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

• Published in: Human mutation Vol. 41; no. 1; pp. 316 - 331
• Main Authors: Wasano, Koichiro, Takahashi, Satoe, Rosenberg, Samuel K., Kojima, Takashi, Mutai, Hideki, Matsunaga, Tatsuo, Ogawa, Kaoru, Homma, Kazuaki
• Format: Journal Article
• Language: English
• Published: United States Hindawi Limited 01.01.2020
• Subjects: Cell Line; Cell lines; Chloride-Bicarbonate Antiporters - genetics; Chloride-Bicarbonate Antiporters - metabolism; DFNB4; DNA sequencing; Doxycycline; Fluorescent Antibody Technique; Gene Expression; Genetic Association Studies - methods; Genetic diversity; Genetic Predisposition to Disease; Genetic Variation; Hearing loss; hereditary hearing loss; Humans; Immunohistochemistry; Inner ear; Models, Molecular; mRNA; Mutation, Missense; Pathogenicity; Pendred syndrome; pendrin; Protein Conformation; RNA Splicing; SLC26A4; Splicing; Structure-Activity Relationship; Sulfate Transporters - chemistry; Sulfate Transporters - genetics; Sulfate Transporters - metabolism; DFNB4; Pendred syndrome; SLC26A4; pendrin; hereditary hearing loss
• DOI: 10.1002/humu.23930
• ISSN: 1059-7794

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 dosage‐dependent 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.