H.; writing—original draft preparation, S.T.; writing—review and editing, S.T. and K.H.; funding acquisition, K.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by NIH grant DC017482 to K. H. and the Hugh Knowles Center.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interests.

References

  1. Choi, B.Y.; Kim, H.M.; Ito, T.; Lee, K.Y.; Li, X.; Monahan, K.; Wen, Y.; Wilson, E.; Kurima, K.; Saunders, T.L.; et al. Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition. J. Clin. Investig. 2011, 121, 4516–4525. [Google Scholar] [CrossRef]
  2. Everett, 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, 153–161. [Google Scholar] [CrossRef]
  3. Zheng, J.; Shen, W.; He, D.Z.; Long, K.B.; Madison, L.D.; Dallos, P. Prestin is the motor protein of cochlear outer hair cells. Nature 2000, 405, 149–155. [Google Scholar] [CrossRef]
  4. Liberman, M.C.; Gao, J.; He, D.Z.; Wu, X.; Jia, S.; Zuo, J. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 2002, 419, 300–304. [Google Scholar] [CrossRef]
  5. Cheatham, M.A.; Huynh, K.H.; Gao, J.; Zuo, J.; Dallos, P. Cochlear function in Prestin knockout mice. J. Physiol. 2004, 560, 821–830. [Google Scholar] [CrossRef]
  6. Dallos, P.; Wu, X.; Cheatham, M.A.; Gao, J.; Zheng, J.; Anderson, C.T.; Jia, S.; Wang, X.; Cheng, W.H.; Sengupta, S.; et al. Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron 2008, 58, 333–339. [Google Scholar] [CrossRef] [PubMed]
  7. Everett, 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, 411–422. [Google Scholar] [CrossRef] [PubMed]
  8. Pryor, 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, 159–165. [Google Scholar] [CrossRef] [PubMed]
  9. Stenson, P.D.; Mort, M.; Ball, E.V.; Chapman, M.; Evans, K.; Azevedo, L.; Hayden, M.; Heywood, S.; Millar, D.S.; Phillips, A.D.; et al. The Human Gene Mutation Database (HGMD((R))): Optimizing its use in a clinical diagnostic or research setting. Hum. Genet. 2020, 139, 1197–1207. [Google Scholar] [CrossRef] [PubMed]
  10. Wasano, K.; Takahashi, S.; Rosenberg, S.K.; Kojima, T.; Mutai, H.; Matsunaga, T.; Ogawa, K.; Homma, K. Systematic quantification of the anion transport function of pendrin (SLC26A4) and its disease-associated variants. Hum. Mutat. 2020, 41, 316–331. [Google Scholar] [CrossRef] [PubMed]
  11. Takahashi, S.; Cheatham, M.A.; Zheng, J.; Homma, K. The R130S mutation significantly affects the function of prestin, the outer hair cell motor protein. J. Mol. Med. 2016, 94, 1053–1062. [Google Scholar] [CrossRef] [PubMed]
  12. Takahashi, S.; Zhou, Y.; Cheatham, M.A.; Homma, K. The pathogenic roles of the p.R130S prestin variant in DFNB61 hearing loss. bioRxiv 2023. [Google Scholar] [CrossRef]
  13. Oza, A.M.; DiStefano, M.T.; Hemphill, S.E.; Cushman, B.J.; Grant, A.R.; Siegert, R.K.; Shen, J.; Chapin, A.; Boczek, N.J.; Schimmenti, L.A.; et al. Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. Hum. Mutat. 2018, 39, 1593–1613. [Google Scholar] [CrossRef] [PubMed]
  14. Ashmore, J.F. Forward and reverse transduction in the mammalian cochlea. Neurosci. Res. Suppl. 1990, 12, S39–S50. [Google Scholar] [CrossRef] [PubMed]
  15. Santos-Sacchi, J. Reversible inhibition of voltage-dependent outer hair cell motility and capacitance. J. Neurosci. 1991, 11, 3096–3110. [Google Scholar] [CrossRef] [PubMed]
  16. Cheng, J.; Novati, G.; Pan, J.; Bycroft, C.; Zemgulyte, A.; Applebaum, T.; Pritzel, A.; Wong, L.H.; Zielinski, M.; Sargeant, T.; et al. Accurate proteome-wide missense variant effect prediction with AlphaMissense. Science 2023, 381, eadg7492. [Google Scholar] [CrossRef] [PubMed]
  17. Liu, Q.; Zhang, X.; Huang, H.; Chen, Y.; Wang, F.; Hao, A.; Zhan, W.; Mao, Q.; Hu, Y.; Han, L.; et al. Asymmetric pendrin homodimer reveals its molecular mechanism as anion exchanger. Nat. Commun. 2023, 14, 3012. [Google Scholar] [CrossRef]
  18. Ge, J.; Elferich, J.; Dehghani-Ghahnaviyeh, S.; Zhao, Z.; Meadows, M.; von Gersdorff, H.; Tajkhorshid, E.; Gouaux, E. Molecular mechanism of prestin electromotive signal amplification. Cell 2021, 184, 4669–4679.e4613. [Google Scholar] [CrossRef]
  19. Bavi, N.; Clark, M.D.; Contreras, G.F.; Shen, R.; Reddy, B.G.; Milewski, W.; Perozo, E. The conformational cycle of prestin underlies outer-hair cell electromotility. Nature 2021, 600, 553–558. [Google Scholar] [CrossRef]
  20. Butan, C.; Song, Q.; Bai, J.P.; Tan, W.J.T.; Navaratnam, D.; Santos-Sacchi, J. Single particle cryo-EM structure of the outer hair cell motor protein prestin. Nat. Commun. 2022, 13, 290. [Google Scholar] [CrossRef]
  21. Futamata, H.; Fukuda, M.; Umeda, R.; Yamashita, K.; Tomita, A.; Takahashi, S.; Shikakura, T.; Hayashi, S.; Kusakizako, T.; Nishizawa, T.; et al. Cryo-EM structures of thermostabilized prestin provide mechanistic insights underlying outer hair cell electromotility. Nat. Commun. 2022, 13, 6208. [Google Scholar] [CrossRef]
  22. Tippett, D.N.; Breen, C.; Butler, S.J.; Sawicka, M.; Dutzler, R. Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange. eLife 2023, 12, RP87178. [Google Scholar] [CrossRef]
  23. Walter, J.D.; Sawicka, M.; Dutzler, R. Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport. eLife 2019, 8, e46986. [Google Scholar] [CrossRef]
  24. Chi, X.; Jin, X.; Chen, Y.; Lu, X.; Tu, X.; Li, X.; Zhang, Y.; Lei, J.; Huang, J.; Huang, Z.; et al. Structural insights into the gating mechanism of human SLC26A9 mediated by its C-terminal sequence. Cell Discov. 2020, 6, 55. [Google Scholar] [CrossRef]
  25. Huang, B.; Han, M.; Wang, G.; Huang, S.; Zeng, J.; Yuan, Y.; Dai, P. Genetic mutations in non-syndromic deafness patients in Hainan Province have a different mutational spectrum compared to patients from Mainland China. Int. J. Pediatr. Otorhinolaryngol. 2018, 108, 49–54. [Google Scholar] [CrossRef]
  26. Carvalho, S.; Grangeiro, C.H.P.; Picanco-Albuquerque, C.G.; Dos Anjos, T.O.; De Molfetta, G.A.; Silva, W.A., Jr.; Ferraz, V.E.F. Contribution of SLC26A4 to the molecular diagnosis of nonsyndromic prelingual sensorineural hearing loss in a Brazilian cohort. BMC Res. Notes 2018, 11, 546. [Google Scholar] [CrossRef]
  27. Rendtorff, N.D.; Schrijver, I.; Lodahl, M.; Rodriguez-Paris, J.; Johnsen, T.; Hansen, E.C.; Nickelsen, L.A.; Tumer, Z.; Fagerheim, T.; Wetke, R.; et al. SLC26A4 mutation frequency and spectrum in 109 Danish Pendred syndrome/DFNB4 probands and a report of nine novel mutations. Clin. Genet. 2013, 84, 388–391. [Google Scholar] [CrossRef]
  28. Turner, T.N.; Wilfert, A.B.; Bakken, T.E.; Bernier, R.A.; Pepper, M.R.; Zhang, Z.; Torene, R.I.; Retterer, K.; Eichler, E.E. Sex-Based Analysis of De Novo Variants in Neurodevelopmental Disorders. Am. J. Hum. Genet. 2019, 105, 1274–1285. [Google Scholar] [CrossRef] [PubMed]
  29. Elsayed, O.; Al-Shamsi, A. Mutation spectrum of non-syndromic hearing loss in the UAE, a retrospective cohort study and literature review. Mol. Genet. Genomic Med. 2022, 10, e2052. [Google Scholar] [CrossRef] [PubMed]
  30. Coyle, B.; Reardon, W.; Herbrick, J.A.; Tsui, L.C.; Gausden, E.; Lee, J.; Coffey, R.; Grueters, A.; Grossman, A.; Phelps, P.D.; et al. Molecular analysis of the PDS gene in Pendred syndrome. Hum. Mol. Genet. 1998, 7, 1105–1112. [Google Scholar] [CrossRef] [PubMed]
  31. Koohiyan, M.; Hashemzadeh-Chaleshtori, M.; Tabatabaiefar, M.A. Molecular diagnosis of SLC26A4-related hereditary hearing loss in a group of patients from two provinces of Iran. Intractable Rare Dis. Res. 2021, 10, 23–30. [Google Scholar] [CrossRef] [PubMed]
  32. Wu, C.C.; Yeh, T.H.; Chen, P.J.; Hsu, C.J. Prevalent SLC26A4 mutations in patients with enlarged vestibular aqueduct and/or Mondini dysplasia: A unique spectrum of mutations in Taiwan, including a frequent founder mutation. Laryngoscope 2005, 115, 1060–1064. [Google Scholar] [CrossRef]
  33. Sakuma, N.; Moteki, H.; Takahashi, M.; Nishio, S.Y.; Arai, Y.; Yamashita, Y.; Oridate, N.; Usami, S. An effective screening strategy for deafness in combination with a next-generation sequencing platform: A consecutive analysis. J. Hum. Genet. 2016, 61, 253–261. [Google Scholar] [CrossRef] [PubMed]
  34. Ladsous, M.; Vlaeminck-Guillem, V.; Dumur, V.; Vincent, C.; Dubrulle, F.; Dhaenens, C.M.; Wemeau, J.L. Analysis of the thyroid phenotype in 42 patients with Pendred syndrome and nonsyndromic enlargement of the vestibular aqueduct. Thyroid. 2014, 24, 639–648. [Google Scholar] [CrossRef]
  35. Albert, 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, 773–779. [Google Scholar] [CrossRef] [PubMed]
  36. Blons, H.; Feldmann, D.; Duval, V.; Messaz, O.; Denoyelle, F.; Loundon, N.; Sergout-Allaoui, A.; Houang, M.; Duriez, F.; Lacombe, D.; et al. Screening of SLC26A4 (PDS) gene in Pendred’s syndrome: A large spectrum of mutations in France and phenotypic heterogeneity. Clin. Genet. 2004, 66, 333–340. [Google Scholar] [CrossRef] [PubMed]
  37. Wang, 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, 245–254. [Google Scholar] [CrossRef] [PubMed]
  38. Park, H.J.; Shaukat, S.; Liu, X.Z.; Hahn, S.H.; Naz, S.; Ghosh, M.; Kim, H.N.; Moon, S.K.; Abe, S.; Tukamoto, K.; et al. Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: Global implications for the epidemiology of deafness. J. Med. Genet. 2003, 40, 242–248. [Google Scholar] [CrossRef]
  39. Miyagawa, M.; Nishio, S.Y.; Usami, S.; Deafness Gene Study, C. Mutation spectrum and genotype-phenotype correlation of hearing loss patients caused by SLC26A4 mutations in the Japanese: A large cohort study. J. Hum. Genet. 2014, 59, 262–268. [Google Scholar] [CrossRef]
  40. Hutchin, T.; Coy, N.N.; Conlon, H.; Telford, E.; Bromelow, K.; Blaydon, D.; Taylor, G.; Coghill, E.; Brown, S.; Trembath, R.; et al. Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK—Implications for genetic testing. Clin. Genet. 2005, 68, 506–512. [Google Scholar] [CrossRef]
  41. Reardon, W.; CF, O.M.; Trembath, R.; Jan, H.; Phelps, P.D. Enlarged vestibular aqueduct: A radiological marker of pendred syndrome, and mutation of the PDS gene. QJM 2000, 93, 99–104. [Google Scholar] [CrossRef] [PubMed]
  42. Choi, 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, 599–608. [Google Scholar] [CrossRef] [PubMed]
  43. Wen, C.; Wang, S.; Zhao, X.; Wang, X.; Wang, X.; Cheng, X.; Huang, L. Mutation analysis of the SLC26A4 gene in three Chinese families. Biosci. Trends 2019, 13, 441–447. [Google Scholar] [CrossRef]
  44. Landa, P.; Differ, A.M.; Rajput, K.; Jenkins, L.; Bitner-Glindzicz, M. Lack of significant association between mutations of KCNJ10 or FOXI1 and SLC26A4 mutations in Pendred syndrome/enlarged vestibular aqueducts. BMC Med. Genet. 2013, 14, 85. [Google Scholar] [CrossRef]
  45. Zhao, J.; Yuan, Y.; Huang, S.; Huang, B.; Cheng, J.; Kang, D.; Wang, G.; Han, D.; Dai, P. KCNJ10 may not be a contributor to nonsyndromic enlargement of vestibular aqueduct (NSEVA) in Chinese subjects. PLoS ONE 2014, 9, e108134. [Google Scholar] [CrossRef]
  46. Chen, D.Y.; Chen, X.W.; Jin, X.; Zuo, J.; Wei, C.G.; Cao, K.L.; Fang, F.D. Screening of SLC26A4 (PDS) gene mutation in cochlear implant recipients with inner ear malformation. Zhonghua Yi Xue Za Zhi 2007, 87, 2820–2824. [Google Scholar]
  47. Van Hauwe, P.; Everett, L.A.; Coucke, P.; Scott, D.A.; Kraft, M.L.; Ris-Stalpers, C.; Bolder, C.; Otten, B.; de Vijlder, J.J.; Dietrich, N.L.; et al. Two frequent missense mutations in Pendred syndrome. Hum. Mol. Genet. 1998, 7, 1099–1104. [Google Scholar] [CrossRef] [PubMed]
  48. Chai, Y.; Huang, Z.; Tao, Z.; Li, X.; Li, L.; Li, Y.; Wu, H.; Yang, T. Molecular etiology of hearing impairment associated with nonsyndromic enlarged vestibular aqueduct in East China. Am. J. Med. Genet. A 2013, 161, 2226–2233. [Google Scholar] [CrossRef]
  49. Gonzalez Trevino, O.; Karamanoglu Arseven, O.; Ceballos, C.J.; Vives, V.I.; Ramirez, R.C.; Gomez, V.V.; Medeiros-Neto, G.; Kopp, P. Clinical and molecular analysis of three Mexican families with Pendred’s syndrome. Eur. J. Endocrinol. 2001, 144, 585–593. [Google Scholar] [CrossRef]
  50. Courtmans, I.; Mancilla, V.; Ligny, C.; Hilbert, P.; Mansbach, A.L.; Van Maldergem, L. Clinical findings and PDS mutations in 15 patients with hearing loss and dilatation of the vestibular aqueduct. J. Laryngol. Otol. 2007, 121, 312–317. [Google Scholar] [CrossRef]
  51. Ji, Y.B.; Han, D.Y.; Wang, D.Y.; Zhou, Y.; Zhao, C.; Wang, H.; Lan, L.; Wang, Q.J. Evaluation of deaf-mute patients with sensitive deafness gene screening in Shandong province. Zhonghua Yi Xue Za Zhi 2009, 89, 2531–2535. [Google Scholar]
  52. Pera, 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, 888–896. [Google Scholar] [CrossRef]
  53. Yuan, Y.Y.; Dai, P.; Zhu, Q.W.; Kang, D.Y.; Huang, D.L. Sequencing analysis of whole SLC26A4 gene related to IVS7-2A > G mutation in 1552 moderate to profound sensorineural hearing loss patients in China. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2009, 44, 449–454. [Google Scholar] [PubMed]
  54. Sloan-Heggen, C.M.; Bierer, A.O.; Shearer, A.E.; Kolbe, D.L.; Nishimura, C.J.; Frees, K.L.; Ephraim, S.S.; Shibata, S.B.; Booth, K.T.; Campbell, C.A.; et al. Comprehensive genetic testing in the clinical evaluation of 1119 patients with hearing loss. Hum. Genet 2016, 135, 441–450. [Google Scholar] [CrossRef]
  55. Scott, D.A.; Wang, R.; Kreman, T.M.; Andrews, M.; McDonald, J.M.; Bishop, J.R.; Smith, R.J.; Karniski, L.P.; Sheffield, V.C. Functional differences of the PDS gene product are associated with phenotypic variation in patients with Pendred syndrome and non-syndromic hearing loss (DFNB4). Hum. Mol. Genet. 2000, 9, 1709–1715. [Google Scholar] [CrossRef] [PubMed]
  56. Yoon, J.S.; Park, H.J.; Yoo, S.Y.; Namkung, W.; Jo, M.J.; Koo, S.K.; Park, H.Y.; Lee, W.S.; Kim, K.H.; Lee, M.G. Heterogeneity in the processing defect of SLC26A4 mutants. J. Med. Genet. 2008, 45, 411–419. [Google Scholar] [CrossRef]
  57. Ishihara, K.; Okuyama, S.; Kumano, S.; Iida, K.; Hamana, H.; Murakoshi, M.; Kobayashi, T.; Usami, S.; Ikeda, K.; Haga, Y.; et al. Salicylate restores transport function and anion exchanger activity of missense pendrin mutations. Hear. Res. 2010, 270, 110–118. [Google Scholar] [CrossRef]
  58. Gillam, M.P.; Bartolone, L.; Kopp, P.; Benvenga, S. Molecular analysis of the PDS gene in a nonconsanguineous Sicilian family with Pendred’s syndrome. Thyroid. 2005, 15, 734–741. [Google Scholar] [CrossRef]
  59. Taylor, J.P.; Metcalfe, R.A.; Watson, P.F.; Weetman, A.P.; Trembath, R.C. 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. [Google Scholar] [CrossRef] [PubMed]
  60. de Moraes, V.C.S.; Bernardinelli, E.; Zocal, N.; Fernandez, J.A.; Nofziger, C.; Castilho, A.M.; Sartorato, E.L.; Paulmichl, M.; Dossena, S. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants. Mol. Med. 2016, 22, 41–53. [Google Scholar] [CrossRef]
  61. Pera, 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, 18608–18613. [Google Scholar] [CrossRef]
  62. Han, J.J.; Nguyen, P.D.; Oh, D.Y.; Han, J.H.; Kim, A.R.; Kim, M.Y.; Park, H.R.; Tran, L.H.; Dung, N.H.; Koo, J.W.; et al. Elucidation of the unique mutation spectrum of severe hearing loss in a Vietnamese pediatric population. Sci. Rep. 2019, 9, 1604. [Google Scholar] [CrossRef]
  63. Cardenas, R.; Prinsley, P.; Philpott, C.; Bhutta, M.F.; Wilson, E.; Brewer, D.S.; Jennings, B.A. Whole exome sequencing study identifies candidate loss of function variants and locus heterogeneity in familial cholesteatoma. PLoS ONE 2023, 18, e0272174. [Google Scholar] [CrossRef]
  64. Mutai, H.; Suzuki, N.; Shimizu, A.; Torii, C.; Namba, K.; Morimoto, N.; Kudoh, J.; Kaga, K.; Kosaki, K.; Matsunaga, T. Diverse spectrum of rare deafness genes underlies early-childhood hearing loss in Japanese patients: A cross-sectional, multi-center next-generation sequencing study. Orphanet J. Rare Dis. 2013, 8, 172. [Google Scholar] [CrossRef]
  65. Toth, T.; Deak, L.; Fazakas, F.; Zheng, J.; Muszbek, L.; Sziklai, I. A new mutation in the human pres gene and its effect on prestin function. Int. J. Mol. Med. 2007, 20, 545–550. [Google Scholar] [CrossRef] [PubMed]
  66. Zhou, X.; Feliciano, P.; Shu, C.; Wang, T.; Astrovskaya, I.; Hall, J.B.; Obiajulu, J.U.; Wright, J.R.; Murali, S.C.; Xu, S.X.; et al. Integrating de novo and inherited variants in 42,607 autism cases identifies mutations in new moderate-risk genes. Nat. Genet. 2022, 54, 1305–1319. [Google Scholar] [CrossRef]
  67. Morgan, A.; Lenarduzzi, S.; Cappellani, S.; Pecile, V.; Morgutti, M.; Orzan, E.; Ghiselli, S.; Ambrosetti, U.; Brumat, M.; Gajendrarao, P.; et al. Genomic Studies in a Large Cohort of Hearing Impaired Italian Patients Revealed Several New Alleles, a Rare Case of Uniparental Disomy (UPD) and the Importance to Search for Copy Number Variations. Front. Genet. 2018, 9, 681. [Google Scholar] [CrossRef] [PubMed]
  68. Koire, A.; Katsonis, P.; Kim, Y.W.; Buchovecky, C.; Wilson, S.J.; Lichtarge, O. A method to delineate de novo missense variants across pathways prioritizes genes linked to autism. Sci. Transl. Med. 2021, 13, eabc1739. [Google Scholar] [CrossRef]
  69. Brandes, N.; Weissbrod, O.; Linial, M. Open problems in human trait genetics. Genome Biol. 2022, 23, 131. [Google Scholar] [CrossRef] [PubMed]
  70. Richards, S.; Aziz, N.; Bale, S.; Bick, D.; Das, S.; Gastier-Foster, J.; Grody, W.W.; Hegde, M.; Lyon, E.; Spector, E.; et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015, 17, 405–424. [Google Scholar] [CrossRef]
  71. Livesey, B.J.; Marsh, J.A. Interpreting protein variant effects with computational predictors and deep mutational scanning. Dis. Model. Mech. 2022, 15, dmm049510. [Google Scholar] [CrossRef]
  72. Tollefson, M.R.; Gogal, R.A.; Weaver, A.M.; Schaefer, A.M.; Marini, R.J.; Azaiez, H.; Kolbe, D.L.; Wang, D.; Weaver, A.E.; Casavant, T.L.; et al. Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome. Hum. Genet. 2023, 142, 819–834. [Google Scholar] [CrossRef]
  73. Fowler, D.M.; Adams, D.J.; Gloyn, A.L.; Hahn, W.C.; Marks, D.S.; Muffley, L.A.; Neal, J.T.; Roth, F.P.; Rubin, A.F.; Starita, L.M.; et al. An Atlas of Variant Effects to understand the genome at nucleotide resolution. Genome Biol. 2023, 24, 147. [Google Scholar] [CrossRef] [PubMed]
  74. Tabet, D.; Parikh, V.; Mali, P.; Roth, F.P.; Claussnitzer, M. Scalable Functional Assays for the Interpretation of Human Genetic Variation. Annu. Rev. Genet. 2022, 56, 441–465. [Google Scholar] [CrossRef] [PubMed]
  75. Keller, J.P.; Homma, K.; Duan, C.; Zheng, J.; Cheatham, M.A.; Dallos, P. Functional regulation of the SLC26-family protein prestin by calcium/calmodulin. J. Neurosci. 2014, 34, 1325–1332. [Google Scholar] [CrossRef] [PubMed]
  76. Xu, J.; Barone, S.; Varasteh Kia, M.; Holliday, L.S.; Zahedi, K.; Soleimani, M. Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney. Front. Mol. Biosci. 2022, 9, 874186. [Google Scholar] [CrossRef]
  77. Zheng, J.; Du, G.G.; Matsuda, K.; Orem, A.; Aguinaga, S.; Deak, L.; Navarrete, E.; Madison, L.D.; Dallos, P. The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting. J. Cell Sci. 2005, 118, 2987–2996. [Google Scholar] [CrossRef]
  78. Zheng, L.; Zheng, J.; Whitlon, D.S.; Garcia-Anoveros, J.; Bartles, J.R. Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model. J. Neurosci. 2010, 30, 7187–7201. [Google Scholar] [CrossRef]
  79. Royaux, I.E.; Suzuki, K.; Mori, A.; Katoh, R.; Everett, L.A.; Kohn, L.D.; Green, E.D. Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells. Endocrinology 2000, 141, 839–845. [Google Scholar] [CrossRef]
  80. Wangemann, 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, 30. [Google Scholar] [CrossRef]
  81. Santos-Sacchi, J.; Kakehata, S.; Takahashi, S. Effects of membrane potential on the voltage dependence of motility-related charge in outer hair cells of the guinea-pig. J. Physiol. 1998, 510 Pt 1, 225–235. [Google Scholar] [CrossRef] [PubMed]
  82. Takahashi, S.; Zhou, Y.; Kojima, T.; Cheatham, M.A.; Homma, K. Prestin’s fast motor kinetics is essential for mammalian cochlear amplification. Proc. Natl. Acad. Sci. USA 2023, 120, e2217891120. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. TM9-10 of pendrin. (A) The homodimeric structure of mouse pendrin (PDB: 7WK1). Protomers are shown in green and gray. Transmembrane and cytosolic domains are indicated in the lateral view (left). TM9 and TM10 are highlighted in cyan and blue, respectively, with connecting residues highlighted in yellow. The bound chlorides are indicated by red spheres. In the extracellular view (right), the core domain of one of the protomers is shown in bright orange. (B) TM9 and TM10 region of the structure (residues 376–420), extracted from the right protomer in (A). TM9 (381–398) is shown in cyan, linker region (399–405) is in yellow, and TM10 (406–416) is in blue. Bound chloride is shown as a red sphere. (C) Partial amino acid sequences of human and mouse pendrin (A4) and prestin (A5) showing the TM9 and TM10 region. Numbers in parentheses indicate the residue numbers at the N- and C-terminal ends of the partial amino acid sequences. TM9 and TM10 are highlighted in cyan and blue, respectively, with connecting residues highlighted in yellow as in (A). The residues with asterisks (*) on top and gray shades indicate the locations of missense changes evaluated in Figure 2.
Figure 1. TM9-10 of pendrin. (A) The homodimeric structure of mouse pendrin (PDB: 7WK1). Protomers are shown in green and gray. Transmembrane and cytosolic domains are indicated in the lateral view (left). TM9 and TM10 are highlighted in cyan and blue, respectively, with connecting residues highlighted in yellow. The bound chlorides are indicated by red spheres. In the extracellular view (right), the core domain of one of the protomers is shown in bright orange. (B) TM9 and TM10 region of the structure (residues 376–420), extracted from the right protomer in (A). TM9 (381–398) is shown in cyan, linker region (399–405) is in yellow, and TM10 (406–416) is in blue. Bound chloride is shown as a red sphere. (C) Partial amino acid sequences of human and mouse pendrin (A4) and prestin (A5) showing the TM9 and TM10 region. Numbers in parentheses indicate the residue numbers at the N- and C-terminal ends of the partial amino acid sequences. TM9 and TM10 are highlighted in cyan and blue, respectively, with connecting residues highlighted in yellow as in (A). The residues with asterisks (*) on top and gray shades indicate the locations of missense changes evaluated in Figure 2.
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Figure 2. HCO3−/Cl− and I−/Cl− antiport assays on pendrin variants. HCO3−/Cl− (A) and I−/Cl− (B) antiport rates were plotted against doxycycline (Dox) concentration (0.1–10 µg/mL) for each mTq2-tagged pendrin variant alongside WT as indicated. Horizontal dotted lines indicate transport rates of uninduced cells. Error bars indicate SD. Solid lines indicate linear regressions (log10 [Dox] vs. transport rates). Sample size information and statistics are provided in Table 1.
Figure 2. HCO3−/Cl− and I−/Cl− antiport assays on pendrin variants. HCO3−/Cl− (A) and I−/Cl− (B) antiport rates were plotted against doxycycline (Dox) concentration (0.1–10 µg/mL) for each mTq2-tagged pendrin variant alongside WT as indicated. Horizontal dotted lines indicate transport rates of uninduced cells. Error bars indicate SD. Solid lines indicate linear regressions (log10 [Dox] vs. transport rates). Sample size information and statistics are provided in Table 1.
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Figure 6. Correlation with AlphaMissense pathogenicity scores. HCO3−/Cl− transport rates of pendrin WT and variants from this study and the previous study [10] were normalized to the WT value and plotted against AlphaMissense (AM) pathogenicity scores [16]. Light blue shades indicate HCO3−/Cl− rate of WT with errors (propagated errors). Gray shade between AM scores 0.34–0.56 marks the “ambiguous” category, with scores lower being “benign” and higher being “pathogenic” as indicated. Red line indicates the linear fit between the HCO3−/Cl− antiport activity and the AM scores. Inset: Region indicated by the broken lines in left are enlarged to visualize variants with little or no HCO3−/Cl− transport activity.
Figure 6. Correlation with AlphaMissense pathogenicity scores. HCO3−/Cl− transport rates of pendrin WT and variants from this study and the previous study [10] were normalized to the WT value and plotted against AlphaMissense (AM) pathogenicity scores [16]. Light blue shades indicate HCO3−/Cl− rate of WT with errors (propagated errors). Gray shade between AM scores 0.34–0.56 marks the “ambiguous” category, with scores lower being “benign” and higher being “pathogenic” as indicated. Red line indicates the linear fit between the HCO3−/Cl− antiport activity and the AM scores. Inset: Region indicated by the broken lines in left are enlarged to visualize variants with little or no HCO3−/Cl− transport activity.
Ijms 25 02759 g006
Table 2. Comparison of experimental data vs. AM scores. The slope values of the Dox-dependent HCO3−/Cl− antiport activities from our previous study [10] and from this study for each variant were normalized to WT. For variants with negative slope values (slope < 0), normalized activity of 0 was assigned (highlighted in yellow) with no error propagations. Variants with WT-like HCO3−/Cl− antiport activity (p ≥ 0.05) were shaded in light blue, and ones with impaired functions (p < 0.05) are shaded in pale pink. AM scores for pathogenicity classification uses the same color shades as in Supplementary Figures S1 and S2. Asterisks (*) indicate variants not found in human patients (as of January 2024).
Table 2. Comparison of experimental data vs. AM scores. The slope values of the Dox-dependent HCO3−/Cl− antiport activities from our previous study [10] and from this study for each variant were normalized to WT. For variants with negative slope values (slope < 0), normalized activity of 0 was assigned (highlighted in yellow) with no error propagations. Variants with WT-like HCO3−/Cl− antiport activity (p ≥ 0.05) were shaded in light blue, and ones with impaired functions (p < 0.05) are shaded in pale pink. AM scores for pathogenicity classification uses the same color shades as in Supplementary Figures S1 and S2. Asterisks (*) indicate variants not found in human patients (as of January 2024).
HCO3–/Cl– Antiport Assay ResultsAM Prediction
%WT ActivityVariant EffectAM ScoreAM Category
Wasano et al., 2020 [10]p.Ser28Gly (c.82A>G)29.2 ± 8.3Function impaired0.1421benign
p.Ser49Arg (c.147C>G)104.1 ± 13.7WT-like0.0831benign
p.Pro76Ser (c.226C>T)30.5 ± 6.3Function impaired0.2937benign
p.Ser90Leu (c.269C>T)0.1 ± 0.9Function impaired0.8985pathogenic
p.Thr99Arg (c.296C>G)0Function impaired0.9802pathogenic
p.Leu117Phe (c.349C>T)95.5 ± 12.5WT-like0.804pathogenic
p.Pro123Ser (c.367C>T)2.9 ± 1.3Function impaired0.7286pathogenic
p.Gly131Val (c.392G>T)0.4 ± 1.7Function impaired0.9925pathogenic
p.Ser133Thr (c.397T>A)2.3 ± 2.0Function impaired0.4642ambiguous
p.Gly139Ala (c.416G>C)0Function impaired0.9105pathogenic
p.Met147Thr (c.440T>C)0.6 ± 2.8Function impaired0.9459pathogenic
p.Met147Val (c.439A>G)6.4 ± 8.5Function impaired0.7909pathogenic
p.Val163Ile (c.487G>A)105.1 ± 23.2WT-like0.0632benign
p.Val186Phe (c.556G>T)0Function impaired0.4777ambiguous
p.Thr193Ile (c.578C>T)0.9 ± 1.3Function impaired0.9082pathogenic
p.Tyr214Cys (c.641A>G)31.4 ± 6.0Function impaired0.8371pathogenic
p.Val239Asp (c.716T>A)4.9 ± 2.3Function impaired0.9612pathogenic
p.Asp266Asn (c.796G>A)106.7 ± 17.7WT-like0.0528ambiguous
p.Thr307Ala (c.919A>G)105.5 ± 15.2WT-like0.4074ambiguous
p.Asn324Tyr (c.970A>T)99.4 ± 19.5WT-like0.1773pathogenic
p.Gly334Val (c.1001G>T)59.5 ± 10.8Function impaired0.7329pathogenic
p.Phe354Ser (c.1061T>C)96.6 ± 17.4WT-like0.5739pathogenic
p.Lys369Glu (c.1105A>G)39.4 ± 4.2Function impaired0.4968ambiguous
p.Ala372Val (c.1115C>T)0.1± 0.9Function impaired0.9528pathogenic
p.Asn392Tyr (c.1174A>T)0.5 ± 0.7Function impaired0.9493pathogenic
p.Ser399Pro (c.1195T>C)78.9 ± 12.2Function impaired0.5658pathogenic
p.Ser408Phe (c.1223C>T)0.9 ± 1.3Function impaired0.9938pathogenic
p.Arg409His (c.1226G>A)2.8 ± 2.6Function impaired0.9499pathogenic
p.Thr410Lys (c.1229C>A)1.9 ± 1.1Function impaired0.9957pathogenic
p.Thr410Met (c.1229C>T)1.4 ± 1.7Function impaired0.9314pathogenic
p.Thr416Pro (c.1246A>C)2.2 ± 0.7Function impaired0.8985pathogenic
p.Gln421Leu (c.1262A>T)14.1 ± 5.9Function impaired0.8205pathogenic
p.Ile426Asn (c.1277T>A)63.8 ± 11.2Function impaired0.8501pathogenic
p.Leu445Trp (c.1334T>G)0Function impaired0.9933pathogenic
p.Asn457Lys (c.1371C>A)36.4 ± 5.5Function impaired0.992pathogenic
p.Arg470His (c.1409G>A)119.6 ± 19.7WT-like0.0709benign
p.Val483Glu (c.1448T>A)70.7 ± 11.6WT-like0.7757pathogenic
p.Gly497Ser (c.1489G>A)9.8 ± 2.9Function impaired0.883pathogenic
p.Thr527Pro (c.1579A>C)42.0 ± 6.8Function impaired0.7253pathogenic
p.Ile529Ser (c.1586T>G)16.1 ± 3.1Function impaired0.7726pathogenic
p.Tyr556Cys (c.1667A>G)6.5 ± 1.3Function impaired0.4069ambiguous
p.Cys565Tyr (c.1694G>A)80.8 ± 10.3WT-like0.1986benign
p.Ser657Asn (c.1970G>A)33.0 ± 6.0Function impaired0.7648pathogenic
p.Val659Leu (c.1975G>C)37.9 ± 8.1Function impaired0.2582benign
p.Ser666Phe (c.1997C>T)17.8 ± 5.3Function impaired0.4588ambiguous
p.Asp669Glu (c.2007C>A)0.0 ± 0.0Function impaired0.973pathogenic
p.Phe683Ser (c.2048T>C)2.8 ± 1.8Function impaired0.9461pathogenic
p.Phe692Leu (c.2074T>C)36.0 ± 9.6Function impaired0.7169pathogenic
p.Leu703Pro (c.2108T>C)0.3 ± 0.9Function impaired0.955pathogenic
p.Thr721Met (c.2162C>T)0.6 ± 1.7Function impaired0.4373ambiguous
p.His723Arg (c.2168A>G)13.4 ± 6.2Function impaired0.684pathogenic
This studyp.Gln383Glu (c.1147C>G)2.5 ± 2.4Function impaired0.4658ambiguous
p.Glu384Gly (c.1151G>C)2.7 ± 1.4Function impaired0.9808pathogenic
p.Ala387Val (c.1160C>T)4.7 ± 1.9Function impaired0.9179pathogenic
p.Gly389Arg (c.1165G>A)1.7 ± 1.5Function impaired0.996pathogenic
p.Gly389Arg (c.1165G>C)2.5 ± 3.1Function impaired0.996pathogenic
p.Ser391Asn (c.1172G>A)0Function impaired0.8762pathogenic
p.Ser391Arg (c.1173C>A)4.3 ± 2.4Function impaired0.9974pathogenic
p.Asn392Ser (c.1175A>G)3.4 ± 2.0Function impaired0.3199benign
p.Gly396Glu (c.1187G>A)1.0 ± 0.2Function impaired0.9866pathogenic
p.Val402Met (c.1204G>A)1.7 ± 2.2Function impaired0.5617pathogenic
p.Thr404Ile (c.1211C>T)1.7 ± 2.2Function impaired0.9734pathogenic
p.Ala406Thr (c.1216G>A)20.0 ± 4.9Function impaired0.8845pathogenic
p.Ser408Asp (c. 1222_3TC>GA) *3.7 ± 1.2Function impaired0.9971pathogenic
p.Ser408Glu (c.1222_4TCC>GAG) *2.0 ± 2.0Function impaired0.9964pathogenic
p.Arg409Cys (c.1225C>T)3.4 ± 4.1Function impaired0.8893pathogenic
p.Arg409Leu (c.1226G>T)12.0 ± 2.8Function impaired0.9763pathogenic
p.Ala411Pro (c.1231G>C)0Function impaired0.9893pathogenic
p.Ala411Thr (c.1231G>A)77.0 ± 9.8WT-like0.5393ambiguous
p.Val412Ile (c.1234G>A)95.8 ± 33.5WT-like0.0792benign
p.Gln413Pro (c.1238A>C)3.9 ± 3.4Function impaired0.9839pathogenic
p.Gln413Arg (c.1238A>G)11.2 ± 10.0Function impaired0.8859pathogenic
p.Glu414Lys (c.1240G>A)11.3 ± 8.5Function impaired0.8309pathogenic
p.Ser415Gly (c.1243A>G)37.9 ± 12.1Function impaired0.1027benign
p.Ser415Arg (c.1245C>A)46.5 ± 6.4Function impaired0.9514pathogenic
p.Ser532Ile (c.1595G>T)26.2 ± 10.8Function impaired0.3304benign
p.Ala104Val (c.311C>T)1.0 ± 2.5Function impaired0.9684pathogenic
p.Ala104Thr (c.310G>A) *9.6 ± 3.8Function impaired0.9153pathogenic
p.Ala451Gly (c.1352C>G) *104.6 ± 11.8WT-like0.1774benign
p.Ala451Ser (c.1351G>T) *96.1 ± 11.6WT-like0.2429benign
p.Ala451Leu (c.1351_2GC>CT) *1.4 ± 2.5Function impaired0.9707pathogenic
Bold font was used to distinguish between amino acid changes in the variants and the nucleotide change in the parentheses.
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Takahashi, S.; Kojima, T.; Wasano, K.; Homma, K. Functional Studies of Deafness-Associated Pendrin and Prestin Variants. Int. J. Mol. Sci. 2024, 25, 2759. https://doi.org/10.3390/ijms25052759

AMA Style

Takahashi S, Kojima T, Wasano K, Homma K. Functional Studies of Deafness-Associated Pendrin and Prestin Variants. International Journal of Molecular Sciences. 2024; 25(5):2759. https://doi.org/10.3390/ijms25052759

Chicago/Turabian Style

Takahashi, Satoe, Takashi Kojima, Koichiro Wasano, and Kazuaki Homma. 2024. "Functional Studies of Deafness-Associated Pendrin and Prestin Variants" International Journal of Molecular Sciences 25, no. 5: 2759. https://doi.org/10.3390/ijms25052759

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