1. © Vertex Pharmaceuticals Incorporated | VXMA-US-92-00008| 04/2017 FOR EDUCATIONAL PURPOSES ONLY

2. 2
© Vertex Pharmaceuticals Incorporated FOR EDUCATIONAL PURPOSES ONLY

3. Key Points
Linkage analysis provides evidence for the existence of a single CFTR locus on human chromosome 7 (region q31)1
There are more than 1900 known mutations of the CFTR gene2
Of the mutations reported so far, 140 meet clinical and functional criteria and are categorized as causing CF2
The loss of CFTR protein activity is a result of mutations in the CFTR gene that lead to decreased quantity and/or function of CFTR protein at the epithelial apical cell surface. This leads to defective Cl– transport through CFTR protein channels and is the underlying defect resulting in CF3
The F508del mutation is the most common cause of CF, present in about 83% of patients worldwide4
About 43% of the CF population is homozygous for the F508del mutation worldwide4
References
Kerem JR et al. Science. 1989;245:
Sosnay PR et al. Nat Genet. 2013;45(10):
Zeilinski J. Respiration. 2000;67:
CFTR2.org.

4. Key Point
CFTR protein is delivered to the apical epithelial membrane in a multi-step process comprising CFTR synthesis, CFTR folding and processing, CFTR trafficking, CFTR turnover and CFTR function at the cell surface
Additional Information
CFTR Synthesis: CFTR gene is transcribed to mRNA in the nucleus. Introns are removed from mRNA in a splicing process. mRNA is translated to immature CFTR protein in the endoplasmic reticulum with the aid of ribosomes
CFTR Folding and Processing: Immature CFTR protein is folded in the endoplasmic reticulum, thereby becoming mature CFTR protein. Any protein that is not properly folded is degraded (processed) by intracellular quality control system
CFTR Trafficking: Mature CFTR protein is trafficked to the Golgi apparatus and then to the cell surface
CFTR Turnover: CFTR channels at the cell surface are eventually removed through the process of turnover
CFTR Function: CFTR at the cell surface functions as a transporter of chloride and other ions
References
Derichs N. Eur Respir Rev 2013;22:58–65
Lommatzsch ST & Aris R. Semin Respir Crit Care Med 2009;30:531–8
Lukacs GL & Durie PR. N Eng J Med 2003;349:1401–4

5. Key Point
CFTR mutations can be grouped based on the type of molecular defect in the CFTR protein
Additional Information
Different CFTR mutations cause disruptions at various stages of CFTR protein synthesis, resulting in a reduction in the amount of protein present at the apical membrane or affecting several aspects of the function of the CFTR protein
Mutations that cause the introduction of a premature stop codon result in defective CFTR protein biosynthesis, with no functional CFTR protein expressed at the apical cell membrane (Class I)
Other CFTR mutations, including the most common one, F508del, affect post-translational folding and transport of the CFTR protein to the cell surface, also known as a trafficking defect. These misfolded proteins fail to reach the Golgi apparatus, and the result is that only a small quantity of the misfolded CFTR protein reaches the cell surface (Class II)
Mutations that cause a splicing defect result in CFTR mRNA that is not properly processed. Although some functional protein is produced, the amount of CFTR at the cell surface is decreased in comparison with normal levels (Class V )
Other mutations affect the function of the CFTR protein, resulting in a CFTR protein that reaches the apical membrane; however, either the channel does not open properly, which is known as a gating defect (class III), or it has impaired chloride movement, also known as conductance defect (Class IV). These mutations result in diminished chloride transport across the cell membrane
Class VI mutations do not impair the biogenesis or function of CFTR, but often cause a base pair C-terminus truncation, leading to degradation of the mature CFTR protein at a rate 5-6 times faster than normal
As previously described, overall these CF-associated mutations in the CFTR gene can result in the cell surface having less, an absence of, or dysfunctional CFTR protein
Some mutations may have defects in more than 1 class
A single CFTR mutation can result in multiple defects in the CFTR protein. Any particular defect as a continuum of severity may lead to a range in Total CFTR Activity
References
Boyle MP & De Boeck K. Lancet Respir Med 2013;1:158–63
Wilschanski M et al. J Pediatr 1995;127:705–10
Ratjen F. Curr Opin Pulm Med 2007;13:541–6
MacDonald KD et al. Paediatr Drugs 2007;9:1–10
Sheppard DN et al. Nature 1993;362:160–4
Castellani C et al. J Cyst Fibros 2008;7:179–96
Rowe SM et al. N Engl J Med 2005;352:1992–2001
Zielenski J. Respiration 2000;67:117–33
Rowntree RK & Harris A. Ann Hum Genet 2003;67:471–85
Welsh MJ & Smith AE. Cell 1993;73:1251–4

6. Three class II mutations show a wide range in protein levels4
Some CFTR mutations produce multiple defects that place them into multiple classes. For example:
The R117H-CFTR mutation is classically thought of as a class IV mutation due to it having low levels of conductance; however, it also displays defective gating placing it into class III1
The most common CFTR mutation, F508del, has strong class II defects in processing and trafficking; however, some of the mutated protein is partially active at the cell membrane having defects in surface stability2 placing it in class VI as well
The E831X mutation results in the introduction of a stop codon and, therefore, it might be categorized as a classic class I mutation. However, alternate splicing of its mRNA has been shown to result in some, albeit reduced, synthesis and therefore, it could also be categorized as a class V mutation3
The class system is further limited due to mutation in the same class exhibiting different degrees on the same defect. For example:
Three different class IV mutations show a wide range in channel open probability1
Three class II mutations show a wide range in protein levels4
References
Sheppard DN et al. Nature. 1993;362(6416):
Gentzsch M et al. Mol Biol Cell. 2004;15(6):
Hinzpeter A et al. PLoS Genet. 2010;6(10):e
Sosnay PR et al. Nature Genetics 2013;45(10)

7. Key Points
Total CFTR activity—the total chloride ion transport mediated by CFTR surface channels—is determined by both CFTR quantity (the number of CFTR channels on the cell surface) and CFTR function (the functional ability of each channel to open and transport chloride)1
The degree to which the CFTR mutation reduces CFTR quantity and/or function determines the total CFTR activity1
Additional Information
CFTR quantity is determined by CFTR synthesis (CFTR gene transcription and mRNA translation), CFTR trafficking (delivery of normally processed, mature CFTR protein to the cell surface), and CFTR surface stability (normal CFTR channels are eventually removed from the cell surface)2
CFTR function is determined by channel open probability (the fraction of time that a specific channel is open and transporting chloride) and channel conductance2
References
Sheppard DN et al. EMBO J. 1995;14(5):
Derichs N. Eur Respir Rev. 2013;22(127):
Sheppard DN et al. Nature. 1993;362(6416):

8. Key Points
The degree to which the CFTR mutation reduces CFTR quantity and/or function determines the total CFTR activity1
A single CFTR mutation can result in multiple defects in the CFTR protein
Additional Information
Total CFTR activity—the total chloride ion transport mediated by CFTR surface channels—is determined by both CFTR quantity (the number of CFTR channels on the cell surface) and CFTR function (the functional ability of each channel to open and transport chloride)1
CFTR quantity is determined by CFTR synthesis (CFTR gene transcription and mRNA translation), CFTR trafficking (delivery of normally processed, mature CFTR protein to the cell surface), and CFTR surface stability (normal CFTR channels are eventually removed from the cell surface)2
CFTR function is determined by channel open probability (the fraction of time that a specific channel is open and transporting chloride) and channel conductance2
References
Sheppard DN et al. EMBO J. 1995;14(5):
Derichs N. Eur Respir Rev. 2013;22(127):58-65.

9. Key Points
Ussing chambers are an extensively used experimental procedure to measure ion transport across epithelia measured as a voltage1
The method consists of the measurement of current (or voltage) between two hemi- chambers separated by a section of epithelium or an epithelial cell monolayer grown on a permeable support1
The classic parameter measured in an epithelium mounted Ussing chamber is the “short-circuit”, ISC, that is the current across the epithelium when it is short circuited1
Additional Information
Fischer rat thyroid (FRT) cells expressing different mutations of CFTR result in lower voltage due to decreased Cl- transport compared with cells expressing wildtype CFTR2
References
Moran O, Zegarra-Moran O. J Cyst Fibros. 2008;7(6):
Li H et al. J Cyst Fibros. 2004;3(Suppl 2):

11. FRT cells lack any endogenous CFTR or other chloride channels1
FRT cells are not of human origin, so interactions with orthologous (genes from different species) proteins, kinases, and ion channels may differ from what occurs in human airway epithelial cells2
Movement of Cl- ions through the cells are measured by the electrodes and can be visually represented by the graph on the far right, which shows some common mutations and their Cl- movement. You can see that R74W is a mutation that retains some residual CFTR protein activity and moves chloride more efficiently than cells expressing F508del or G551D, but less efficiently than cells with wild-type protein.
Sheppard et al. Am J Physiol ( 4 Pt 1): L405-13
Gottschalk et al. J Cyst Fibros 2017; 15:3:
Yu et al. J Cyst Fibros, :

12. Sosnay PR et al. Nat Genet. 2013;45(10):1160-1167.
Chloride transport was measured in FRT cells engineered to express one of 63 CFTR mutations (61 missense and 2 in-frame deletion)
Ussing chamber studies were used to measure chloride transport, which was expressed as a percentage of the mean transepithelial current in FRT cell lines expressing normal CFTR (% normal CFTR)
Reference
Sosnay PR et al. Nat Genet. 2013;45(10):
© Vertex Pharmaceuticals Incorporated FOR EDUCATIONAL PURPOSES ONLY

13. CFTR genotype is correlated with clinical phenotype, including pancreatic status, lung function, gastrointestinal complications and mortality
However, individuals with CF can show substantial individual variation in clinical outcomes
This variation is influenced by individual CFTR alleles, non-genetic factors, and other modifier genes
Non-genetic factors, such as level of care, environmental factors (e.g. passive smoking and polluting factors have been linked with pulmonary function), and age of onset of lung infection, contribute significantly to clinical outcomes
Genes other than CFTR also affect disease course and lung function. These are known as modifier genes:
For example, genotypes with insufficient MBL2 are associated with worse lung function
The majority of published studies investigated associations between candidate modifier genes and some aspect of the CF phenotype – a definitive modifier gene for CF is yet to be identified
References
Zielenski J. Respiration 2000;67:117–33
Castellani C et al. J Cyst Fibros 2008;7:179–96
Wilschanski M et al. Gut 2007;56:1153–63
Cutting GR. Ann NY Acad Sci 2010;1214:57–69
Collaco JM et al. Curr Opin Pulm Med 2008;14:559–66

14. Key Point
CFTR genotype of both alleles is a determinant of total CFTR activity and this variation in total CFTR activity (below carrier levels) is a factor that contributes to variation in CF phenotype among different individuals with CF
Additional Information
Overall, 2 mutations with little to no total CFTR activity are associated with a more typical CF phenotype
However, the presence of a complex allele may also contribute to a reduction in Total CFTR Activity
As previously mentioned, in addition to the CFTR genotype, phenotype is also affected by modifier genes and environmental factors; these will affect lung function and significantly affect disease course
For further information refer to the CFTR2 website. This, among other information, provides details on whether specific combinations of mutations cause CF when combined, as well as material on sweat chloride, lung function, pancreatic status and Pseudomonas infection rates in individuals with CF
References
Boyle MP & De Boeck K. Lancet Respir Med 2013;1:158–63
Griesenbach U et al. Thorax 1999;54(Suppl 2):S19–23
Zielenski J. Respiration 2000;67:117–33
Cutting GR. Annu Rev Genomics Hum Genet 2005;6:237–60
Davis PB. Am J Respir Crit Care Med 2006;173:475–82
Wilschanski M & Durie PR. Gut 2007;56:1153–63
Castellani C et al. J Cyst Fibros 2008;7:179–96

15. Key Point
CFTR genotype of both alleles determines total CFTR activity. Total CFTR activity, in turn, contributes to the expression of the clinical phenotype
Additional Information
Both CFTR alleles contribute to the expression of CFTR protein in epithelial cells. Some CFTR mutations result in little to no CFTR activity while other mutations result in some residual CFTR activity
The specific mutations on the 2 CFTR alleles and the respective levels of total CFTR activity associated with each mutation together determines the total CFTR activity in a specific individual
The total CFTR activity, in turn, influences the expression of the clinical phenotype
Reference
Zielenski J. Respiration 2000;67:117–33

16. Key Point
The expression of the CF clinical phenotype in a specific individual is affected by total CFTR activity (determined by CFTR genotype, dependent on the variation of the 2 alleles) as well as by modifier genes and non-genetic, environmental factors
Additional Information
Genes other than CFTR also affect disease course and lung function. These are known as modifier genes:
For example, genotypes with insufficient MBL2 and TGFβ1 affect lung function and disease course
The majority of published studies investigated associations between candidate modifier genes and some aspect of the CF phenotype – a definitive modifier gene for CF is yet to be identified
Non-genetic factors, such as level of care, environmental factors (e.g. passive smoking and polluting factors have been linked with pulmonary function), and age of onset of lung infection also contribute significantly to clinical outcomes
References
Castellani C et al. J Cyst Fibros 2008;7:179–96
Cutting GR. Annu Rev Genomics Hum Genet 2005;6:237–60
Wilschanski M & Durie PR. Gut 2007;56:1153–63

17. Key Point
Different organs may be more or less sensitive to reductions in CFTR activity, resulting in variation of disease severity in each organ
Additional Information
Air trapping and airway thickening are early pathological changes that are followed by bronchiectasis, increased mucus plugging, and peribronchial thickening. Chronic bacterial colonization occurs due to the failure of the body to clear the thick, sticky mucus that traps inhaled microbes from the airways. Other sinopulmonary manifestations of CF include atelectasis, bronchial cysts, pneumothorax, and opacification of the sinuses.
More than 85% of individuals with CF are born with pancreatic insufficiency. Malfunction or absence of CFTR protein and the accompanying breakdown in the flow of luminal chloride ions results in reduced volumes of more acidic pancreatic secretions, leading to obstruction and parenchymal destruction. Idiopathic chronic pancreatitis can also be CFTR-protein related.
Hepatic manifestations of CF, including cirrhosis, portal hypertension, cholelithiasis, and steatosis, occur with much less frequency than pulmonary and pancreatic manifestations. Up to 30% of individuals with CF have liver disease attributable to the underlying CFTR protein defect. However, decreasing mortality in CF has drawn more attention to CF-related liver disorders. As with pulmonary and pancreatic disease, liver disease associated with CF is related to CFTR protein.
The absence of normal CFTR protein function in the gastrointestinal tract can result in intestinal obstruction, known as meconium ileus in neonates and distal intestinal obstruction syndrome in older individuals.
Reproductive CFTR protein-related disorders include congenital absence of the vas deferens and reduced fertilty.
References
Davis PB. Am J Respir Crit Care Med 2006;173:475–82
Ramsey BW. Proc Am Thorac Soc 2007;4:359–63
Tiddens HA. & de Jong PA. Proc Am Thorac Soc 2007;4:343–6
Walkowiak J, et al. Eur J Gastroenterol Hepatol 2008;20:157–60
Colombo C, et al. J Pediatr Gastroenterol Nutr 2006;43(suppl 1):S49–55

19. Mutations in the CFTR gene that lead to defective Cl– and other ion transport through CFTR protein channels are the underlying defect of CF
CFTR gene mutations affect the total activity of the CFTR protein channel at the apical cell surface, by affecting the quantity of CFTR channels at the cell surface and/or function of CFTR as an ion channel
The reduction in total CFTR protein activity leads to pathophysiological changes in epithelial cells that affect the airway, pancreas, gastrointestinal tract and sweat glands, among other systems
References
MacDonald KD et al. Paediatr Drugs 2007;9:1–10
Rowe SM et al. N Engl J Med 2005;352:1992–2001
Lommatzsch ST & Aris R. Semin Respir Crit Care Med 2009;30:531–8
Davis PB. Am J Respir Crit Care Med 2006;173:475–82

20. Key Point
Total CFTR Activity as low as 50% of normal can be associated with no CF phenotype
Additional Information
In CFTR carriers, 1 normal CFTR allele produces normal CFTR protein and the second allele has a CFTR mutant defective protein with reduced CFTR quantity or function
This results in total CFTR activity that is 50–90% of normal levels but is sufficient to be associated with no CF disease phenotype
Some carriers may have an increased risk of certain pulmonary conditions (e.g. asthma, pancreatitis, sinusitis or bronchiectasis)
References
Boyle MP & De Boeck K. Lancet Respir Med 2013;1:158–63
Griesenbach U et al. Thorax 1999;54(Suppl 2):S19–23
Zielenski J. Respiration 2000;67:117–33
Davis PB. Am J Respir Crit Care Med 2006;173:475–82
Wilschanski M & Durie PR. Gut 2007;56:1153–63
Castellani C et al. J Cyst Fibros 2008;7:179–96

22. Chloride transport was measured in FRT cells engineered to express one of 63 CFTR mutations (61 missense and 2 in-frame deletion)
Ussing chamber studies were used to measure chloride transport, which was expressed as a percentage of the mean transepithelial current in FRT cell lines expressing normal CFTR (% normal CFTR)
Reference
Sosnay PR et al. Nat Genet. 2013;45(10):

23. Key Point
Total CFTR Activity as low as 50% of normal can be associated with no CF phenotype
Additional Information
In CFTR carriers, 1 normal CFTR allele produces normal CFTR protein and the second allele has a CFTR mutant defective protein with reduced CFTR quantity or function
This results in total CFTR activity that is 50–90% of normal levels but is sufficient to be associated with no CF disease phenotype
Some carriers may have an increased risk of certain pulmonary conditions (e.g. asthma, pancreatitis, sinusitis or bronchiectasis)
References
Boyle MP & De Boeck K. Lancet Respir Med 2013;1:158–63
Griesenbach U et al. Thorax 1999;54(Suppl 2):S19–23
Zielenski J. Respiration 2000;67:117–33
Davis PB. Am J Respir Crit Care Med 2006;173:475–82
Wilschanski M & Durie PR. Gut 2007;56:1153–63
Castellani C et al. J Cyst Fibros 2008;7:179–96

27. Chloride transport was measured in FRT cells engineered to express one of 63 CFTR mutations (61 missense and 2 in-frame deletion)
Ussing chamber studies were used to measure chloride transport, which was expressed as a percentage of the mean transepithelial current in FRT cell lines expressing normal CFTR (% normal CFTR)
Reference
Sosnay PR et al. Nat Genet. 2013;45(10):

28. References
Boyle MP & De Boeck K. Lancet Respir Med 2013;1:158–63
Griesenbach U et al. Thorax 1999;54(Suppl 2):S19–23
Zielenski J. Respiration 2000;67:117–33
Davis PB. Am J Respir Crit Care Med 2006;173:475–82
Wilschanski M & Durie PR. Gut 2007;56:1153–63
Castellani C et al. J Cyst Fibros 2008;7:179–96

29. Comparing clinical data from patients with 6 different residual function mutations show variability in measurements such as:
Age of diagnosis ranges from a mean of 61 to 402
Sweat chloride levels range from a mean of 573 to 1034
ppFEV1 levels range from a mean of 595 to 851
Pancreatic insufficiency percentages range from 136 to 501
References
De Braekeleer M et al. Hum Genet. 1997;101(2):
Castaldo G et al. J Cystic Fibros. 2006;5(3):
Gilfillan A et al. J Med Genet. 1998;35(2):
Masvidal L et al. Eur J Hum Genet. 2014;22(6):
Antinolo G et al. J Med Genet. 1997;34(2):89-91.
The Cystic Fibrosis Genotype –Phenotype Consortium. N Engl J Med. 1993;329(18):

30. This study compared ppFEV1 in patient with CF with CFTR-R117H vs CFTR-F508del
A retrospective cohort of patients with CF reported in the US CFF Patient Registry from 2006 to 2010 was used to compare patients with ≥1 R117H mutation with patients homozygous for the F508del mutation
In total, 156 R117H and 6251 homozygous F508del patients (from an original 14,450 patients) met the selection criteria and were included in the analysis
The ppFEV1 rate of change (slope) was estimated for the R117H and F508del cohorts, adjusted for age group (6-12, 13-17, 18-24, and ≥25 years)
Younger patients (6-12 years of age) with an R117H mutation had an overall improving ppFEV1 slope; R117H and F508del patients in the oldest two age groups (18-24 and ≥25 years of age) had similar slopes (rates of decline)
Reference
Wagener JS et al. NACFC Poster 415.

31. This study focused on survival of patients with CF in the US CFF database based on the severity of their genotypes1
Analysis was limited to CFTR genotypes that were recorded in the database, had a known functional class, and whose allele frequency was >0.1%1
Genotypes were classified as “severe” if the mutations on both alleles fell into class I, II, or III, and classified as “mild” if at least one mutation on one allele fell into class IV or V1
Patients were followed between 1993 and 20021
Median follow-up was 8.6 years for patients with a high-risk CFTR genotype vs 5.1 years for patients with a low-risk CFTR genotype1
There were a total of 1672 deaths during the 10-year follow-up period1
Reference
McKone EF et al. Chest. 2006;130(5):