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In memory of a kind and gentle soul, I would like to share this brief and basic overview of the genetics of cystic fibrosis [CF].
While CF had been known to be an autosomal recessive gene since 1949 – given its inheritance pattern – it was in 1989 when researchers at the venerable Hospital for Sick Children in Toronto, Canada located the gene responsible for CF; they named the protein translated from the isolated gene the cystic fibrosis transmembrane receptor [CFTR]. The CFTR gene lies on chromosome 7 and there are currently over 1900 mutations of CFTR known to cause disease. These mutations are classified into 5 categories, as described below. Typically, the first 3 classes of genetic aberrations result in the most severe phenotypes; however, pulmonary phenotype correlates poorly with genotype, in general. The function of CFTR is intricate, but ultimately serves a role in chloride transport.
The Central Dogma
The DNA, within the nucleus of the cell, is unwound in a mesmerizingly, complex choreograph and transcribed into messenger RNA [mRNA]. This chemical creature is then translated into a protein, comprised of amino acids, which is modified and trafficked to its appropriate position within the cell. The CFTR gene contains over 180,000 base-pairs and the protein has 1480 amino acids. Abnormalities in any of these aforementioned steps – from DNA to fully functional protein – can result in irregularity of CFTR activity and therefore the CF phenotype.
These sorts of mutations are commonly denoted with an ‘X’ because they are a consequence of an early truncation in the mRNA. The ultimate result is a total deficiency of the CFTR protein. Class I mutations are uncommon making up less than 5% of all known mutations. In the Ashkenazi Jewish population, however, class I mutations can be quite common due to founder effects.
If the CFTR is generated, but incorrectly processed, a class II mutation has resulted. The most common mutation in Caucasians [delta F508] is within this sub-category. Delta F508 is the consequence of the deletion of phenylalanine, which normally occurs at amino acid position 508. Interestingly, if the delta F508 CFTR is placed into cell-free lipid membranes, the flawed protein is able to maintain ample chloride conductance. Unfortunately, in vivo, the protein is recognized as misfolded and degraded. In totality, nearly 1 in 2 patients with the CF phenotype will be homozygous for delta F508 and 90% of those with CF will have at least one copy.
When the CFTR is appropriately trafficked to the cell membrane, chloride transport depends on ATP binding to one of two intra-cellular nuclear binding folds [NBFs]. ATP-NBF interaction plus ATP-protein kinase A production of cAMP are both required for chloride conductance. Class III mutations usually occur within one of the NBFs such that continuous ATP activity is impaired and so too is chloride conductance. A change from glycine to aspartate at position 551 [G551D] is a common cause of diminished ATP activity in an appropriately-positioned CFTR at the cellular membrane.
Unlike class III defects, mutations of the class IV subtype result in adequate chloride transport in response to ATP stimulation. The difference is that the absolute amount of chloride efflux through the CFTR is diminished.
This subtype is characterized by normal and fully functional CFTR protein, but in diminished absolute levels at the cell membrane. Notably, such mutations may be the result of abnormalities in any of the steps of the central dogma listed above. For example, instability of the mRNA due to a splicing defect may result in an absolute reduction in normal CFTR. Some classify post-translational CFTR instability mutations as a sixth category.
Small molecules known as potentiators and correctors are showing some clinical promise. Potentiators are drugs which improve chloride conductance, most notably in Class III mutations and potentially Class IV. Ivacaftor is a potentiator and was the first drug approved for the treatment of CF with molecular specificity for the CFTR. In patients with at least one copy of the class III G551D mutation, ivacaftor, compared to placebo improved FEV1 by about 10%, reduced exacerbation frequency and led to a 3 kg weight gain.
The only approved corrector is the small molecule lumacaftor which is known to improve the folding defects of the Class II mutation [e.g. delta F508]. Importantly, lumacaftor can increase delta F508 maturation and chloride conduction by 5 fold. However, monotherapy with lumacaftor proved disappointing likely because of the functional complexity of the delta F508 protein. Importantly, the potentiator ivacaftor had previously shown some augmentation in chloride transport in the delta F508 mutation. Thus, the corrector-potentiator combination of lumacaftor-ivacaftor was attempted more recently. This combination drug, compared to placebo – in homozygotes of delta F508, with baseline FEV1 between 40 and 60% - proved to carry clinical merit. There was a about a 3-5% increase in FEV1 and diminished occurrence of pulmonary exacerbation.
While exciting, there is still much to be achieved with respect to molecular manipulation of the aberrant CFTR. The cornerstone of CF therapy remains good airway clearance, aggressive treatment of pulmonary exacerbations, a sophisticated group of health care providers knowledgeable in the management of CF and supportive loved ones. Even with all of the aforementioned, CF remains a persistent, challenging and – at times – devastating disease.