Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which forms a protein that transports chloride and helps cells maintain a constant external environment. Shortly after the gene was identified, the CF Mutation Database was established to catalog all variants in the gene (www.genet.sickkids.on.ca). Currently there are almost 2000 different variants described on this website. Some have been identified in individuals with CF, but only a few variants show clear evidence that they either cause CF or represent a genetic difference that does not cause disease. Several innovations require that we have a greater understanding of the genetic epidemiology of CF, the mechanism by which the mutations disrupt CFTR function, and the range of phenotypes displayed in patients who carry a mutation. This newsletter issue and next month's podcast will illustrate how the genetics of CF can be used to make a diagnosis or identify a CF carrier, predict how individuals with a given mutation will do clinically and allow clinicians to select currently available and hopefully future therapies that directly address the defect caused by their individual CFTR mutations.

Cystic Fibrosis is an autosomal recessive disorder; CFTR genes carrying CF-causing mutations must be present in both parents and passed on to the offspring to result in a baby with CF. A single CFTR copy is sometimes referred to as an allele. The two alleles that we all possess are called a CFTR genotype. The terms mutation and variant are commonly used to describe changes in a gene that make it different from the widely recognized most common version of the gene (geneticists refer to this as the "wild-type" version). It is important to note that not all differences from wild-type CFTR cause CF. Some genetic variations have no effect on protein function, others cause the protein to be completely disabled or not produced at all, while others fall somewhere in between. In general, there is good evidence that the more severely a variation disrupts or disables CFTR, the more likely patients carrying a copy of that mutation on both of their CFTR gene copies will have the disease CF.

Genetic variants can be labeled for the change they produce in the protein (F508del, G551D, or W1282X are all examples of this) or for the change they cause in the nucleotides (the DNA base pain sequence, for example 1717-1G->A or 3849+10kbC->T). A few uncommon mutations are named for changes they produce in the exons, pieces of DNA that will eventually be assembled into a mature protein (an example of this is CFTRdel2,3). As part of a movement in the genetics fields, some mutation names that CF researchers have used since the gene was found may be reported differently on diagnostic lab reports or in scientific studies — for example, F508del can be referred to as p.Phe508del or as c.1521_1523delCTT). The CF mutation database website (www.genet.sickkids.on.ca) and the CFTR2 site (www.cftr2.org) can be used to search between the legacy (traditional) and new nomenclature.

Cystic Fibrosis mutation analysis is commonly used to aid in the diagnosis of CF and in screening for carriers. The most recent guidelines on CF diagnosis require that a patient have both clinical symptoms (which could be the typical organ system manifestations of CF disease, or a newborn test for the immunoreactive trypsinogen, or a sibling with CF), and evidence of CFTR dysfunction.¹ The criteria of CFTR dysfunction can be either an elevated sweat chloride or the presence of two CF-causing mutations. In carriers who do not have symptoms as well as in newborns who may not yet have developed organ system disease, CFTR genetic analysis plays a greater role. At this time, genetic testing technologies have become less expensive and more widely available. Increasingly, the technique of sequencing the gene and looking at each nucleotide is replacing panel testing that looks only for specific mutations. The advantage of sequencing is that it can identify infrequently seen mutations that may not be contained in a panel of common CFTR mutations; however, the consequence is that variants with uncertain disease consequence may be identified.

A more challenging way to correlate CFTR genotype with phenotype extends beyond using CFTR genotype to diagnose CF toward using CFTR genotype to predict the disease course in a person with CF. Scientists have identified several ways that CFTR mutations could disrupt the CFTR protein. It was recognized that classes of mutation that retained some residual CFTR function were more likely to be associated with pancreatic-sufficient CF.² Lung function, which is most important to CF patients' morbidity and mortality, has a less clear correlation between genotype and phenotype. Lung function varies tremendously, even among patients with the same genotype.³ That variability is related to other genes as well as to environmental factors.⁴ The variability and lack of clear genotype-phenotype correlation for lung disease means that for any given individual, there is no reliable way to predict lung function over time.

Since the identification of CFTR, the CF field has searched for ways to directly correct the defects caused by CFTR mutation. The first therapy to improve CFTR function is ivacaftor, which is effective for individuals with at least one G551D CFTR mutation, is discussed in this newsletter. Experimental evidence shows that ivacaftor may also benefit other mutations that affect CFTR gating.⁵ The concept of classes of CFTR mutations will hopefully be replaced with mutation "theratypes" that are grouped by their response to a given type of drug rather than molecular defect. As we move toward this era, the priority will be finding therapies for the mutations that affect the most patients. It makes sense to be able to try these therapies in as many mutations as possible. For that reason, there is a greater need to understand the mechanism of all CFTR mutations, even the rarely seen ones.

Throughout medicine there is a shift toward tailoring therapies to an individual patient. Even in a Mendelian disease such as CF, this personalized approach is appropriate. Great strides have been made in CF care in the last several decades, with an encouraging increase in life expectancy. The improvements in airway clearance/maintenance, nutrition, and antimicrobials, as well as centralized care and attention to adherence, have all had great benefit. Bringing about a dramatic change will require a new type of therapy that alleviates defective CFTR function and can be given safely for a long time. This individualized approach to therapy will require the mutations that contribute to the differences in our CF patients to be well characterized.


Commentary References

1.	Farrell PM, Rosenstein BJ, White TB, et al. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report. J Pediatr. 2008;153:S4-S14.
2.	Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell. 1999;73(7):1251-1254.
3.	Kerem E, Corey M, Kerem BS, et al. The relation between genotype and phenotype in cystic fibrosis--analysis of the most common mutation (delta F508). N Engl J Med. 1990;323(22):1517-1522.
4.	Vanscoy LL, Blackman SM, Collaco JM. Heritability of lung disease severity in cystic fibrosis. Am J Respir Crit Care Med. 2007;175(10):1036-1043.
5.	Yu H, Burton B, Huang CJ, Worley J, et al. Ivacaftor potentiation of multiple CFTR channels with gating mutations. J Cyst Fibros. 2012;11(3):237-245.

Flume PA, Liou TG, Borowitz DS, et al; VX 08-770-104 Study Group. Ivacaftor in subjects with cystic fibrosis who are homozygous for the F508del-CFTR mutation. Chest. 2012 Sep;142(3):718-724. (For non-subscribers to this journal, an additional fee may apply to obtain full-text articles.)

View journal abstract View full article

This article summarizes a randomized trial comparing ivacaftor to placebo in patients with CF who are homozygous for the most common mutation, F508del. The rationale for this study was that in vitro experiments showed increased CFTR activity in cells carrying CFTR with an F508del mutation that are treated with ivacaftor. The magnitude of response was not as great as in cells that carried CFTR with the G551D mutation. It was not known whether these smaller improvements in CFTR function would translate to clinically meaningful improvements for CF patients with F508del mutations.

The F508del mutation and the G551D mutation disrupt CFTR in different ways. F508del prevents correct folding of the newly synthesized protein; as a result, very little CFTR protein can reach the cell surface, although the small amount that does is functional. G551D-CFTR, however, folds normally and can be found at the cell surface but is cannot be "turned on" to act as a chloride channel. The drug ivacaftor aids in "turning on" the mutant G551D-CFTR. Because a small fraction of F508del reaches the cell surface, it was considered worthwhile to see whether a drug that enhances chloride conductance, such as ivacaftor, would be beneficial.

The results of the trial demonstrated in that ivacaftor had no substantial benefit to individuals with CF due to two copies of the F508del CFTR mutation. Of the 140 patients, 112 were randomized to receive ivacaftor at the same dose as the study by Ramsey et al (150 mg twice daily), and all were offered the drug as part of the open label extension. A 2.9 mmol/L lowering of sweat chloride was seen in the patients treated with ivacaftor compared to patients receiving placebo at 16 weeks (95% CI 5.6 - 0.2, P = 0.04). However, this evidence of improved CFTR function was not associated with any statistically significant difference in lung function, quality of life (once again assessed by CF specific questionnaire), pulmonary exacerbations, or weight gain.

If larger improvements in CFTR function were associated with clinically relevant and statistically significant improvements in outcomes such as lung function, why do small improvements in CFTR function not show at least some translation to clinical outcomes? Two explanations are possible. First, the study was not designed or powered to detect small differences in lung function. It is likely that the small magnitude of improvement in CFTR function, as evidenced by the lowered sweat chloride, would have to be present for a much longer time before any difference in lung function were apparent. Second, it is also possible there is a threshold to which CFTR function must be restored before a difference in organ function is realized. These considerations will be important as future therapies are investigated.

Importantly, this trial provides further evidence that ivacaftor has an acceptable safety profile. However, no drug, especially not a new drug, has zero risk of adverse events. The most commonly seen side effects were symptoms that could be considered part of the spectrum of CF disease, such as cough, sinus congestion, and pulmonary exacerbation. All of these events occurred at similar rates with the placebo group. There were slightly higher occurrences of rash and contact dermatitis in the ivacaftor group, and these symptoms should be monitored in patients receiving ivacaftor.