Mapping pathogenic mutations suggests an innovative structural model for the pendrin (SLC26A4) transmembrane domain

Accepted Manuscript Mapping pathogenic mutations suggests an innovative structural model for the pendrin (SLC26A4) transmembrane domain Claudio Bassot, Giovanni Minervini, Emanuela Leonardi, Silvio C.E. Tosatto PII:

S0300-9084(16)30232-2

DOI:

10.1016/j.biochi.2016.10.002

Reference:

BIOCHI 5072

To appear in:

Biochimie

Received Date: 8 August 2016 Accepted Date: 3 October 2016

Please cite this article as: C. Bassot, G. Minervini, E. Leonardi, S.C.E. Tosatto, Mapping pathogenic mutations suggests an innovative structural model for the pendrin (SLC26A4) transmembrane domain, Biochimie (2016), doi: 10.1016/j.biochi.2016.10.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Mapping pathogenic mutations suggests an innovative structural model for the pendrin (SLC26A4) transmembrane domain Claudio Bassot1, Giovanni Minervini1, Emanuela Leonardi2, Silvio C.E. Tosatto1,3,*

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1 – Dept. of Biomedical Sciences and CRIBI Biotechnology Center, University of Padua, 2 – Dept. of Woman and Child Health, University of Padua, 3 – CNR Institute of Neuroscience, Padua * – To whom correspondence should be addressed at [email protected]

Abstract:

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Human pendrin (SLC26A4) is an anion transporter mostly expressed in the inner ear, thyroid and kidney. SLC26A4 gene mutations are associated with a broad phenotypic spectrum, including

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Pendred Syndrome and non-syndromic hearing loss with enlarged vestibular aqueduct (ns-EVA). No experimental structure of pendrin is currently available, making phenotype-genotype correlations difficult as predictions of transmembrane (TM) segments vary in number. Here, we propose a novel three-dimensional (3D) pendrin transmembrane domain model based on the SLC26Dg transporter. The resulting 14 TM topology was found to include two non-canonical transmembrane segments crucial for pendrin activity. Mutation mapping of 147 clinically

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validated pathological mutations shows that most affect two previously undescribed TM regions.

Keywords: pendrin, SLC26A4, Pendred Syndrome, non-syndromic hearing loss with enlarged

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Highlights:

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vestibular aqueduct (ns-EVA), homology modeling, mutation mapping, transmembrane protein.

A homology model was built for the transmembrane region of pendrin. 147 known pathogenic mutations were mapped on the pendrin model and analyzed.

Functional effects are suggested for the mutations.

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Introduction Pendrin Pathophysiology Pendrin is an anion exchanger of the apical cell membrane and member of the SLC26A family (SLC26A4) [1,2]. It mediates the transport of Cl-, HCO3-, OH-, I- ions, as well as formate, nitrate

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and thiocyanate. Reduction in pendrin functionality causes endolymph acidification and is thought to be responsible for Ca2+ re-absorption inhibition, yielding auditive sensory transduction defects [3]. The SLC26A4 gene is mostly expressed in the inner ear, thyroid and kidney, while different tissue-specific specializations were reported in the literature [3–6]. In the inner ear,

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pendrin was found in endolymphatic sac and hair cells [4], where it is involved in pH homeostasis, acting as bicarbonate/chloride exchanger [3]. In the thyroid, pendrin is expressed in

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follicular cells [7], as electroneutral iodide/chloride exchanger allowing iodide efflux from cell to follicular lumen [5]. In kidney, pendrin was found in both type B and non-A-non-B cells of cortical collectin [8], either as chloride/hydroxide or chloride/bicarbonate exchanger [6]. Mutations of the SLC26A4 gene yield Pendred Syndrome, as well as non-syndromic hearing loss with enlarged vestibular aqueduct (ns-EVA) [9]. Biallelic SLC26A4 mutations are thought to affect iodide efflux, promoting a localized defect of iodide organification, which is believed to be

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one the causes of typical Pendred syndrome, characterized by congenital fluctuating and progressive hearing loss associated with vertigo and/or goiter [10]. A number of monoallelic mutations have been associated with ns-EVA [11]. However, previous experiments aimed at shedding light on ns-EVA pathogenesis suggested that a minimal retained

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transport ability is sufficient to prevent thyroid dysfunction but not sensorineural deafness [12]. ns-EVA patients with no or biallelic mutations in SLC26A4 have been reported [9,13–15]. On the other hand, cases of Pendred syndrome and ns-EVA associated to a more complex genetic

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scenario are described in the literature, i.e. double heterozygosity between SLC26A4 and other genes, such as FOXI1 [16] and KCNJ10 [17].

1.2. Domain organization

Experimental information of protein structure is still missing, although SLC26A4 mutations are estimated to be the second most common genetic cause of human deafness. Some have been analyzed for their effects on anion transport (Table 1), however the precise molecular mechanisms underlying pendrin function remain largely unknown. Like the SulP transporters, 2

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pendrin comprises a transmembrane domain and an intracellular Sulfate Transporter – AntiSigma factor agonist (STAS) domain, regulating anion trafficking, stability and transport (16). The transmembrane domain of 780 amino acids presents a conserved “SulP sulfate transport signature” at the N-terminus of the transmembrane (TM) region [18], and a “Saier motif”,

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conserved among all SLC26A family members, located at the C-terminus of the TM domain [1,19]. Finally, two glycosylation sites at Asparagines N167 and N172, probably located on an extracellular loop of the TM domain, have been experimentally determined [20].

In 1997 Everett et al. proposed a topology model of the transmembrane region characterized by

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11 transmembrane (TM) segments [21], while Royaux et al. demonstrated the cytosolic localization of the N- and C- termini suggesting 12 TM segments [22] [23]. Recently, Gorbunov

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et al. proposed an innovative 14 TM model for prestin (SLC26A5), a member of the SLC26/SulP family and paralog of pendrin [24]. This model was constructed using a high-resolution structure of the bacterial uracil transporter UraA [25]. The authors predicted the two conserved SulP and Saier motifs to be in direct contact, overlapping two functionally relevant regions distant in sequence, termed Non Linear Charge domains NLC1 and NLC2, respectively. Very recently, the structure of SLC26Dg, a prokaryotic member of the SLC26 family, was solved. SLC26Dg adopts

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the same fold observed for UraA, with a slightly higher sequence identity with pendrin (SLC26Dg 19%, UraA 14%, respectively [26]).

Here, we present a novel pendrin homology model for the transmembrane domain (residues 86 – 509) based on the SLC26Dg protein. The model was used to map known pathogenic mutations

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onto the TM domain. These locate mostly between residues 89-142 and 393-448, corresponding to the prestin NLC domains, supporting a 14 TM topology and also suggesting a relevant functional role for the NLC domains in pendrin. The model was finally used to provide a

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molecular explanation of the loss of function effect of 37 functionally tested point mutations.

2. Materials and Methods

2.1. Sequence analysis and homology modeling The human pendrin sequence was retrieved from Uniprot [27] (accession code: O43511-1) and a topology analysis was conducted to define the TM regions. A meta-prediction was built based on the consensus between the TM region predictions of HMMTOP [28], DAS-TM filter [29], Phobius [30], TMHMM [31] and TOPCONS [32]. Pendrin homolog sequences were retrieved 3

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with three iterations of PSI-Blast [33] on the non-redundant database and aligned using T-Coffee [34] (both used with standard parameters). The alignment was manually refined using the consensus TM region map. A template search for homology modelling was performed with HHpred [35] from the manually curated alignment using default parameters. The pendrin

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structure was modeled using SwissModel [36], with an alignment between pendrin and SLC26Dg obtained by HHPred and minimized using the Chimera [37] minimization module to avoid structural artifacts. The same sequence alignment and structure template were used to perform further 3D predictions with I-Tasser [38] and Modeller [39], while QMEAN [40,41] was used to

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evaluate the model structure. 2.2. Mutation study

collected

from

the

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Non conservative missense point mutations causing both Pendred and DFNB4 syndromes were MORL

Deafness

(URL:http://www.deafnessvariationdatabase.org/)

on the 27

th

Variation

Database

of July 2015. A panel of 147

clinically validated pathological missense mutations was compiled and used to infer the structurefunction relationship. A subset of 37 mutations with experimental evidence on pendrin function

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were collected from literature [42,43,15,44–46] and used to derive mechanistic insights. Energy variation induced by mutations was estimated using NeEMO [47] for the inner-facing and Bluues [48] for the outward facing mutations.

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3. Results 3.1. The pendrin model

The TM domain of pendrin remains the structurally most elusive part, as only the STAS domain

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of the paralogous protein prestin (with 38% sequence identity to pendrin) was solved [49]. The TM region topology was therefore predicted with several methods. The results vary greatly, with predictions ranging from 9 to 14 TM and a majority for 12 TM segments. Eleven of the TM regions were consistently predicted by the majority of the methods, although single predictions disagree on predicted TM number and length (Figure 1). A consensus TM region map was used to manually refine the multiple sequence alignment of pendrin homologs, with the C-terminus being the most difficult part to align. A HHpred search returned SLC26Dg (PDB code: 5DA0) [26], a prokaryotic homolog of the SLC26 family, as the sole template for pendrin in the interval 4

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between residues 83 and 514 (19% sequence identity). From the alignment, it appears that pendrin contains two sequence insertions not present in SLC26Dg, corresponding to two loops between TM 3-TM4 and TM5-TM6 (Figure S1 Supplementary Material). The resulting pendrin model is characterized by 14 TMs of which 12 are classic transmembrane α-helices, while the

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third and tenth TM present a peculiar structure (Figure 2). In these segments, the α-helix are shorter and preceded by loops. Similar results were obtained using I-Tasser and Modeller. All generated models are in agreement on the number and composition of the TM domains, with only the conformations of

loops 154-186 and 242-251 showing significant differences (see

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Supplementary Figure S2). Analysis of model quality with QMEAN returned an overall quality score of 0.493, which is mainly due to variability in the loops. A QMEAN residue error of less

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than 4Å was estimated for the TM domain.

Although predicted TM residues are very similar in the 3D model and the TM consensus map, all prediction methods failed to predict TM10 and the majority of methods predicted TM13 and TM14 as a single TM domain. In our model, the peculiar fold of TM3 and TM10 contributes to form a central cavity in the structure. The same cavity was experimentally determined in the SLC26Dg template and in UraA, where the antiparallel β-strands plays a fundamental role in the

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uracil transport mechanism [25]. In UraA, the E290 residue located in TM10 is known to directly mediate uracil transport [25], while in SLC26Dg residues E38, E241 and Q287 located in TM1 and TM8, are supposed to form the fumarate binding site [26]. Coherently with the different substrates specificity, these residues are not conserved in pendrin.

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Indeed, the pendrin model presents a Serine-Arginine pair (S408, R409) at the position corresponding to the SLC26Dg G286 and Q287, which are suspected to mediate anion in/out translocation through the transporter [24].

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A core and gate domain were found in both the prestin model and SLC26Dg crystal structure (Figure 2). In pendrin, the core domain is composed of TM1-TM4 and TM8-TM11, while TM5TM7 and TM12-TM14 form the “gate” domain. We believe that changes in the arrangement of the core and the gate domains allow the substrate binding site to be exposed to the two sides of the membrane, as also suggested for SLC26Dg [26] and UraA [50]. The pendrin TM domain shows a tertiary structure pseudo-symmetry, with two groups of seven TM segments facing opposed sides of the membrane. Sequence conservation mapped on the pendrin model shows how structurally conserved regions are close to the exchanger core. (Figure 3). Indeed, as seen in 5

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prestin, the SulP and Saier motifs are close in structure and TM2 and TM9 are in direct contact. A sequence alignment between pendrin (UniProt accession: O43511-1) and prestin (UniProt accession: P58743) shows an overall global identity of 38%, raising to 58% and 41% when restricted to residues 89-142 and 393-448, corresponding to the functionally relevant prestin

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NLC1 and NLC2 domains [24,51]. This suggests that the structurally peculiar TM3 and TM10 as much as the SulP and Saier motifs are involved in pendrin anion transport. As expected for a TM protein, amino acid composition for the membrane-facing surface is highly hydrophobic compared to the cytosolic and luminal portions (Figure 4). Furthermore, the two experimentally

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determined glycosylation sites N167 and N172 are located on an extracellular loop between TM3 and TM4 [20].

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3.2. Pendred Syndrome and NSHL associated mutation analysis

We mapped 147 pathological missense point mutations, found in patients with Pendred syndrome or ns-Eva, on the 3D TM domain model to gain insight about functionally relevant protein regions. The mutations were mostly found to affect residues oriented towards the TM center clustering between residues 89-142 and 393-448 (Figure 3 and Figure 5), forming three different

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clusters. The ratio of pathogenic mutations within the region homologous to the prestin NLC domains and the entire TM domain was analyzed to investigate the functional role of these specific regions. We found 1 pathological mutation every 1.7 residues affecting NLCs, against a background of 1 mutation every 3.0 residues for the entire TM domain. As expected, structural

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positions of the mutation cluster vary depending on the considered topology. In the classical 12 TM topology model, mutations cluster at TM3 and in an extracellular loop between TM9 and TM10, as well as at the end of TM10 and the following cytosolic loop (Figure S2, Supplementary

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Material). In our 14 TM topology model, the same clusters were found in TM3, TM10 and at the C-terminus of TM11 (Figure 3 and Figure 5). This class of mutations are suspected to promote disease onset by affecting anion transport between TM3 and TM10, as both TM regions were suggested to be fundamental SLC26 protein family [24,26]. In particular, 35% of Pendred Syndrome-causative variants localize within TM3 and TM10, suggesting that both regions are also functionally relevant for pendrin activity (Figure 5). A subset of 37 missense mutations have been previously tested for their ability to affect anion transport. We used the 3D model to analyze possible structural effects deriving from these amino 6

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acid substitutions to gain insight in the structure-function relationship (Table 1). 29 of the 37 tested missense mutations occur at conserved positions. Functional alteration is predicted to occur mainly through the following mechanisms: destabilization of the central core (e.g. L236P), steric hindrance or destabilization of the central loops (e.g. V138F), alteration of substrate

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binding site (e.g. R409H), altered local structural flexibility of TMs or loops involved in conformational changes (e.g. P123S), disruption of the GxxxG motif on TM14 (e.g. G497S). The energy variation induced by mutations was tested with NeEMO and BLUUES (see Table 1 and Table 2). NeEMO predicts change in internal folding energy and can be useful to identify highly

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destabilizing mutations. BLUUES estimates the change in solvation energy for mutations on the protein surface, which can serve to estimate the effect on the surrounding lipids. The majority of

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the mutations tested show an increase in free energy which is consistent with reduced folding. A significant number of inner facing mutations cause a slight reduction in total free energy, which may suggest functional impairment due to unfolding.

4. Discussion

The X-ray based structure determination of transmembrane proteins is challenging due to the

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peculiar structural and physiochemical proprieties and only a limited number of structures is available [52]. This work aims to generate a novel structural model, to gain insight in the pendrin structure-function relationship, and to study the pathogenic role of mutations present in Pendred Syndrome and ns-EVA patients. In particular, we have characterized the topology of the TM

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domain by in silico structural analysis. One of the most critical aspects of current pendrin models is the exact assignment of TM segments. Experimental procedures (e.g. x-ray crystallography) generally fail to solve transmembrane proteins due to their hydrophobic nature and TM prediction

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tools still have difficulty to predict the correct TM topology [32]. Furthermore, TM topology does not provide information on 3D TM arrangement relative to the membrane. Several different pendrin models were presented in the literature [7,22,42]. The most used model counts 12 TM segments, while several authors proposed alternative topologies [21,42], all generated by single TM predictors. Our novel pendrin TM domain homology model uses the very recent SLC26Dg protein crystal structure as template [26]. The approach we adopted to build the model included construction of a TM consensus map of pendrin, based on the convergence of at least five different predictors. This was used to refine a multiple sequence alignment for both template 7

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search and target-template alignment in homology modeling. SLC26Dg shows only 19% sequence identity with the pendrin TM region, even though it is a member of the SLC26/SulP protein family. Comparison between the predictor consensus and our homology model revealed a good agreement in the definition of the TM-forming residues, confirming a large part of the

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model, except for a discrepancy in TM segment number. TM13 and TM14 are predicted as a single TM, while TM10 is not predicted. Our model shows a short loop separating the TM13 from TM14, while TM10 was predicted as a non-canonical TM helix, instead of forming a “loop” followed by a short α-helix. The short coil between TM13 and TM14, as well as the unusual

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topology of TM10, are probably the reason why predictors fail to recognize the two TM domains [53].

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Pathological mutations were mapped on our novel 14 TM model. We found known pathological mutations to cluster in two regions corresponding to the functionally relevant prestin NLC domains. Based on the proposed topology, pathological pendrin mutations are gathered in TM3 and TM10, as well as in TM11, suggesting their relevance for pendrin activity. TM3 and TM10 are also found to be non-canonical TM α-helices. Discontinuous helices are also found in other ion transporters, such as NhaA [54] and LeuTAa [55]. In these proteins, the discontinuity has a

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fundamental role for the recognition of different ions and substrate binding, conferring the required flexibility at a lower energy cost than an α-helix [56]. SLC26Dg and UraA, the only two experimentally solved structures related to the SLC26 family, have a short antiparallel β-strand in TM3 and TM10 instead of the loops predicted by our model. Future studies should clarify the

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exact structure of these two regions, in particular, whether absence of the central β-strands in pendrin is a modeling artifact or a real peculiarity of pendrin. This may be due to P142 being located in correspondence of the β-strands present in the homologous structures. The structural

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rigidity of proline may alter the conformation yielding the two loops predicted by our model. The TM segments show an inverted repetition. Pseudo-symmetry is a common feature of TM proteins as half of the all known transmembrane proteins contain internal symmetrical repeats [57]. In particular for ion transporters, it was suggested that inverted repeats help the protein to assume the inward and outward conformations [58]. A “gate “ and “core” domain are distinguished in the structure and involved in conformational change. Using the model, we discuss the role of 37 functionally tested pathological mutations (Table 1). For most mutations it was possible to propose a molecular mechanism explaining the observed 8

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altered anion transport. As expected, pathological mutations were found mostly facing towards the protein interior, due to the higher probability for a mutated residue to have a pathological effect when altering the protein core than by contacting lipids. Lipid facing mutations, tested with BLUUES, promote relevant changes in solvation energy, confirming a disruptive effect on

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hydrophobic interactions with lipid bilayer. Most loss of function mutations are predicted by NeEMO to destabilize the protein and cause unfolding, coherently with different papers suggesting retention of improperly folded pendrin mutants in the endoplasmic reticulum as the major pathological mechanism for Pendred syndrome [59,60]. The remaining inner facing

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mutations showed slighted reduction in total free energy, probably affecting the protein functionality, but with weaker effects on protein stability. In SLC26Dg the central cavity residues

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E38, E241 and Q287 (located in TM1, TM8 and TM10) are supposed to form the fumarate binding site, while their role in proton symport remains to be elucidated [26]. These residues are not conserved in pendrin which presents Q101 and V367 at the same position, which may explain the absence of fumarate-proton symport. Q287 is substituted by R409 in the pendrin model. Based on the anion binding mechanism previously proposed for prestin [24], we believe that R409 is located at the cavity center, where it mediates anion entrance. For this reason, the

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substitution with three different amino acids (R409H, R409C and R409P, respectively) are of particular interest, which if mutated are causative of both Pendred syndrome and ns-EVA. Functional assays performed on R409H show a detectable activity reduction not coupled with complete functional impairment [42]. R409H does not abolish the positive charge, but may

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introduce a relevant pH dependency yielding a reduction in activity. It also suggests that a positive charge in this position is an important factor for pendrin function. Our data suggests that regions regulating core and gate mobility are mutation hotspots. Indeed, mutations affecting

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residues S93, T105, F141, P141, V412, E414, N457 are located in the central cavity at the cytosolic site. Similarly, pathogenic mutations were found at the C-terminus of TM11 at the extracellular membrane side between the gate and core domains (Fig. 2). These two regions were suggest to have a relevant role in anion binding as well as core and gate gating regulation. The non-canonical transmembrane segments formed by TM3 and TM10 are delimited by TM2 and TM9, where the conserved Y127 residue belonging to the “Saier motif” and E384 in the SUL1 domain interact with each other to stabilize the protein fold. Mutation E384G is known to cause Pendred syndrome, yielding a phenotype characterized by complete loss of Cl- and I9

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uptake [42], confirming the structural role of this residue in maintaining activity. Five pathogenic mutations are located at the C-terminus of TM11, between the gate and core domains, and their position suggests a specific role in regulating opening/closing. Mutations in the GxxxG region suggest this specific motif to play a functional/structural role in

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pendrin dimerization. In membrane proteins, the GxxxG motif is known to facilitate oligomerization and to help proteins reaching the correct fold [61]. In the case of pendrin, GxxxG containing domain was associated with protein dimerization [42]. While pendrin appears to be mostly monomeric under physiological conditions, a dimeric form was observed by sucrose

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gradient centrifugation [62]. Of note, oligomerization was also repoted for the bacterial SLC26 protein structures [63,64]. Although prestin C415 plays a role in protein oligomerization [65], we

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did not find relevant conservation in the phylogenetic tree for the four pendrin cysteines located in the TM domain, and only mutation C466Y is reported as pathogenic. Finally, considering the good fit between our novel 3D model, the location of mutations and hydrophobicity profile, we believe that this study will be useful to future works aimed to shed light on pendrin function.

Acknowledgements

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S.T. is grateful to Alessandra Murgia for introducing him to pendrin and to members of the BioComputingUP group for insightful discussions. Funding

This work was supported by Italian Ministry of Health [GR-2011-02346845 to S.T., GR-2011-

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02347754 to E.L. and S.T.].

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Competing financial interests

The authors declare no competing financial interests.

Contributions

C.B., E.L. and S.C.E.T. conceived and designed the study and experiments. C.B. performed the experiments. C.B., G.M., E.L. analyzed the data. C.B., G.M., E.L., S.C.E.T. wrote the paper. All authors read, reviewed and approved the final manuscript. 10

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TE D

112

EP

111

AC C

110

hearing impairment with ipsilateral enlarged vestibular aqueduct. Int. J. Pediatr. Otorhinolaryngol. 74, 1049–1053. Kim, M., Kim, J., Kim, S. H., Kim, S. C., Jeon, J. H., Lee, W. S., Kim, U.-K., Kim, H. N. and Choi, J. Y. (2011) Hemorrhage in the endolymphatic sac: a cause of hearing fluctuation in enlarged vestibular aqueduct. Int. J. Pediatr. Otorhinolaryngol. 75, 1538–1544. Adato, A., Raskin, L., Petit, C. and Bonne-Tamir, B. (2000) Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus. Eur. J. Hum. Genet. 8, 437–442. Han, B., Dai, P., Qi, Q., Wang, L., Wang, Y., Bian, X., Wang, Q., Zhang, X., Kang, D., Wang, G., et al. (2007) [Prenatal diagnosis for hereditary deaf families assisted by genetic testing]. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 42, 660–663. Hu, H., Wu, L., Feng, Y., Pan, Q., Long, Z., Li, J., Dai, H., Xia, K., Liang, D., Niikawa, N., et al. (2007) Molecular analysis of hearing loss associated with enlarged vestibular aqueduct in the mainland Chinese: a unique SLC26A4 mutation spectrum. J. Hum. Genet. 52, 492–497. Cama, E., Alemanno, M. S., Bellacchio, E., Santarelli, R., Carella, M., Zelante, L., Palladino, T., Inches, I., di Paola, F., Arslan, E., et al. (2009) Identification of a novel mutation in the SLC26A4 gene in an Italian with fluctuating sensorineural hearing loss. Int. J. Pediatr. Otorhinolaryngol. 73, 1458–1463. Volo, T., Sathiyaseelan, T., Astolfi, L., Guaran, V., Trevisi, P., Emanuelli, E. and Martini, A. (2013) Hair phenotype in non-syndromic deafness. Int. J. Pediatr. Otorhinolaryngol. 77, 1280–1285. Kahrizi, K., Mohseni, M., Nishimura, C., Bazazzadegan, N., Fischer, S. M., Dehghani, A., Sayfati, M., Taghdiri, M., Jamali, P., Smith, R. J. H., et al. (2009) Identification of SLC26A4 gene mutations in Iranian families with hereditary hearing impairment. Eur. J. Pediatr. 168, 651–653. Madden, C., Halsted, M., Meinzen-Derr, J., Bardo, D., Boston, M., Arjmand, E., Nishimura, C., Yang, T., Benton, C., Das, V., et al. (2007) The influence of mutations in the SLC26A4 gene on the temporal bone in a population with enlarged vestibular aqueduct. Arch. Otolaryngol. Head Neck Surg. 133, 162–168. Rendtorff, N. D., Schrijver, I., Lodahl, M., Rodriguez-Paris, J., Johnsen, T., Hansén, E. C., Nickelsen, L. a. A., Tümer, Z., Fagerheim, T., Wetke, R., et al. (2013) SLC26A4 mutation frequency and spectrum in 109 Danish Pendred syndrome/DFNB4 probands and a report of nine novel mutations. Clin. Genet. 84, 388–391. Wu, C.-C., Yeh, T.-H., Chen, P.-J. and Hsu, C.-J. (2005) 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 115, 1060–1064. Courtmans, I., Mancilla, V., Ligny, C., Hilbert, P., Mansbach, A. L. and Van Maldergem, L. (2007) Clinical findings and PDS mutations in 15 patients with hearing loss and dilatation of the vestibular aqueduct. J Laryngol Otol 121, 312–317. Gonzalez Trevino, O., Karamanoglu Arseven, O., Ceballos, C. J., Vives, V. I., Ramirez, R. C., Gomez, V. V., Medeiros-Neto, G. and Kopp, P. (2001) Clinical and molecular analysis of three Mexican families with Pendred’s syndrome. Eur. J. Endocrinol. 144, 585–593. Kühnen, P., Turan, S., Fröhler, S., Güran, T., Abali, S., Biebermann, H., Bereket, A., Grüters, A., Chen, W. and Krude, H. (2014) Identification of PENDRIN (SLC26A4) mutations in patients with congenital hypothyroidism and “apparent” thyroid dysgenesis. J. Clin. Endocrinol. Metab. 99, E169-176. Banghova, K., Al Taji, E., Cinek, O., Novotna, D., Pourova, R., Zapletalova, J., Hnikova, O. and Lebl, J. (2008) Pendred syndrome among patients with congenital hypothyroidism detected by neonatal screening: identification of two novel PDS/SLC26A4 mutations. Eur. J. Pediatr. 167, 777–783.

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124 Ogawa, A., Shimizu, K., Yoshizaki, A., Sato, S., Kanda, Y., Kumagami, H., Takahashi, H. and Usami, S. (2013) A case of palmoplantar lichen planus in a patient with congenital sensorineural deafness. Clin. Exp. Dermatol. 38, 30–32. 125 Suzuki, H., Oshima, A., Tsukamoto, K., Abe, S., Kumakawa, K., Nagai, K., Satoh, H., Kanda, Y., Iwasaki, S. and Usami, S. (2007) Clinical characteristics and genotype-phenotype correlation of hearing loss patients with SLC26A4 mutations. Acta Otolaryngol. 127, 1292–1297. 126 Mercer, S., Mutton, P. and Dahl, H.-H. M. (2011) Identification of SLC26A4 mutations in patients with hearing loss and enlarged vestibular aqueduct using high-resolution melting curve analysis. Genet Test Mol Biomarkers 15, 365–368. 127 Bogazzi, F., Russo, D., Raggi, F., Ultimieri, F., Berrettini, S., Forli, F., Grasso, L., Ceccarelli, C., Mariotti, S., Pinchera, A., et al. (2004) Mutations in the SLC26A4 (pendrin) gene in patients with sensorineural deafness and enlarged vestibular aqueduct. J. Endocrinol. Invest. 27, 430–435.

19

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Tables Nucleotide Exon change

Residue change

Protein location

Cons.

NeEMO BLUUES stability solvatation

Ref.

Pathogenic Cellular effect localization

Functionality

Predicted molecular effect Loss of charge at membrane interface

3

p.Asp87Tyr

TM1 (Inw)

+++

-0.61

[43,66]

NSHL

PM

Reduction of formate uptake

c.279T>A

3

p.Ser93Arg

TM1 (inw)

++

-0.67

[43]

EVA

PM

Reduction of formate uptake

Insertion of charged residue at interface between gate and core

c.296C>G

3

p.Thr99Arg

TM1 (Inw)

+

+0.5

[15]

EVA

Normale iodide transport

??

c.304G>A

3

p.Gly102Arg

TM1 (Inw)

+++

+0.03

[67]

PS

ER

Loss of Iodide efflux

Insertion of charged residue in a hydrophobic region at interface between TM1 and TM11

c.367C>T

4

p.Pro123Ser

TM2 (Inw)

+++

+1.22

[13,44,68]

NSHL

Intracell.

Loss of Cl-/I-exchange activity

Removal of proline-kink in TM2

c.412G>T

4

p.Val138Phe

TM3 (Inw)

+

-0.63

[67,69–71]

PS NSHL

ER

Loss of iodide efflux

Interference with transport mechanism

c.419C>A

5

p.Pro140His

TM3 (Inw)

++

+1.77

[72,73]

PS

Loss of iodide and cloride transport

Substitution of proline in central loops. Disruption of the local structure

++

-0.65

[14,74]

EVA

Intracell.

Loss of Cl-/HCO3 -exchange activity

Substitution of proline in the loops, disruption of the local structure

+0.32

[13,44,74– 76]

NSHL

Intracell.

Loss of chloride and iodide transport

Partial loss of local hydrophobic interaction

M AN U

SC

RI PT

c.259G>T

5

p.Pro142Arg

c.439A>G

5

p.Met147Val

TM3 (Inw)

+++

c.440T>C

5

p.Met147Thr

TM3 (Inw)

+++

+11.07

[15]

EVA

c.497G>A

5

p.Ser166Asn

Extracell. Loop

-

+0.12

[14,77]

EVA

c.554G>C

5

p.Arg185Thr

Extracell. Loop

++

-0.19

[45,78,79]

PS

Intracell.

Reduction of Iodite transport

c.626G>T

6

p.Gly209Val

Amphipa. helix

c.665G>T

6

p.Gly222Val

TM5 (Inw)

EP

TE D

c.425C>T

TM3 (Inw)

Loss of local hydrophobic interaction

Intracell. Normal Cl-/HCO3 exchange

Loss of a salt bridge with Asp182

+0.73

[67,69,71]

EVA, NSHL, PS

PM

Severe reduction of Iodide transport

+++

+1.17

[43]

EVA

PM

Reduction of formate uptake

Insertion of a side chain at the interaction interface of TM13

[12,69,74,8 0,81]

PS NSHL

ER

Loss of iodide transport and Cl/I- amd Cl-/HCO3 exchange activity

Insertion of proline in α-helix Disruption of hydrophobic interaction between TM5 and TM6.

AC C

+++

c.707T>C

6

p.Leu236Pro

TM5 (Lip)

c.707T>C

6

p.Val239Asp

TM5 (Inw)

++

-0.05

[11,82,83]

PS NSHL

ER

Severe reduction of chloride and iodide transport

c.907G>C

7

p.Glu303Gln

TM7 (Inw)

+++

+0.17

[84,85]

EVA

PM

Loss of Cl-/I- amd Cl-/HCO3 exchange activity

c.941C>T

8

p.Ser314Leu

TM7 (Inw)

+++

-0.04

[43]

NSHL

Intracell.

Reduction of formate uptake

c.1003T>C

9

p.Phe335Leu

External loop

+

-0.5

[86,87]

PS

PM

Reduction of Cl-/I- amd Cl/HCO3

+++

+0.08

20

ACCEPTED MANUSCRIPT

exchange activity 9

p.Ala360Val

TM8 (Inw)

+++

+0.7

[43,76,88,8 9]

PS

Reduction of formate uptake

Larger side chain, destabilization of the central loops

c.1105A>G

9

p.Lys369Glu

TM8 (Inw)

-

-0.43

[13,44,68]

EVA

Normal Cl-/I- exchange

Maintenance of charged reside at the cytosolic interface.

c.1115C>T

9

p.Ala372Val

TM8 (Inw)

++

+0.73

[13,44,68]

EVA

Reduction in the Cl-/Iexchange

c.1151A>G

10

p.Glu384Gly

TM9 (Inw)

+++

+1.82

[12,74,80,8 1,90]

PS NSHL

ER Intracell.

Loss of chloride and iodide uptake

Loss of H bond between Glu384 and Tyr127. Necessary for TM9 and TM2 interaction

c.1174A>T

10

p.Asn392Tyr

TM9 (Inw)

+++

-0.32

[44,76,82,9 1,92]

NSHL

Intracell.

Reduction in the Cl-/Iexchange

Clashes in the protein core

c.1204G>A

10

p.Val402Met

TM10 (Inw)

+

+0.29

[87]

EVA

Intracell.

Loss of Cl-/I- and Cl-/HCO3 exchange activity

Clashes in the protein core

c.1225C>T

10

p.Arg409Cys

TM10 (Inw)

+++

+0.48

[43]

EVA

Intracell.

Loss of formate uptake

Loss of Arg409 putatively involved in anion binding

c.1226G>A

10

p.Arg409His

TM10 (Inw)

+++

+1.43

[23,42,69,9 1,93]

PS

c.1229C>T

10

p.Thr410Met

TM10 (Inw)

++

-1.02

[67,71,76,8 4,90,91,94, 95]

PS NSHL

c.1238A>C

10

p.Gln413Pro

TM10 (Inw)

++

-0.61

[72,73]

PS

c.1246A>C

10

p.Thr416Pro

Cytosolic Interface

+++

-0.49

[12,69,71,7 4,80,81]

EVA

c.1271G>A

11

p.Gly424Asp

TM11 (Inw)

+++

+0.42

[72,73]

PS

c.1334T>G

11

p.Leu445Trp

External Loop

+++

+1.31

[69– 71,75,87]

PS NSHL

c.1337A>G

11

p.Gln446Arg

External Loop

+

+0.10

[67,96]

EVA

c.1439T>A

13

p.Val480Asp

TM13 (Lip)

+

[12]

c.1454C>G

13

p.Thr485Arg

TM13 (Inw)

c.1468A>C

13

p.Ile490Leu

TM13 (Lip)

++

c.1517T>G

13

p.Leu506Arg

TM14 (Lip)

++

SC

M AN U

Reduction of chloride and Partially PM iodide transport loss of iodide efflux

Loss of cloride and iodide transport

Insertion of a proline in α helix

Loss of chloride and Iodide uptake

Insertion of a proline in loop

Reduction of Chloride and Iodide transport

Salt bridge with His135 and destabilization of internal loops

Intracell.

Loss of Chloride and Iodide transport

Change in steric hindrance

ER

Loss of iodide efflux

Insertion of a charged residue

PS

Reduction of chloride and iodide uptake

Charged residue lipid facing

[72,73]

PS

Reduction of chloride and iodide transport

Disruption of interaction between TM13 and TM5

-0.49

[12,97]

EVA

Mild reduction of chloride and iodide uptake

-0.11

[43]

EVA

TE D

Alteration of the anion binding site

EP

-18.89

-0.46

ER

Loss of Arg409 but partial conservation of positive charge

Loss of iodide efflux

AC C

+

RI PT

c.1079C>T

ER Intracell.

PM

Reduction of formate uptake

Insertion of lipid facing charged residue

Table 1. Summary of the 37 mutations tested experimentally. Missense mutations are listed with their affected nucleotide, exon and amino acid change followed by their location on the 14 TM model, conservation of the mutated residues (Cons.), and (where possible) predictions for stability with NeEMO and solvation with BLUUES. References are provided together with the cellular localization and functionality of each pathogenic variant. Where possible, a prediction of 21

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

the molecular effect of the mutation based on the 14 TM model completes the information. The location column shows the position of the residue change in the 14 TM model, indicating for affected transmembrane segments whether the position is inward-looking (Inw) or lipid-exposed (Lip). The degree of conservation shown ranges from “-“ (unconserved) to “+++” (highly conserved). Both NeEMO and BLUUES predict ∆∆G energies in Kcal/mol. The following acronyms are used for pathogenic effect: Pendred Syndrome (PS), enlarged vestibular aqueduct (EVA), non-syndromic hearing loss (NSHL). The cellular localization can be plasma membrane (PM), endoplasmic reticulum (ER) or intracellular (Intracell.).

22

ACCEPTED MANUSCRIPT

Residue change

Protein location

Cons.

NeEMO stability

BLUUES solvation

Reference

Pathogenic effect

Predicted molecular effect

+0.74

[11,72]

Benign

Maintenance of lipid facing Hydrophobic residue Loss of H bond with Asp87

3

p.Val88Ile

TM1 (Lip)

++

c.269C>T

3

p.Ser90Leu

TM1 (Inw)

+++

-0.84

[82]

NSHL

c.281C>T

3

p.Thr94Ile

TM1 (Inw

+++

-1.17

[85]

EVA

c.311C>T

4

p.Ala104Val

TM1 (Inw)

+++

+0.28

[98]

NSHL

c.314A>G

4

p.Tyr105Cys

TM1 (Inw)

++

+1.45

[86]

PS

c.317C>A

4

p.Ala106Asp

TM1 (Inw)

+++

+1.08

[86]

c.334C>T

4

p.Pro112Ser

Extracellu lar Loop

+++

+0.09

[85]

c.340G>A

4

p.Gly114Arg

TM2 (Inw)

-

-0.73

c.347G>T

4

p.Gly116Val

TM2 (Inw)

+++

-0.63

c.349C>T

4

p.Leu117Phe

TM2 (Inw)

+++

1.37

c.392G>T

4

p.Gly131Val

Cytoplas mic Loop

+++

-0.01

c.395C>T

4

p.Thr132Ile

Cytoplas mic Loop

+++

-0.58

c.397T>A

4

p.Ser133Thr

Cytoplas mic Loop

+++

c.398C>A

4

p.Ser133Stop

Cytoplas mic Loop

+++

c.404A>G

4

p.His135Arg

TM3 (Inw)

+++

c.409T>C

4

p.Ser137Pro

TM3 loop (Inw)

c.412G>C

4

p.Val138Leu

c.413T>A

4

c.416G>C

5

c.416G>T

5

c.422T>C

5

c.425C>G

c.441G>A

Loss of H bond with Ser427 of TM12

Clash with Val454

Formation of Hydrogen bond with Gly102

SC

c.262G>A

RI PT

Nucleotide Exon change

Disruption of the interaction between TM2 and TM3

EVA

Loss of conserved proline

M AN U

PS

EVA

Addition of a charge in a hydrophobic pocket

[100]

NSHL

Disruption of Gly-Gly interaction with Gly92 of TM1

[101]

Benign (in silico)

Maintenance of the hydrophobicity

[102]

NSHL

Clash with Tyr 127

[103]

NSHL

Loss of H bond with Asp380

[104]

PS

[71,105]

PS

Protein truncation

+1.62

[91]

EVA

Putative anion binding site

+++

-0.67

[105]

PS

Loss of anion binding cooperation?

TM3 loop (Inw)

+

-1.14

[106]

PS

Change in the lateral chain steric hindrance in the central loops.

p.Val138Asp

TM3 loop (Inw)

+

+0.01

[66]

NSHL-EVA

Destabilization of the central loops.

p.Gly139Ala

TM3 (Inw)

+++

-0.15

[69]

PS

Change in the lateral chain steric hindrance in the central loops.

p.Gly139Val

TM3 (Inw)

+++

+0.25

[107]

NSHL

Change in the lateral chain steric hindrance in the central loops.

p.Phe141Ser

TM3 (Inw)

+++

+3.12

[108]

NSHL

Loss of Pi stack between Phe141 and Pro142

5

p.Pro142Leu

TM3 (Inw)

++

-1.20

[70]

EVA

Loss of Pi stack between Phe141 and Pro142

5

p.Met147Ile

TM3 (Inw)

+++

+0.02

[109]

EVA

TE D

[99]

AC C

EP

+0.62

23

ACCEPTED MANUSCRIPT

5

p.Gly149Glu

TM3 (Inw)

+++

-0.75

[70]

EVA

Disruption of hydrophobic interaction between TM3 and TM1

c.487G>A

5

p.Val163Ile

Extracellu lar Loop

-

-0.48

[102]

EVA

Interference with the glycosylation site Asn 167N?

c.487G>C

5

p.Val163Leu

Extracellu lar Loop

-

-0.27

[77]

NSHL

Interference with the glycosylation Asn 167N?

c.532A>C

5

p.Thr178Pro

Extracellu lar Loop

-

+0.35

[110]

EVA

Insertion of a proline, interference with glycosylation site Asn 172N?

c.554G>C

5

p.Ile188Thr

TM4 (Inw)

++

+1.38

[76]

EVA

c.349C>T

5

p.Ala189Ser

TM4 (Inw)

++

+1.30

[101]

Benign

c.578C>T

5

p.Thr193Ile

TM4 (Inw)

+

-1.15

[70,111]

PS NSHL

c.589G>A

5

p.Gly197Arg

TM4 (Inw)

+++

-0.11

[76,85]

c.596T>C

5

p.Ile199Thr

TM4 (Inw)

++

+0.90

[15]

c.611G>T

6

p.Gly204Val

TM4

+++

-0.26

c.626G>A

6

p.Gly209Glu

Amphipat ic helix

+++

+0.47

c.644T>C

6

p.Leu215Ser

Intracellul ar loop

+++

+0.42

c.664G>A

6

p.Gly222Ser

TM5 (Inw)

+++

+0.72

c.665G>T

6

P.Gly222Ala

TM5 (Inw)

+++

+0.57

c.668T>C

6

p.Phe223Ser

TM5 (Inw)

+++

c.679G>C

6

p.Ala227Pro

TM5 (Inw)

c.691G>A

6

p.Val231Met

c.697G>C

6

c.749T>C

6

c.754T>C

6

c.757A>G

6

c.811G>C

7

c.812A>G

RI PT

c.446G>A

Insertion of a polar residue in a hydrophobic pocket.

Tolerate insertion of a polar residue in a hydrophobic pocket

SC

Loss of H bond with Phe401 Ile383

Disruption of the interaction between TM5 and TM9

EVA

Disruption of hydrophobic interaction with TM8

[85]

EVA

Disruption of the Gly interaction between TM4 and TM10

[85]

EVA

Clashes with TM11 and TM12

[102]

EVA

Formation of H bond with Ala131

[77]

NSHL

Formation of hydrogen bond withThr485 or Cys486

[102]

EVA

Insertion of a side chain at the interaction interface of TM13

+2.22

[112]

NSHL

Insertion of a polar residue in a hydrophobic pocket.

+++

+0.67

[92]

NSHL

Insertion of a proline in the α helix

TM5 (Inw)

++

-0.32

[107]

NSHL

Clashes in the protein core.

p.Val233Leu

TM5 (Inw)

+

-0.40

[113]

NSHL

p.Val250Ala

External loop

+

+0.38

[84]

NSHL

p.Ser252Pro

External loop

++

+0.38

[76,82,92]

NSHL

p.Ile253Val

External loop

++

+0.52

[43,77]

NSHL

p.Asp271His

TM6 (Inw)

-

+0.16

[69]

PS

Loss of a salt bridge with Lys362

7

p.Asp271Gly

TM6 (Inw)

-

+0.30

[85]

EVA

Loss of a salt bridge with Lys362

c.841G>A

7

p.Val281Ile

TM6 (Inw)

++

-0.41

[78]

NSHL

?

c.849G>C

7

p.Met283Ile

TM6 (Lip)

++

[101]

Benign (in silico)

Maintenance of a hydrophobic lipid facing residue.

AC C

EP

TE D

M AN U

EVA

+1.13

24

Reduction of the loop flexibility

ACCEPTED MANUSCRIPT

7

p.Pro297Gln

Intracell. loop

-

-0.20

[68]

EVA

Loss of a Proline in the loop

c.917T>G

7

p.Val306Gly

TM7

++

+ 3.55

[110]

NSHL

Insertion of a Gly in alpha helix

c.920C>T

8

p.Thr307Met

TM7 (Inw)

+

-1.06

[15]

EVA

c.941C>A

8

p.Ser314Stop

[114]

NSHL

Protein truncation

c.946G>T

8

p.Gly316Stop

[76,84]

PS

Protein truncation

c.964A>G

8

p.Asn322Asp

External loop

-

+0.34

[101]

Benign (in silico)

c.970A>T

8

p.Asn324Tyr

External loop

++

+0.63

[101]

Benign (in silico)

c.983T>G

8

p.Val328Gly

External loop

++

+0.71

[102]

EVA

c.1000G>T

8

p.Gly334Trp

External loop

+++

+1.95

[99]

c.1001G>C

8

p.Gly334Ala

External loop

+++

+2.31

[115]

c.1001G>T

8

p.Gly334Val

External loop

+++

+2.86

c.1004T>C

9

p.Phe335Ser

External loop

+

+7.49

c.1061T>C

9

p.Phe354Ser

TM8 (Lip)

+

c.1102G>T

9

p.Gly368Stop

c.1124A>G

9

p.Tyr375Cys

c.1147C>G

9

p.Gln383Glu

TM9 (Lip)

+++

c.1160C>T

10

p.Ala387Val

TM9 (Inw)

+++

c.1165G>C

10

p.Gly389Arg

TM9 (Inw)

c.1172G>A

10

p.Ser391Asn

TM9 (Inw)

c.1173C>A

10

p.Ser391Arg

TM9 (Inw)

+

c.1174A>T

10

p.Asn392Ser

TM9 (Inw)

+++

c.1187G>A

10

c.1195T>C

10

c.1211C>T

10

c.1226G>C

SC

RI PT

c.890C>A

EVA

M AN U

NSHL

[116]

NSHL

[117]

EVA

[101]

Benign (in silico)

[93]

NSHL

Protein truncation

[84]

NSHL

Loss of pi stack with Tyr 371

[118]

PS NSHL

Insertion of a negative charge at the cytosolic membrane interface

+1.11

[119]

EVA

Larger side chain cause clashes with TM2

+++

-0.35

[71]

EVA

Disruption of the interaction site between TM9 and TM4

+

-0.32

[105]

PS

Formation of a H-bond with Thr126

-0.53

[15]

EVA

Charged residue in a hydrophobic pocket. Disruption of the arrangement of the central loops

+0.38

[85]

EVA

Loss of H-bond with Ala403

-0.58

AC C

EP

TE D

++

p.Gly396Glu

TM9 (Inw)

++

-0.72

[68]

EVA

Disruption of the interaction between TM9 and TM4

p.Ser399Pro

Extracell. loop

-

-1.28

[23]

NSHL

Change of the local flexibility

p.Thr404Ile

TM10 loop (Inw)

++

-0.84

[99]

EVA

Clashes in the protein core.

10

p.Arg409Pro

TM10 (Inw)

+++

+0.53

[82]

NSHL

Loss of Arg409 putatively involved in anion binding

c.1226G>T

10

p.Arg409Leu

TM10 (Inw)

+++

-0.37

[102]

EVA

Loss of Arg409 putatively involved in anion binding

c.1231G>A

10

p.Ala411Thr

TM10

+

-0.26

[120]

EVA

Formation of a new H-bond with Leu407.

25

ACCEPTED MANUSCRIPT

(Inw) c.1231G>C

10

p.Ala411Pro

TM10 (Inw)

c.1238A>G

10

p.Gln413Arg

TM10 (Inw)

++

-0.56

[76,85,122]

NSHL

Loss of a H bonds with TM1, Ser432 and Tm12, Ser90. Disruption of local folding

c.1240G>A

10

p.Glu414Lys

TM10 (Inw)

+++

-0.02

[43]

EVA

Charge inversion loss of Salt bridge with Lys414

c.1245C>A

10

p.Ser415Arg

TM10 (Inw)

+++

-0.08

[43]

EVA

c.1259C>T

10

p.Thr420Ile

Cytosolic loop

+++

+0.27

[116]

NSHL

c.1261C>A

10

p.Gln421Lys

TM11 (Inw)

+++

-0.44

[123]

PS

c.1262A>C

10

p.Gln421Pro

TM11 (Inw)

+++

-0.51

[71,85]

EVA

c.1262A>G

10

p.Gln421Arg

TM11 (Inw)

+++

-0.52

[98]

c.1262A>T

10

p.Gln421Leu

TM11 (Inw)

+++

-0.57

[15]

c.1265T>A

11

p.Val422Asp

TM11 (Lip)

++

c.1277T>A

11

p.Ile426Asn

TM11 (Lip)

-

c.1286C>A

11

p.Ala429Glu

TM11 (Lip)

++

c.1300G>A

11

p.Ala434Thr

TM11 (Lip)

+

-0.14

c.1315G>A

11

External p.Gly439Arg anphipath ic helix

+++

+0.25

c.1318A>T

11

External p.Lys440Stop anphipath ic helix

-

c.1322T>C

11

External p.Leu441Pro anphipatic helix

+++

c.1327G>C

11

p.Glu443Gln

External Loop

-

c.1336C>T

11

p.Gln446Stop

External Loop

+

c.1343C>T

12

p.Ser448Leu

TM12 (Inw)

++

c.1363A>T

12

p.Ile455Phe

TM12 (Lip)

c.1367C>A

12

p.Ala456Asp

c.1369A>G

12

c.1369A>T c.1370A>T

-0.51

[121]

PS

Insertion of a proline in the α helix

RI PT

+

Charge insertion

Insertion of a proline in the α helix

SC NSHL

Charge insertion

EVA

M AN U +15.17

Charge insertion

[123]

PS

Charged residue lipids facing

[66]

PS

Polar residue Lipid facing

[76]

EVA

Charged residue Lipid facing

[68,124]

SDPLP

Polar residue Lipid facing

[68,125]

NSHL

Charged residue in hydrophobic region.

[85]

EVA

Protein truncation

[117]

EVA

Insertion of a proline in the α helix

[43]

EVA

Loss of salt bridge with Lys440

[85]

NSHL PS

Protein truncation

-0.94

[119]

EVA

Loss of H-bond with Asp271

++

+0.18

[82]

NSHL

TM12 (Inw)

++

-0.15

[77]

NSHL

Disruption of the interaction with TM6

p.Asn457Asp

TM12 (Inw)

+++

-0.02

[113]

NSHL

Residue putatively involve in the movement of the gate domain

12

p.Asn457Tyr

TM12 (Inw)

+++

-0.41

[108]

NSHL

Residue putatively involve in the movement of the gate domain

12

p.Asn457Ile

TM12 (Inw)

+++

-1.29

[77]

NSHL

Residue putatively involve in the movement of the gate domain

+2.01

TE D

+22.44

AC C

EP

+0.16

-0.02

26

ACCEPTED MANUSCRIPT

12

p.Asn457Lys

TM12 (Inw)

+++

-0.25

[82]

NSHL

Residue putatively involve in the movement of the gate domain

c.1397G>A

12

p.Cys466Tyr

TM12 (Inw)

-

+0.25

[77]

NSHL

Formation of H bond with Arg 470 and Glu 414

c.1409G>A

12

p.Arg470His

TM12 (Inw)

-

+0.05

[46]

NSHL

c.1415G>A

12

p.Trp472Stop

TM12

-0.05

[77]

NSHL

c.1454C>T

13

p.Thr485Met

TM13 (Inw)

+

-1.22

[126]

EVA

c.1472T>C

13

p.Ile491Thr

TM 13

+

-0.30

[46]

NSHL

c.1477G>T

13

p.Gly493Trp

Loop

+++

-0.08

[118]

PS NSHL

c.1489G>A

13

p.Gly497Ser

TM14 (Inw)

+++

0.17

[12,74,97]

EVA PS

c.1489G>C

13

p.Gly497Arg

TM14 (Inw)

+++

0.l3

[127]

c.1522A>G

13

p.Thr508Ala

TM14

+++

-0.12

[102]

c.1523C>A

13

p.Thr508Asn

TM14

+++

-0.09

RI PT

c.1371C>A

Protein truncation

Loss of the Glycine

Disruption of GxxxG motif

SC EVA

Disruption of GxxxG motif

EVA

M AN U [127]

Disruption of the interaction between TM13 and TM5

PS

Disruption of interaction between TM13 e TM14

AC C

EP

TE D

Table 2. Summary of SLC26A4 missense variants without experimentally validated functional information. Missense mutations are listed with their affected nucleotide, exon and amino acid change followed by their location on the 14 TM model, conservation of the mutated residues (Cons.), and (where possible) predictions for stability with NeEMO and solvation with BLUUES. References are provided for the origin of each variant and their pathogenic effect is indicated. Where possible, a prediction of the molecular effect of the mutation based on the 14 TM model completes the information. The location column shows the position of the residue change in the 14 TM model, indicating for affected transmembrane segments whether the position is inward-looking (Inw) or lipid-exposed (Lip). The degree of conservation shown ranges from “-“ (unconserved) to “+++” (highly conserved). Both NeEMO and BLUUES predict ∆∆G energies in Kcal/mol. The following acronyms are used for pathogenic effect: Pendred Syndrome (PS), enlarged vestibular aqueduct (EVA), non-syndromic hearing loss (NSHL), sensorineural deafness with palmoplantar lichen planus (SDPLP).

27

ACCEPTED MANUSCRIPT

Figure Legends

RI PT

Figure 1. Schematic representation of TM predictions for pendrin. Grey segments represent the position and length of predicted TM domains according to different methods. The consensus is established between at least 5 predictors and compared to the homology model. Amino acid positions are shown on top.

M AN U

SC

Figure 2. Cartoon representation of the pendrin model. The NLC domains are shown in green, the rest of the core domain in light blue and the gate domain in deep blue. (A) Zoom in on TM3 and T10, with TM11 hidden. (B) Transmembrane segments (TM) are labelled and numbered from the N- to C- terminus. Figure 3. (A) 14 TM model colored using the Consurf schema. Highly conserved residues are shown in purple, and less conserved ones in light blue. Spheres show the location of mutations in clusters indicated with the letters A,B, C. (B) Mutated residues lining the central cavity (S93, T105, F141, P141, R409, V412, E414, N457) colored using Consurf schema on the surface of the structure.

TE D

Figure 4. Hydrophobicity profile of pendrin. Red lines delimit the putative lipid bi-layer. Pendrin hydrophobic and hydrophilic surfaces are shown in red and blue respectively.

AC C

EP

Figure 5. Mutation map on the 14 TM model. α-helices are indicated as rectangles, with the gate domain in blue, the core domain in light blue, and the NLC1 and NLC2 domains (part of the core domain) in green. Point mutations are indicated with stars while stop codon mutations are indicated with a square. Colors change with the caused pathology: red for mutations found in Pendred syndrome, yellow for mutations found in ns-EVA, orange for mutations found in both, purple for benign mutations. Mutation clusters are indicated with the letters A,B,C.

28

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT