Welcome to Advancing CF Science

Exploring the impact of CFTR mutations on protein biosynthesis and CFTR activity

Welcome to advancingCFscience.com, where we explore how mutations in the CFTR (the cystic fibrosis transmembrane conductance regulator) gene cause impairment in protein translation, the earliest stage of CFTR protein biosynthesis. These defects may also lead to functional consequences on chloride transport activity at the cell surface. Such mutations also have significant impact on the course of cystic fibrosis (CF). This website provides general information about CF with a focused look at the earliest steps in CFTR protein biosynthesis, protein translation. Impacting protein biosynthesis at this early step has the potential to provide increased levels of CFTR, which can improve CFTR activity. Enhancing CFTR translation, in addition to improving trafficking, processing and chloride transport, has the promise to advance the science of CF. We hope that you will find this website to be a valuable resource for understanding CF, and that it will serve to provide ongoing education on the progress of CF science.

What Is Cystic Fibrosis?

Genetics of Cystic Fibrosis

Inherited Pattern in Cystic Fibrosis
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Cystic fibrosis (CF) is an autosomal recessive genetic disorder in which individuals who carry one copy of the affected gene are carriers for the mutation but do not have the disease. Individuals with CF must inherit two defective alleles of the CFTR gene, one from each parent. Geneticists refer to the combination of CF-causing alleles by using one of two terms. If the individual has two identical CF-causing alleles, that is referred to as “homozygosity,” whereas having two different CF-causing alleles is referred to as “compound heterozygosity.” For example, if both parents are carriers of CF-causing alleles (eg, father is a carrier of a G551D-mutated allele and mother is a carrier of a F508del-mutated allele), a father could pass on the G551D mutation and the mother an F508del mutation to their child. The child would then be compound heterozygous (G551D; F508del) for the CFTR mutation. When two carriers for the mutated CFTR gene have children, each child has a 25% risk of having two abnormal genes and being at risk for CF (affected child), a 50% risk of being a carrier, and a 25% chance of having two normal genes (unaffected child).1

Cause of CF: CFTR Mutations

CFTR Protein in Epithelial Cell Membrane
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CFTR Mutation Classification3
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CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes for a protein that functions as a chloride channel and regulates ion flow across the apical surface of epithelial cells.2 CFTR is a complex, multidomain, membrane-spanning protein that undergoes tightly regulated steps during its biosynthesis to achieve a correctly localized and functional mature protein that mediates chloride transport.3 CF carriers require, at most, only 50% of functional CFTR to be healthy, and CF patients exhibit clinical and symptomatic manifestations of the disease if they have even less CFTR activity than this.4,5 Generally, residual CFTR function correlates with CF disease severity—for example, patients with higher CFTR activity (10%) may present with absence of the vas deferens, whereas patients with lower CFTR function (<1%) may exhibit pancreatic insufficiency.4,6

In 1989, the CF locus (where the gene is located) was localized through linkage analysis to the long arm of human chromosome 7, band q31.7 Since then, more than 2,000 CFTR mutations have been identified.6 Some mutations result in hardly any CFTR function, while others are associated with some residual function.8 Researchers have created 6 classes to aid in categorizing the CF disease-causing mutations based on their resulting impact on CFTR.8-11 F508del is the most prevalent mutation, which affects up to 90% of CF patients worldwide (~50% homozygous and another ~40% compound heterozygous).12,13

Genetics of Cystic Fibrosis

Genetics of Cystic Fibrosis

Inherited Pattern in Cystic Fibrosis
h

Cystic fibrosis (CF) is an autosomal recessive genetic disorder in which individuals who carry one copy of the affected gene are carriers for the mutation but do not have the disease. Individuals with CF must inherit two defective alleles of the CFTR gene, one from each parent. Geneticists refer to the combination of CF-causing alleles by using one of two terms. If the individual has two identical CF-causing alleles, that is referred to as “homozygosity,” whereas having two different CF-causing alleles is referred to as “compound heterozygosity.” For example, if both parents are carriers of CF-causing alleles (eg, father is a carrier of a G551D-mutated allele and mother is a carrier of a F508del-mutated allele), a father could pass on the G551D mutation and the mother an F508del mutation to their child. The child would then be compound heterozygous (G551D; F508del) for the CFTR mutation. When two carriers for the mutated CFTR gene have children, each child has a 25% risk of having two abnormal genes and being at risk for CF (affected child), a 50% risk of being a carrier, and a 25% chance of having two normal genes (unaffected child).1

Cause of CF: CFTR Mutations

Cause of CF: CFTR Mutations

CFTR Protein in Epithelial Cell Membrane
h
CFTR Mutation Classification3
h

CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes for a protein that functions as a chloride channel and regulates ion flow across the apical surface of epithelial cells.2 CFTR is a complex, multidomain, membrane-spanning protein that undergoes tightly regulated steps during its biosynthesis to achieve a correctly localized and functional mature protein that mediates chloride transport.3 CF carriers require, at most, only 50% of functional CFTR to be healthy, and CF patients exhibit clinical and symptomatic manifestations of the disease if they have even less CFTR activity than this.4,5 Generally, residual CFTR function correlates with CF disease severity—for example, patients with higher CFTR activity (10%) may present with absence of the vas deferens, whereas patients with lower CFTR function (<1%) may exhibit pancreatic insufficiency.4,6

In 1989, the CF locus (where the gene is located) was localized through linkage analysis to the long arm of human chromosome 7, band q31.7 Since then, more than 2,000 CFTR mutations have been identified.6 Some mutations result in hardly any CFTR function, while others are associated with some residual function.8 Researchers have created 6 classes to aid in categorizing the CF disease-causing mutations based on their resulting impact on CFTR.8-11 F508del is the most prevalent mutation, which affects up to 90% of CF patients worldwide (~50% homozygous and another ~40% compound heterozygous).12,13

Multi-System Impact of CF

Impact of CF on Lungs and Other Organs14-16

Click on each circle for a detailed description of CF's impact.

Mutations resulting in CFTR disruption can cause multisystem dysfunctions. Today, the most life-limiting of these are in the lungs: the repetitive, infectious respiratory exacerbations that lead to destruction of airway architecture, acute and chronic inflammatory changes, and deterioration in lung function.2,17-19

Further clinical manifestations in CF affect the gastrointestinal (digestive tract), hepatobiliary (liver), exocrine/endocrine (pancreas), and reproductive systems, and also include salt-wasting syndromes.2,17,18

While substantial progress has been made in the management of CF in the last 30 years, with a steady increase in the median predicted survival age of patients,8 the disease still carries a significant burden of symptoms and early mortality.8,20 Additionally, the increased proportion of CF patients who are living longer today are faced with new complications stemming from their disease, complications which were previously rare.17

Median Predicted Survival Age of Patients With CF in the Last 30 Years
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CFTR Mutations Impact CFTR Activity

It is well understood that CFTR activity in the epithelial cell is a product of CFTR quantity (the amount of CFTR protein produced) and CFTR function (the ion channel gating and conductance at the cell surface).21,22

CFTR Quantity and Function

The quantity of CFTR protein produced is a product of the amount of RNA transcribed, the efficiency of RNA splicing, the amount of protein correctly translated and folded, and the overall stability of the protein that is made.21

The function of CFTR is dependent on the amount of ion channels present at the cell surface and on the efficiency of conductance of ions through each channel.21,22

CFTR Quantity and Function: A Product of a Multistep Process From mRNA Transcription to Functional Chloride Channel
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Impacting CFTR Quantity, Function or Both Affect CFTR Activity

The level of immature CFTR protein produced during CFTR biosynthesis plays an essential role in CFTR quantity and overall CFTR activity.21 If considering immature CFTR protein in the equation for expressing total CFTR activity, modifications to this calculation may include the following:

Impacting CFTR Quantity, Function, or Both Affects CFTR Activity
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Decrease in immature CFTR protein resulting from impaired protein translation results in reduced levels of CFTR activity.

Addressing a Range of Intracellular Defects

Advances in identifying the genetic basis of CF have elucidated the multiple steps involved in CFTR biosynthesis and function at the cell membrane, as well as demonstrating a correlation between CFTR mutational variants and CF disease manifestations.17,21 Based on this knowledge, strategies have been developed that augment the function of mutant CFTR by addressing CFTR mutation-induced defects at the levels of protein folding, processing, and trafficking, as well as at ion channel gating and conductance.17 However, CFTR mutation-imposed defects can begin even earlier in CFTR biosynthesis. To address the range of defects induced by mutation of CFTR it is necessary to consider transcription, the synthesis and regulation of mRNA in the nucleus and its transport to the cytosol and ribosome engagement; translation of that mRNA into CFTR protein on the surface of the endoplasmic reticulum; and post-translational modification of folding and glycosylation that allow for increased transit of more functional protein to the membrane.23-25

The Role of Potentiators and Correctors

Potentiators and correctors launched a new era in CF by targeting the CFTR protein defects that underlie the disease in select genotype-specific patients.26-29 Potentiators improve chloride transport by increasing the channel-open probability of CFTR proteins at the cell surface,17 and are approved for use in patients with CF with select gating and conductance mutations.26,27,29 Correctors enhance the folding and stability of CFTR harboring the F508del mutation, resulting in more efficient protein folding, as well as facilitating the normal processing and trafficking of CFTR proteins.17 The CFTR combination of corrector/potentiator is approved for use in patients homozygous for the F508del mutation in the CFTR gene.30 Potentiators and correctors work to counteract the defects of CFTR biosynthesis induced by CFTR mutations, providing a means to restore CFTR channel conductance and rescue CFTR protein trafficking and folding. However, there is still an unmet need in many patients who do not carry the variants for which these agents are indicated, and for those patients with limited clinical benefit to current corrector/potentiator combinations.

CFTR Quantity and Function

CFTR Quantity and Function

The quantity of CFTR protein produced is a product of the amount of RNA transcribed, the efficiency of RNA splicing, the amount of protein correctly translated and folded, and the overall stability of the protein that is made.21

The function of CFTR is dependent on the amount of ion channels present at the cell surface and on the efficiency of conductance of ions through each channel.21,22

CFTR Quantity and Function: A Product of a Multistep Process From mRNA Transcription to Functional Chloride Channel
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Impacting CFTR Activity

Impacting CFTR Quantity, Function or Both Affect CFTR Activity

The level of immature CFTR protein produced during CFTR biosynthesis plays an essential role in CFTR quantity and overall CFTR activity.21 If considering immature CFTR protein in the equation for expressing total CFTR activity, modifications to this calculation may include the following:

Impacting CFTR Quantity, Function, or Both Affects CFTR Activity
h

Decrease in immature CFTR protein resulting from impaired protein translation results in reduced levels of CFTR activity.

Intracellular Defects

Addressing a Range of Intracellular Defects

Advances in identifying the genetic basis of CF have elucidated the multiple steps involved in CFTR biosynthesis and function at the cell membrane, as well as demonstrating a correlation between CFTR mutational variants and CF disease manifestations.17,21 Based on this knowledge, strategies have been developed that augment the function of mutant CFTR by addressing CFTR mutation-induced defects at the levels of protein folding, processing, and trafficking, as well as at ion channel gating and conductance.17 However, CFTR mutation-imposed defects can begin even earlier in CFTR biosynthesis. To address the range of defects induced by mutation of CFTR it is necessary to consider transcription, the synthesis and regulation of mRNA in the nucleus and its transport to the cytosol and ribosome engagement; translation of that mRNA into CFTR protein on the surface of the endoplasmic reticulum; and post-translational modification of folding and glycosylation that allow for increased transit of more functional protein to the membrane.23-25

Potentiators & Correctors

The Role of Potentiators and Correctors

Potentiators and correctors launched a new era in CF by targeting the CFTR protein defects that underlie the disease in select genotype-specific patients.26-29 Potentiators improve chloride transport by increasing the channel-open probability of CFTR proteins at the cell surface,17 and are approved for use in patients with CF with select gating and conductance mutations.26,27,29 Correctors enhance the folding and stability of CFTR harboring the F508del mutation, resulting in more efficient protein folding, as well as facilitating the normal processing and trafficking of CFTR proteins.17 The CFTR combination of corrector/potentiator is approved for use in patients homozygous for the F508del mutation in the CFTR gene.30 Potentiators and correctors work to counteract the defects of CFTR biosynthesis induced by CFTR mutations, providing a means to restore CFTR channel conductance and rescue CFTR protein trafficking and folding. However, there is still an unmet need in many patients who do not carry the variants for which these agents are indicated, and for those patients with limited clinical benefit to current corrector/potentiator combinations.

Critical Role of CFTR Protein Translation in Advancing the Science of CF

Inefficient translation is also a contributor to impaired CFTR activity2,17,23,25,31

Requisite amounts of CFTR, critical for sufficient CFTR activity, result from a complex, multistep biosynthetic process that includes successful transcription and processing of mRNA, protection from mRNA degradation processes, and efficient signal sequence recognition that targets CFTR to the endoplasmic reticulum during CFTR protein translation.2,31,32

Following transcription, splicing, and nuclear export, the stability of mRNA—while in the process of translation—is dictated by a balance of specific cellular factors that interact with the newly made protein chain at the ribosome exit site. At this early point in translation, selective reduction in the amount of defective/mutated proteins can occur by specifically degrading their mRNA.33 This in turn leads to decreased levels of the mature CFTR protein that can successfully reach the cell surface to act as functioning ion channels.2,32

When these key early phases of CFTR biosynthesis are negatively impacted by CFTR mutations, disruption of CFTR protein translation also leads to reduced levels of CFTR protein. This results in a decrease in total CFTR activity and, ultimately, an increased severity of CF disease manifestations.2,17,23,25,31,34

CFTR Translation, Folding, and Channel Opening Time

In ciliated epithelial cells, immature CFTR (orange) is secreted in vesicles to the Golgi apparatus where it undergoes maturation. The now mature CFTR (purple) travels on to the cell surface where its acts as a channel for chloride (blue).

Click on each circle within the cell for a detailed description.

Importance of Increasing CFTR Protein Translation and Decreasing CFTR mRNA Degradation

Directing the successful signal sequence recognition of the early portion of CFTR to the endoplasmic reticulum is expected to both enhance CFTR translation and decrease CFTR mRNA degradation. This can improve the quantity of CFTR made earlier in CFTR biosynthesis and augment the activity of potentiators and correctors with a positive impact on the ultimate CFTR activity.2,17,23

Impacting CFTR During its Multistep Process—From Protein Synthesis to Functional Chloride Channel—Has Potential to Improve CFTR Activity.
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