Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

Moraes, Vanessa Cristine Sousa de; Bernardinelli, Emanuele; Zocal, Nathalia; Fernández, Jhonathan Angel Araujo; Nofziger, Charity; Castilho, Arthur Menino; Sartorato, Edi Lúcia; Paulmichl, Markus; Dossena, Silvia

doi:10.2119/molmed.2015.00226

Cited by 18 publications

(33 citation statements)

References 49 publications

(45 reference statements)

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“…Consistent with previous observations [ 27 ], expression levels of pendrin variants in the plasma membrane region determined on a single-cell level ( Figure 5 ) mirrored total expression levels. Expression levels of pendrin variants are summarized in Table 3 .…”

Section: Resultssupporting

confidence: 92%

“…SLC26A4 variants with no reduction in ion transport function are found in the general [ 59 , 60 ], deaf with no EVA [ 59 , 60 ] and deaf with EVA [ 27 , 61 , 62 , 63 , 64 ] populations. Including these variants into genotype-phenotype correlation analyses may lead to false conclusions [ 65 ].…”

Section: Discussionmentioning

confidence: 99%

“…The pEYFPN1 vector (Clontech, Mountain View, CA, USA), containing the cDNA of wild type or mutated human pendrin, was used for co-localization and determination of pendrin expression levels via imaging. Following transfection of this construct in cells, pendrin is produced with the enhanced yellow fluorescent protein (EYFP) fused to its C-terminus [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

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Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Roesch

Bernardinelli

Nofziger³

et al. 2018

IJMS

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Abstract:The prevalence and spectrum of sequence alterations in the SLC26A4 gene, which codes for the anion exchanger pendrin, are population-specific and account for at least 50% of cases of non-syndromic hearing loss associated with an enlarged vestibular aqueduct. A cohort of nineteen patients from Austria with hearing loss and a radiological alteration of the vestibular aqueduct underwent Sanger sequencing of SLC26A4 and GJB2, coding for connexin 26. The pathogenicity of sequence alterations detected was assessed by determining ion transport and molecular features of the corresponding SLC26A4 protein variants. In this group, four uncharacterized sequence alterations within the SLC26A4 coding region were found. Three of these lead to protein variants with abnormal functional and molecular features, while one should be considered with no pathogenic potential. Pathogenic SLC26A4 sequence alterations were only found in 12% of patients. SLC26A4 sequence alterations commonly found in other Caucasian populations were not detected. This survey represents the first study on the prevalence and spectrum of SLC26A4 sequence alterations in an Austrian cohort and further suggests that genetic testing should always be integrated with functional characterization and determination of the molecular features of protein variants in order to unequivocally identify or exclude a causal link between genotype and phenotype.

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Roesch

Bernardinelli

Nofziger³

et al. 2018

IJMS

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“…In this study, we focused on 51 exonic single nucleotide variants found in the pendrin gene, which are presumed to cause missense changes (Table ). Among these, the effects of 22 variants (p.Leu117Phe, p.Pro123Ser, p.Met147Val, p.Thr193Ile, p.Val239Asp, p.Asp266Asn, p.Phe354Ser, p.Lys369Glu, p.Ala372Val, p.Asn392Tyr, p.Ser408Phe, p.Arg409His, p.Thr410Met, p.Thr416Pro, p.Leu445Trp, p.Gly497Ser, p.Tyr556Cys, p.C565Tyr, p.Ser657Asn, p.Ser666Phe, p.Thr721Met, and p.His723Arg) on the anion transport function of pendrin have been previously studied (Choi et al, ; Dai et al, ; de Moraes et al, ; Dossena, Bizhanova et al, ; Dossena, Nofziger et al, ; Gillam, Bartolone, Kopp, & Benvenga, ; Ishihara et al, ; Jung et al, ; Kuwabara et al, ; Lee et al, ; Muskett et al, ; Scott et al, ; Taylor, Metcalfe, Watson, Weetman, & Trembath, ; Yoon et al, ), whereas the remaining 29 await experimental characterization. All these missense variants, except for p.Ser408Phe that was identified in mice in a mutagenesis screen (Dror et al, ), were found in human patients (Stenson et al, ), of which p.Tyr214Cys, p.Thr410Lys, p.Val483Glu, and p.Leu703Pro are novel pendrin missense variants described for the first time in this report (Tables S1 and S2).…”

Section: Resultsmentioning

confidence: 99%

Systematic quantification of the anion transport function of pendrin (SLC26A4) and its disease‐associated variants

et al. 2019

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Thanks to the advent of rapid DNA sequencing technology and its prevalence, many disease-associated genetic variants are rapidly identified in many genes from patient samples. However, the subsequent effort to experimentally validate and define their pathological roles is extremely slow. Consequently, the pathogenicity of most disease-associated genetic variants is solely speculated in silico, which is no longer deemed compelling. We developed an experimental approach to efficiently quantify the pathogenic effects of disease-associated genetic variants with a focus on SLC26A4, which is essential for normal inner ear function. Alterations of this gene are associated with both syndromic and nonsyndromic hereditary hearing loss with various degrees of severity. We established HEK293T-based stable cell lines that express pendrin missense variants in a doxycycline-dependent manner, and systematically determined their anion transport activities with high accuracy in a 96-well plate format using a high throughput plate reader. Our doxycycline dosagedependent transport assay objectively distinguishes missense variants that indeed impair the function of pendrin from those that do not (functional variants). We also found that some of these putative missense variants disrupt normal messenger RNA splicing. Our comprehensive experimental approach helps determine the pathogenicity of each pendrin variant, which should guide future efforts to benefit patients. K E Y W O R D SDFNB4, hereditary hearing loss, Pendred syndrome, pendrin, SLC26A4

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“…Control cells were co-transfected with pEYFP H148Q;I152L N1 and the empty pTARGET vectors. The functional test was performed 48 hours after transient transfection as already described [34][35][36][37], with adaptations allowing for the use of a multiplate reader [38][39][40][41] (Table 1). Then, the fluorescence intensity was measured again (1 measurement/sec for 16 sec).…”

Section: Pendrin Functional Testmentioning

confidence: 99%

Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4) Activity in a Human Kidney Cell Line

Bernardinelli¹,

Costa²,

Nofziger³

et al. 2016

Cell Physiol Biochem

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Background/Aims: Pendrin is a Cl^-/I^-/HCO₃^- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport) to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compound and/or the nature of the model system used in the study. Methods: Pendrin activity was evaluated by measuring pendrin-dependent iodide influx following overexpression of the transporter in a human kidney cell line, in the presence of selected test compounds or the respective vehicles. Results: Pendrin activity was significantly hampered by 0.1 mM 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB), niflumic acid and tenidap, but was resistant to 0.1 mM 4, 4′-diisothiocyano-2, 2′-stilbene-disulfonic acid (DIDS), furosemide and probenecid. Conclusions: The results of the present study indicate that clinically effective non-steroidal anti-inflammatory drugs (niflumic acid and tenidap) directly inhibit pendrin activity.

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Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

Cited by 18 publications

References 49 publications

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Systematic quantification of the anion transport function of pendrin (SLC26A4) and its disease‐associated variants

Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4) Activity in a Human Kidney Cell Line

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