Hypo-functional SLC26A4 variants associated with nonsyndromic hearing loss and enlargement of the vestibular aqueduct: genotype-phenotype correlation or coincidental polymorphisms?

Abstract

Hearing loss with enlargement of the vestibular aqueduct (EVA) can be associated with mutations of the SLC26A4 gene encoding pendrin, a transmembrane Cl(-)/I(-)/HCO(3)(-) exchanger. Pendrin's critical transport substrates are thought to be I(-) in the thyroid gland and HCO(3)(-) in the inner ear. We previously reported that bi-allelic SLC26A4 mutations are associated with Pendred syndromic EVA whereas one or zero mutant alleles are associated with nonsyndromic EVA. One study proposed a correlation of nonsyndromic EVA with SLC26A4 alleles encoding pendrin with residual transport activity. Here we describe the phenotypes and SLC26A4 genotypes of 47 EVA patients ascertained since our first report of 39 patients. We sought to determine the pathogenic potential of each variant in our full cohort of 86 patients. We evaluated the trafficking of 11 missense pendrin products expressed in COS-7 cells. Products that targeted to the plasma membrane were expressed in Xenopus oocytes for measurement of anion exchange activity. p.F335L, p.C565Y, p.L597S, p.M775T, and p.R776C had Cl(-)/I(-) and Cl(-)/HCO(3)(-) exchange rate constants that ranged from 13 to 93% of wild type values. p.F335L, p.L597S, p.M775T and p.R776C are typically found as mono-allelic variants in nonsyndromic EVA. The high normal control carrier rate for p.L597S indicates it is a coincidentally detected nonpathogenic variant in this context. We observed moderate differential effects of hypo-functional variants upon exchange of HCO(3)(-) versus I(-) but their magnitude does not support a causal association with nonsyndromic EVA. However, these alleles could be pathogenic in trans configuration with a mutant allele in Pendred syndrome.

FIGURE 1

Intracellular trafficking of SLC26A4/pendrin variants in COS-7 cells. Merged representative images show GFP-tagged wild type or missense allele products (green) and concanavalin A staining (red) of plasma membranes. Co-localization (yellow) indicates targeting of pendrin to the plasma membrane. Green intracellular reticular fluorescence reflects retention of pendrin in the endoplasmic reticulum. Scale bars = 20 μm.

FIGURE 2

SLC26A4/pendrin variants exhibit altered Cl⁻/Cl⁻ and Cl⁻/I⁻ exchange. A: Time course of ³⁶Cl⁻ efflux from representative individual oocytes expressing wild type or variant pendrin. Oocytes were sequentially exposed to 96 mM Cl⁻, then to Cl⁻-free bath first containing and then lacking 5 mM I⁻. B: ³⁶Cl⁻ efflux rate constants (means ± S.E.) for wild type pendrin and the indicated variants in the presence of 96 mM Cl⁻ (black bar), 0 mM Cl⁻/5 mM I⁻ (light gray bar) and 0 Cl⁻/0 I⁻ (dark gray bar), as derived from experiments such as shown in (A). C: Rate constants of Cl⁻/I⁻ exchange normalized to the corresponding rate constants of Cl⁻/Cl⁻ exchange. Mean values ± S.E. for each group of (n) oocytes expressing wild type pendrin or the indicated missense variant differed by one-way ANOVA. P<0.05 compared to wild type pendrin as calculated by Student’s unpaired t-test (gray bars) or by Tukey’s t-test for multiple comparisons (asterisk).

FIGURE 3

SLC26A4/pendrin variants exhibit altered Cl⁻/HCO₃⁻ exchange. A: Time course of ³⁶Cl⁻ efflux from representative individual oocytes expressing wild type or variant pendrin. Oocytes were sequentially exposed to 96 mM Cl⁻, then 24 mM HCO₃⁻ in the presence of Cl⁻ followed by removal of Cl⁻ in the continued presence of HCO₃⁻ and finally by removal of HCO₃⁻. B: ³⁶Cl⁻ efflux rate constants for wild type pendrin and indicated missense variants in the presence of 96 mM Cl⁻ (black bar), 76 mM Cl⁻/24 mM HCO₃⁻ (light gray bar) and 0 Cl⁻/24 mM HCO₃⁻ (dark gray bar). C: Rate constants of Cl⁻/HCO₃⁻ exchange normalized to corresponding rate constants of Cl⁻/Cl⁻ exchange. Mean values ± S.E. for groups of (n) oocytes expressing wild type pendrin or the indicated missense variant differed by one-way ANOVA. P<0.05 compared to wild type pendrin as calculated by Student’s unpaired t-test (gray bars) or Tukey’s t-test for multiple comparisons (asterisks).