Mutations Causing Cystic Fibrosis

A number of different mutations in the CFTR protein can underlie the cystic fibrosis disease phenotype in humans. Five different classes of CFTR mutations have been described based on the nature of the molecular defect, and have been outlined by Proesmans et al. (Proesmans et al., 2008). Class I CFTR mutants are truncated and non-functional due to frameshift or nonsense mutations that prevent proper protein formation. Class II CFTR mutants do not reach the cell surface due to missense mutations or single amino acid deletions that result in protein misfolding. Misfolded CFTR is detected by ER quality control mechanisms, leading to CFTR degradation. Class III CFTR mutants reach the cell surface, however they are non-functional due to some form of missense mutation resulting in a disease phenotype. Class IV CFTR mutants also reach the cell surface, however they are partially functional and only result in improper chloride conductance. Class V CFTR mutants are functionally normal CFTR proteins which are transcribed at a lower rate due to some disruption of synthesis or processing (Proesmans et al., 2008).

Experimental Approaches

In order to determine which class of CFTR mutation underlies the disease-causing phenotype of a novel CFTR mutant cDNA, the first step would be the proper sequencing of this cDNA. This sequencing can be accomplished using any of a number of commercially available sequencing technologies following the PCR-based amplification of the disease-causing cDNA. The cDNA sequence can then be compared to the sequence of WT CFTR in order to establish what mutations are present in the protein sequence. If nonsense or frameshift mutations are present, it is likely that this mutant will be of a class I phenotype, whereas if missense mutations are present only in intronic reasons it is likely that there will be some splicing defect which will result in a class V mutation.

After sequence comparisons are complete, the next step would be to assess whether the mutant CFTR protein exits the ER. To do this, cells transfected with either WT-CFTR or the mutant cDNA should be pulse labeled with radiolabeled methionine for 30 minutes, CFTR should be immunoprecipitated after either 0 or 3 hours chase, and immunoprecipitated CFTR should be run on a gel (Wang, et al., 2004). This will allow for an assessment of the relative levels of B-band (ER-associated) and C-band (golgi-associated) CFTR, as the heavily glycosylated C-band will migrate at a higher molecular weight than will the less glycosylated B-band. If the mutant CFTR protein is present in only the B-band form after 3 hours chase time, this suggests that it is never entering the golgi and is thus of the class II phenotype.

If mutant CFTR is present in the C-band, it should next be established to what extent the protein is present on the cell surface. This can be done by treating mutant- or WT-CFTR expressing cells with cyclohexamide (CHX) to prevent protein synthesis and then fixing (but not permeabilizing) cells at various points following this treatment. Surface levels of CFTR can then be established using fluorescently labeled anti-CFTR antibodies specific to the extracellular domain of the CFTR protein. If there is a significantly decreased amount of mutant-CFTR on the cell surface compared to WT-CFTR, particularly following CHX treatment, then it is likely that this particular mutant is of the class V phenotype. This can be confirmed by assessing the functionality of the CFTR chloride channel using previously described iodide influx assays (Pedemonte, et al., 2005). If the mutant CFTR is present at reduced levels on the cell surface and remains functional, this will confirm that this mutant is of a class V phenotype.

If the abovementioned experiments have not established the class of CFTR mutant, then the mutant cDNA must encode for either a class III or class IV mutant CFTR which reaches the cell surface but is partially non-functional. Class III mutants are typically non-repsonsive to cAMP stimulation, whereas class IV mutants are responsive to such stimulation but generate reduced Cl- channel activity (Proesmans, et al., 2008). To test whether mutant CFTR is amenable to some degree of cAMP-activation, PKA-treated or untreated mutant CFTR should be assessed for chloride conductance using previously described methods (Cai, et al., 2006). If there is some increase in chloride conductance following PKA treatment (but less so than in PKA-treated WT-CFTR expressing cells) then this implies a mutant of the class IV phenotype. If, on the other hand, there is no increase in conductance following PKA-treatment, then the mutant CFTR is likely of the class III phenotype.

Ultimately, the abovementioned techniques should allow for the efficient categorization of the class of CFTR mutant encoded by the novel cDNA. Knowledge of this classification will allow for both investigations of potential corrective treatments and studies of altered CFTR function in vitro.


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