Functional Study of CFTR Mutations Using CFTR-eGFP Expression in MDCK Cells Author: Fiorella Maldonado
Location: Quito, Ecuador
Course: HTGAA (How To Grow (Almost) Anything)
SECTION 1: ABSTRACT Cystic fibrosis (CF) is a life-threatening autosomal recessive disorder caused by mutations in the CFTR gene located on chromosome 7q31.2. While modulator therapies such as Trikafta have transformed care for CF patients carrying the common F508del mutation, these treatments are not accessible or effective for patients with uncharacterized rare variants. In Ecuador, unpublished data from our team reveal that F508del is rare and that H609R has emerged as the most prevalent variant, highlighting a critical gap in knowledge that prevents precision medicine for Ecuadorian CF patients. The broad objective of this project is to establish a functional platform to characterize the trafficking and localization of Ecuadorian CFTR variants using a CFTR-eGFP fusion expression system in polarized MDCK cells. The central hypothesis is that uncharacterized variants such as H609R cause measurable defects in CFTR trafficking to the apical membrane, and these defects can be quantified using fluorescence microscopy and biochemical assays. To achieve this objective, three specific aims will be pursued. Aim 1 (completed) involved identification of the most common CFTR variants in Ecuador and in silico design of a CFTR-eGFP fusion plasmid using SnapGene. Aim 2 will construct mutant plasmids via restriction enzyme cloning, transiently transfect MDCK cells, and evaluate protein localization using confocal microscopy and cell surface biotinylation followed by Western blot. Aim 3 will apply these functional data to classify Ecuadorian variants and guide modulator therapy decisions. This project addresses an urgent need for region-specific genetic evidence to enable equitable CF care.