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

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.

Keywords: SLC26A4; enlarged vestibular aqueduct; non-syndromic hearing loss.

Figure 1

Functionality of wild type pendrin and four pendrin variants identified in Austrian deaf patients with enlarged vestibular aqueduct (EVA). (a) Representative measurements of intracellular fluorescence intensity in HEK 293 Phoenix cells transfected with wild type (WT) or mutated pendrin and the iodide sensor enhanced yellow fluorescent protein (EYFP) H148Q;I152L or EYFP H148Q;I152L alone (control) and bathed in chloride- or iodide-containing solutions. The arrow indicates the addition of iodide to the extracellular solution. Fluorescence intensity was normalized for the average of fluorescence intensity in the chloride-containing solution; (b) Percentage of fluorescence decrease (ΔF%) determined over the experimental period (19 s) in cells expressing the indicated pendrin variants and in control cells. Error bars represent SEM. *** p < 0.001, ** p < 0.01, n.s.: not statistically significant compared to wild type; ^### p < 0.001 compared to control, one-way ANOVA with Bonferroni’s multiple comparison post-test. 36 ≤ n ≤ 107 independent measurements collected in 3–9 independent experiments.

Figure 2

Co-localization of wild type pendrin and its variants with the plasma membrane. (a) From left to right: fluorescent signal of EYFP (green) fused to the C-terminus of the indicated pendrin variants and plasma membrane (magenta) of living HeLa cells 72 h after transfection, corresponding merge image and scatter plot. Scale bar: 12 µm; (b) Pearson’s correlation coefficient, overlap coefficient and co-localization rate referred to the co-localization of wild type (WT) pendrin and the indicated variants with the plasma membrane. Error bars represent SEM. ** p < 0.01 compared to wild type and therefore excluded from the plasma membrane, n = 12, one-way ANOVA with Dunnet’s multiple comparison post-test. (n) refers to the number of cells from 3 independent experiments.

Figure 3

Co-localization of wild type pendrin and its variants with the endoplasmic reticulum (ER). (a) From left to right: fluorescent signal of EYFP (green) fused to the C-terminus of the indicated pendrin variants and ER (red) of living HeLa cells 72 h after transfection, corresponding merge image and scatter plot. Scale bar: 12 µm; (b) Pearson’s correlation coefficient, overlap coefficient and co-localization rate referred to the co-localization of wild type (WT) pendrin and the indicated variants with the ER. Error bars represent SEM. ** p < 0.01 compared to wild type and therefore retained within the ER, n = 12, one-way ANOVA with Dunnet’s multiple comparison post-test. (n) refers to the number of cells from 3 independent experiments.

Figure 4

Total cellular levels of wild type pendrin and its variants in intact cells. (a) From left to right: nuclei counterstained with 4’,6-diamidino-2-phenylindole (DAPI, cyan), fluorescent signal of EYFP (yellow) fused to the C-terminus of the indicated pendrin variants expressed in HeLa cells for 72 h and corresponding merge image. Scale bar: 30 µm; (b) Wild type (WT) and mutated pendrin total expression levels expressed as fluorescence intensity (levels of grey) normalized for the cell density. Error bars represent SEM. n = 24, ** p < 0.01 compared to wild type, one-way ANOVA with Dunnet’s multiple comparison post-test. (n) refers to the number of imaging fields from 6 independent experiments.

Figure 5

Abundance of wild type pendrin and its variants in the plasma membrane region of living cells. (a) From left to right: fluorescent signal of the transfection marker enhanced cyan fluorescent protein (ECFP, cyan), EYFP fused to the C-terminus of the indicated pendrin variants expressed in HeLa cells for 72 h (green), and plasma membrane (magenta). Scale bar: 15 µm; (b) Wild type (WT) and mutated pendrin fluorescence intensity in three regions of interest of the plasma membrane of a single cell were expressed as levels of grey, averaged and normalized for the fluorescence intensity of ECFP in the cytosol of the same cell. Error bars represent SEM. n = 24, ** p < 0.01 compared to wild type, one-way ANOVA with Bonferroni’s multiple comparison post-test. (n) refers to the number cells from 6 independent experiments.

Figure 6

Allelic discrimination plots from custom genotyping assays. The original scatter plots generated from the custom genotyping assays for c.343T>G (p.Y115D) (a) and c.1301C>A (p.A434D) (b). Little to no fluorescence from either fluorophore in either assay was generated in the no template controls (NTC). Raw endpoint fluorescence data were analyzed with QuantStudio 12K Flex software v1.2.2 and scatter plots were generated with Taqman Genotyping software v1.3 (Thermo Fisher Scientific, Waltham, MA, USA).