Abstract
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.
Original language | English (US) |
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Pages (from-to) | 316-331 |
Number of pages | 16 |
Journal | Human mutation |
Volume | 41 |
Issue number | 1 |
DOIs | |
State | Published - Jan 1 2020 |
Funding
We thank Dr. Jing Zheng for providing laboratory resources for generating the pendrin constructs used in this study. This study is supported by NIH grants (DC014553 and DC017482 to K. H., DC011813 to J. Z.), JSPS Grant-in-Aid for Scientific Research (18K16869 to K. W.), and the Hugh Knowles Center. Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. We thank Dr. Jing Zheng for providing laboratory resources for generating the pendrin constructs used in this study. This study is supported by NIH grants (DC014553 and DC017482 to K. H., DC011813 to J. Z.), JSPS Grant‐in‐Aid for Scientific Research (18K16869 to K. W.), and the Hugh Knowles Center. Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center.
Keywords
- DFNB4
- Pendred syndrome
- SLC26A4
- hereditary hearing loss
- pendrin
ASJC Scopus subject areas
- Genetics
- Genetics(clinical)