Cystic Fibrosis Flashcards

Genetics, Mechanism, and Treatments of Cystic Fibrosis

Introduction to Cystic Fibrosis

Cystic fibrosis (CF) is an inherited, life-threatening disorder that primarily affects the lungs and digestive system. A core concept is understanding the link between genetic mechanisms, disease function changes, and the development of effective treatments.

CF is one of the most common autosomal recessive genetic disorders in Caucasians, leading to a decreased lifespan. Data indicates that while the median age for the general US population approaches the mid-70s, for CF patients, it is around 35 to 40 years. The life expectancy of CF patients has increased over the past 40-50 years, but it remains a major genetic disorder that reduces lifespan.

Prevalence and Cause

CF affects approximately one in every 2,500 live births, with an estimated one in three Caucasian individuals being carriers (a 3-4% carrier rate). It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

CF Disease Characteristics

CF is a chronic, progressive, and life-limiting disease characterized by chronic respiratory diseases and infections, pancreatic insufficiency, elevated sweat electrolytes, male infertility, and affects various other organs. These issues stem from the structure and function of the CFTR gene.

CFTR Gene and Protein

The CFTR protein is a regulated chloride ion channel located in the apical epithelial membrane of most epithelial cells. It mediates the transport of chloride ions from inside the cell to the outside. Loss-of-function mutations in the CFTR gene disrupt this chloride ion transport, leading to abnormal fluid and mucus secretions. This lack of chloride transport is an early diagnostic feature of CF disease, noted as high salt concentrations in sweat since the 1600s.

Sweat Chloride Concentrations

Normally, sweat glands secrete a high ionic concentration of approximately 40 \, \text{mmol} of sodium chloride. CFTR genes in sweat glands reabsorb chloride, reducing sweat chloride concentration to about 20 \, \text{mmol}. In individuals with CF, the inhibited chloride reabsorption results in high salt chloride concentrations of around 100 \, \text{mmol} in their sweat.

CFTR Gene Structure and Function

The CFTR gene encodes a large integral membrane protein of about 170 \, \text{kDa}. It contains 27 exons and spans approximately 190 \, \text{kb} of DNA, comprising five major structural domains:

1. Two Membrane-Spanning Domains (MSD1 and MSD2): Each contains six membrane-spanning sections. These domains form a pore that allows chloride ions to pass through.
2. Two Nucleotide-Binding Domains (NBD1 and NBD2): These domains bind and hydrolyze ATP, which is essential for opening and closing the CFTR pore.
3. Regulatory Domain: Contains multiple phosphorylation sites and regulates the function and activity of the gene.

There are over 2,000 identified CFTR mutations, varying in frequency.

The location of mutations within the CFTR gene has significant implications for understanding CF disease.

Classes of CFTR Mutations and Dysfunction

Six major classes of dysfunctional CFTR proteins correlate with CF disease:

Class 1: No Protein Production

These mutations result in no CFTR protein being produced due to premature stop codons. This class typically leads to the most severe form of the disease.

Class 2: Impaired Protein Folding

These mutations impair the folding of the CFTR protein, causing it to be retained in the endoplasmic reticulum (ER) and subsequently degraded. As a result, no CFTR protein reaches the cell surface, rendering it non-functional. Delta F508 is a major mutation in this class.

Class 3: Non-Functional Protein at Cell Surface

The protein is produced and reaches the cell surface but cannot open the pore for chloride transport. These are typically gating mutations.

Class 4: Reduced Function

Functional CFTR protein is produced and present at the cell surface but has reduced function due to mutations in the nucleotide-binding or regulatory domains, resulting in a small amount of chloride ion transport. These mutations lead to a less severe disease phenotype.

Class 5: Reduced Number of CFTR Proteins

Functional CFTR genes are produced but in reduced numbers, leading to a reduction in chloride ion transport across the cells.

Class 6: Less Stable CFTR Proteins

CFTR proteins make it to the cell surface and actively transport chloride ions, but due to mutations causing instability, they constantly cycle in and out of the cell membrane resulting in reduced chloride ion transport.

Classes 1-3 cause more severe disease due to minimal to no CFTR function, while classes 4-6 result in less severe disease due to some residual chloride ion transport.

Specific mutations, such as G542X and W1282X (Class 1), G551D (Class 3), and Delta F508 (Class 2), are significant and targeted by specific drugs.

Delta F508 Mutation

The Delta F508 mutation, the most common CF mutation, involves the deletion of phenylalanine at amino acid residue position 508. It is a Class 2 mutation in the NBD1 domain, leading to a misfolded protein that cannot exit the endoplasmic reticulum.

Approximately 50% of CF patients are homozygous for Delta F508. An additional 40% are genetic compounds with one copy of Delta F508 and another mutant allele. Approximately 70% of CF carriers have a single copy of this mutation, making it a prominent focus in CF research.

Pathogenicity and Disease Manifestation

Mutations in the CFTR gene lead to abnormal CFTR function, causing two major classes of pathogenicity:

Pancreatic Dysfunction

CFTR misfunction in the pancreas leads to maldigestion and a lack of digestive enzymes, resulting in poor growth and stunting. This pathogenicity is treatable with increased caloric intake or enzyme supplements.

Abnormal Mucus Secretion

Abnormal mucus secretion leads to plugging of the lungs, causing bacterial and viral infections, inflammation, and immune system response. This is a major cause of morbidity and mortality in CF patients.

The CFTR genotype is a poor predictor of lung disease outcome. However, patients who are homozygous for Delta F508 mutations have variable stages of lung disease.

Mechanism of Lung Disease

Defective CFTR protein prevents chloride ion transport from inside the cell to the outside, causing a buildup of chloride ions in the cytoplasm. This drives a hypersecretion of sodium ions (cations) into the cell, mediated by the ENaC transporter, to balance the negative charge. This leads to an increased concentration of salt (cations and anions) inside the cell.

Subsequently, water is drawn into the cell to dilute the salt concentration, dehydrating the extracellular environment (the mucus in the lungs). This results in a thick, sticky mucus secretion that is difficult to clear, leading to obstructions and bronchiectasis. This thick mucus creates an ideal environment for bacterial infections and biofilm formation, triggering an immune response and causing scarring and damage to the epithelial tissue.

This cycle of bacterial infection, immune activation, and scarring leads to progressive loss of lung function.

Role of Cilia in Healthy Lungs

Cilia, hair-like filaments on lung epithelial cells, clear mucus from the lungs by beating in a wave-like motion, moving mucus to the back of the esophagus and into the stomach. Impaired mucus secretion prevents cilia from functioning properly.

Mucus Viscosity and Infections

The mucus is a gel-like structure of biopolymers tightly controlled by water concentration. If the water content changes (either too much or too little), the system fails. In CF, hyperabsorption of sodium and chloride ions dehydrates the mucus, compacting its structure and suppressing the cilia. This compacted mucus cannot be cleared, leading to obstructions and bacterial infections.

Lung Microbiome in CF Patients

CF patients have a diverse and unique lung microbiome, often typified by Pseudomonas aeruginosa. The microbiome diversity varies among patients and changes over time, which is one reason why the CFTR genotype is a poor predictor of end-stage lung disease. The variability is also driven by adherence to different treatments.

Summary of the Disease Mechanism

The steps leading to sever lung disease and death in CF patients is as follows:

1. Defective CFTR proteins prevent chloride ion transport out of the cell.
2. Hypersecretion of sodium ions into the cell increases intracellular salt concentration.
3. Water is drawn into the cell, dehydrating the lung mucus.
4. Dehydrated mucus becomes thick and sticky, obstructing airways.
5. Mucus obstructions lead to bacterial and viral infections.
6. Infections trigger an immune response, recruiting neutrophils that damage lung epithelium.
7. Lung tissue is scarred, leading to irreversible loss of lung function.

This cycle of infection and immune response is known as an exacerbation.

Measuring Lung Function

Lung function is monitored using a spirometry test known as FEV1 (forced expiratory volume), which measures the volume of air a patient can expire in one second. With each infection and exacerbation, the patient's FEV1 value decreases.

The FEV1 is measured as the percentage compared to normal people in the population. The value is expected to gradually drop over time do to reoccurring infections. Eventually the patient will die of respiratory failure or receive a lung transplant.

Treatments for Cystic Fibrosis

By understanding the genetics, mechanism, and function interplay, various treatments are designed to prevent and interfere with different stages of CF disease progression:

Antibiotics

Inhaled antibiotics, such as tobramycin, are administered during exacerbations. They reduce bacterial burdens in the lung, slowing lung damage. However, antibiotic resistance remains a significant issue.

Hypertonic Saline

Inhaling a hypertonic saline solution (saltwater) deposits sodium and chloride onto the apical surface of epithelial cells. This draws water out of the cells and into the dehydrated lung mucus, rehydrating it and allowing patients to cough up the mucus. It also disrupts bacterial growth and biofilm production.

These treatments are preventative and designed to control pulmonary infection and reduce mucus buildup.

Small Molecule Treatments targeting CFTR

Vertex has pioneered the development of small molecule drugs that target the genetic basis of CF, offering revolutionary approaches to treating genetic diseases.

Ivacaftor

Ivacaftor is a CFTR potentiator that interacts with the CFTR protein to increase chloride ion transport by improving the gating of the CFTR protein at the cell surface. It works in patients with Class 3 CFTR mutations (gating mutations), allowing the pore to open and permit chloride ions to exit the cell. The G551D mutation is a specific target of ivacaftor.

Lumacaftor

Lumacaftor improves the conformational stability of Delta F508-homozygous mutations (Class 2 mutations). It interacts with the unfolded CFTR protein, allowing it to refold and escape the endoplasmic reticulum, making its way to the cell surface. However, the Delta F508 mutation typically still has gating problems once it reaches the cell membrane.

Orkambi (Lumacaftor and Ivacaftor Combination)

Orkambi combines the activities of Lumacaftor and Ivacaftor, increasing both the quantity and gating capacity of Delta F508 CFTR protein, resulting in increased chloride ion transport. This treatment is revolutionary because Delta F508 accounts for over half of the cystic fibrosis population.

Limitations

Small molecule treatments do not cure the underlying genetic disease, and patients can experience symptoms if they stop taking the medicine. In addition, the annual cost is relatively high.

Learning Objectives

Key takeaways from learning about CF:

1. Define cystic fibrosis as a genetic disease associated with CFTR protein dysfunction.
2. Understand the function of the CFTR protein as a chloride ion transporter.
3. Describe the six general classes of CFTR mutations, specifically including the Delta F508 mutation.
4. Sketch how disrupting CFTR function leads to progressive lung disease and infection.
5. Demonstrate how particular treatments can alleviate cystic fibrosis disease or its symptoms.