Genetic heterogeneity in patients with enlarged vestibular aqueduct and Pendred syndrome

Pathogenic variants in the SLC26A4 gene, encoding for Cl^-/HCO₃^- and I^- anion transporter pendrin, are associated with non-syndromic hearing loss with enlarged vestibular aqueduct (NSEVA) and Pendred syndrome (PDS). In the Caucasian population, up to 75% of patients fail to identify a genetic cause through biallelic mutations in the SLC26A4 gene. The CEVA haplotype could therefore play an important role in the diagnostics of NSEVA. The aim of the study was to determine the genetic etiology of hearing loss with EVA or with fully developed PDS in 37 probands and the functional characterization of novel variants identified in the SLC26A4 gene.

To determine the genetic etiology, Sanger sequencing, WES and KASP genotyping assay were used. Functional characterization of SLC26A4 variants c.140G>A (p.R47Q), c.415G>A (p.G139R), c.441G>A (p.M147I), c.481T>A (p.F161I), c.1589A>C (p.Y530S) and c.2260del (p.D754Ifs*5) involved determination of iodide influx, total and plasma membrane pendrin expression level and subcellular localization of pendrin by confocal imaging. The nanopore sequencing of nasopharyngeal swab samples was performed to confirm the pathogenic effect of potential splice site variant c.415G>A.

Biallelic variants in the SLC26A4 gene (M2 genotype) were identified in ten probands and a complete CEVA haplotype was confirmed in three probands harbouring SLC26A4 monoallelic variants (M1 genotype). Fifteen variants in the SLC26A4 gene were identified in total, three of which are novel. The functional characterization of the novel variants and variants which were not yet functionally characterized confirmed the pathogenic potential of five out of six tested variants (p.G139R, p.M147I, p.Y530S, p.D754Ifs*5, and p.F161I). Analysis of nasopharyngeal swab samples confirmed exon 4 skipping due to novel variant SLC26A4:c.415G>A. Probands with biallelic SLC26A4 variants had significantly larger thyroid volume per m² of body surface area than subjects with monoallelic SLC26A4 variants and the CEVA haplotype.

The genetic aetiology was determined in 13 out of 37 probands (35%), seven manifested with PDS and six with NSEVA. The present study highlights the importance of functional testing to confirm the pathogenicity of SLC26A4 variants and the phenotype-genotype correlation in SLC26A4-related disorders.