Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function

The number of complex cystic fibrosis transmembrane conductance regulator ( CFTR) genotypes identified as having double-mutant alleles with two mutatio ns inherited in cis has been growing. We investigated the structure-functio n relationships of a severe cystic fibrosis (CF)-associated double mutant ( R347H-D979A) to evaluate the contribution of each mild mutation to the phen otype, CFTR mutants expressed in HeLa cells were analyzed for protein biosy nthesis and Cl- channel activity. Our data show that R347H is associated wi th mild defective Cl- channel activity and that the D979A defect leads to m isprocessing. The mutant R347H-D979A combines both defects for a dramatic d ecrease in Cl- current. To decipher the molecular mechanism of this phenoty pe, single and double mutants with different charge combinations at residue s 347 and 979 were constructed as charged residues were involved in this co mplex genotype. These studies revealed that residue 979, located in the thi rd cytoplasmic loop, is critical for CFTR processing and Cl- channel activi ty highlighting the role of charged residues. These results have also impor tant implications for CF, as they show that two mutations in cis can act in concert to alter dramatically CFTR function contributing to the wide pheno typic variability of CF disease.