Title: Role of SLC6A14 as a Modifier of Cystic Fibrosis Phenotype
Abstract: Cystic Fibrosis (CF) is considered to be a single gene disorder, most commonly the result of a F508del mutation in the CFTR gene. However, patients homozygous for F508del mutation show a considerable variation in disease severity. Consistent with this observation, recent genome wide association studies identified secondary genes that modify disease. A SNP in the putative promoter region of SLC6A14 gene showed the highest significance (p=1.28x10−12). SLC6A14 is a cationic/neutral amino acid transporter, which transports 2Na+, 1 Cl− and a basic/neutral amino-acid into the cell from the apical surface of intestinal/bronchial epithelium. We hypothesize that expression of SLC6A14 is protective against meconium ileus, a CF phenotype detected at birth. To test this hypothesis we over-expressed SLC6A14 in BHK cells stably expressing either WT or F508del-CFTR. We measured CFTR channel activity using the halide sensitive fluorophore, SPQ. We found that CFTR function is enhanced with over-expression of SLC6A14 in cells expressing WT CFTR or F508del CFTR (after rescue of its trafficking defect), (p<0.05). This suggests a possible functional interaction between SLC6A14 and CFTR. We also monitored the functional interaction of SLC6A14 and CFTR in Caco-2 cells, an intestinal cell line that endogenously expresses both CFTR and SLC6A14. Caco-2 cells form three-dimensional, cyst-like cultures wherein CFTR mediated secretion determines the volume of its internal cavity. We found that the area of the cyst cavity increases upon cAMP stimulation of CFTR stimulation by 5.5 fold and that this expansion was decreased by 44% in the presence of the SLC6A14 inhibitor (α-methyltryptophan). These results suggest that SLC6A14 exerts a positive effect on regulated CFTR function and future studies will explore the possibility that agonists of SLC6A14 could constitute a means for therapeutic intervention. (Studies supported by grants to C.E.B and J.M.R. from CFC, CIHR)