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Image-based ?-adrenergic sweat rate assay captures minimal cystic fibrosis transmembrane conductance regulator function.

ABSTRACT: BACKGROUND:There is a need to prognosticate the severity of cystic fibrosis (CF) detected by newborn screening (NBS) by early assessment of CF transmembrane conductance regulator (CFTR) protein function. We introduce novel instrumentation and protocol for evaluating CFTR activity as reflected by ?-adrenergically stimulated sweat secretion. METHODS:A pixilated image sensor detects sweat rates. Compounds necessary for maximum sweat gland stimulation are applied by iontophoresis, replacing ID injections. Results are compared to a validated ?-adrenergic assay that measures sweat secretion by evaporation (evaporimetry). RESULTS:Ten healthy controls (HC), 6 heterozygous (carriers), 5 with CFTR-related metabolic syndrome (CRMS)/CF screen-positive, inconclusive diagnosis (CFSPID), and 12 CF individuals completed testing. All individuals with minimal and residual function CFTR mutations had low ratios of ?-adrenergically stimulated sweat rate to cholinergically stimulated sweat rate (?/chol) as measured by either assay. CONCLUSIONS:?-Adrenergic assays quantitate CFTR dysfunction in the secretory pathway of sweat glands in CF and CRMS/CFSPID populations. This novel image-sensor and iontophoresis protocol detect CFTR function with minimal and residual function and is a feasible test for young children because it is insensible to movement and it decreases the number of injections. It may also assist to distinguish between CF and CRMS/CFSPID diagnosis.

SUBMITTER: Salinas DB

PROVIDER: S-EPMC6962560 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Image-based β-adrenergic sweat rate assay captures minimal cystic fibrosis transmembrane conductance regulator function.

Salinas Danieli Barino DB Peng Yu-Hao YH Horwich Brian B Wee Choo Phei CP Frisbee Eric E Maarek Jean-Michel JM

Pediatric research 20190725 1

<h4>Background</h4>There is a need to prognosticate the severity of cystic fibrosis (CF) detected by newborn screening (NBS) by early assessment of CF transmembrane conductance regulator (CFTR) protein function. We introduce novel instrumentation and protocol for evaluating CFTR activity as reflected by β-adrenergically stimulated sweat secretion.<h4>Methods</h4>A pixilated image sensor detects sweat rates. Compounds necessary for maximum sweat gland stimulation are applied by iontophoresis, rep ...[more]

PMID: 31344706

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin–proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

Image-based ?-adrenergic sweat rate assay captures minimal cystic fibrosis transmembrane conductance regulator function.

Publications

Image-based β-adrenergic sweat rate assay captures minimal cystic fibrosis transmembrane conductance regulator function.

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