- Genetic disorders
- Autosomal recessive
Cystic fibrosis (CF) is an aggressive life-limiting autosomal recessive disease with a high rate of mortality. Mutation in the cystic fibrosis conductance regulator (CFTR) gene is the sole origin of CF. CFTR gene encodes a protein that functions cAMP-activated chloride (Cl-) channel. Mutation in CFTR gene leads to less or no production of CFTR protein that results in the defective or reduced transport of Cl- and bicarbonate (HCO3-) ions in multiple organs. Organs affected due to mutation include gut, lungs, liver, pancreas and reproductive organs, however, the lungs are mostly affected organs. CF lung disease is characterized by the accumulation of viscous mucus, chronic inflammation, and persistent bacterial infections in the lungs. The thick, viscous mucus leads to clogging in the respiratory linings resulting in stuffy nose, wheezing and breathlessness. Persistent infection-directed inflammation leads to permanent structural damages of the respiratory linings that eventually results in respiratory failure and death. CF is highly prevalent among the Caucasian population from developed countries including UK, Europe, North America and Australasia. Until 2005, the life expectancy of patients with CF was around 28 years, however, with the advancement of diagnosis and treatment, the median survival time of patients with CF increased to 37 years over the past decade.Cystic Fibrosis
In the past, a diagnosis of CF was based primarily on clinical signs and symptoms. However, with the introduction of CF newborn screening (NBS) in many countries including the United States, a majority of newly diagnosed cases are minimally symptomatic or asymptomatic infants. Screening involves procedures such as immunoreactive trypsinogen (IRT) biomarker testing on dried blood spots as well as testing for one or more CFTR gene mutations. In many cases, the diagnosis can be confirmed by a high chloride concentration in a follow up sweat test.
Given the variation in disease severity and age of onset, CF diagnosis can be a challenge. Patients with CF can present with manifestations such as respiratory symptoms, chronic sinusitis, pancreatitis and male infertility. Moreover, patients with CF lung disease present with persistent cough with viscous mucus, wheezing, repeated airway infection, leading to bronchiectasis, gas trapping, hypoxaemia, and hypercarbia.
CF is a monogenic disease caused by mutation in CFTR gene. Patients with CF suffer from intense systemic inflammation characterized by increased serum acute phase proteins, high antibody titres to respiratory pathogens, atopy and heightened Th2 responses. Initially, it was thought that CFTR was largely expressed by the airway epithelial cells (AECs), however, expression and function of CFTR had been reported to other inflammatory cell types such as monocytes, macrophages, T cells, mast cells. Molecular studies suggest a negative feedback loop between functional CFTR and inflammation. However, it is still unknown why CF inflammation never stops.
Airway epithelial cells (AECs)
The airway epithelium acts as structural and immunological barriers to the inhaled pathogens. Airway surface liquid (ASL) is a low viscous mucus layer containing a number of proteins including mucin and anti-microbial peptides that tightly regulate antimicrobial activity, mucociliary transport and trapping and clearance of the inhaled pathogens. Mutation in CFTR gene not only distort the structural integrity of the airway but also abolish Cl- ion transport leading to the production of viscous mucus which trap the respiratory pathogens resulting in chronic inflammation in the respiratory linings. Airway epithelial cells (AECs) of the CF lungs release enormous amount of pro-inflammatory cytokines those chemoattract neutrophils and macrophages.
Neutrophils play crucial role in CF disease pathogenesis. Higher neutrophil counts had been observed in the bronchoalveolar (BAL) fluid and CF specimens of CF patients. Microbial killing ability of neutrophils had been compromised with non-functional CFTR. Elevated neutrophil elastase activity in CF airway is thought not only to be the underlying reason for structural damage of airway linings but also compromise activation of adaptive immune cells.
Monocytes maintain a circulatory pool of immune effector cells. During infection, they migrate to the tissues and differentiate into either macrophages or dendritic cells to mount innate and adaptive immune responses respectively. Macrophages with their pro-inflammatory (M1) and anti-inflammatory (M2) subsets maintain balance between inception and resolution of respiratory inflammation. Truncated form of CFTR protein had been observed in both CF monocytes and macrophages. Monocytes from CF patients demonstrate enhanced inflammatory cytokine profile with defective phagocytic ability. Macrophages in CF had been found highly pro-inflammatory without their anti-inflammatory attributes.
Repeated and chronic infection with bacteria, particularly with Staphylococcus aureus and Pseudomonas aeruginosa are common in CF. Episodes of infection increase with age and are associated with neutrophilic inflammation leading to lung damage, bronchiectasis that leads to fibrosis during adulthood.
CFTR protein is expressed at the apical membrane of almost all cell types, including epithelial cells, monocytes, neutrophils, mast cells. Following synthesis, CFTR protein enters into the endoplasmic reticulum (ER) and golgi apparatus for checking the proper folding and glycosylation of the protein. The fully matured and functional CFTR protein is then transported to the plasma membrane to acts as Cl- ion channel. Mutation in CFTR gene leads to less or no production, trafficking and function of CFTR protein. Cellular effects due to the mutation vary with cell types. For instance, mutation in CFTR leads to aberrant Cl- ion channel activity in airway epithelial cells, defective bacterial clearance by monocytes and macrophages, excessive inflammatory mediator release by neutrophils and mast cells.
Excessive neutrophil counts along with reduced antimicrobial activity and dysregulated cytokine production by neutrophils are the hallmark features of CF lung inflammation. An inverse relationship was observed between dysfunctional CFTR protein and cellular redox state, ER stress and mucin overproduction by AECs. Enhanced release of CCL20 (macrophage recruitment chemokine) and higher macrophage count had been observed in CF airways. Reduced acidification of lysosomes in macrophages leads to defective clearance of bacteria from the respiratory linings. Failure of bacterial clearance in the lungs results in chronic infections that later lead to remodelling and thickening of airway lining and eventually failure of lung functions.
Types of mutations in CFTR
The mutation in CFTR gene was first reported in 1989. From then, more than 2000 mutations have so far been reported in human CFTR gene which are broadly categorized into six different classes according to their effects on the maturation and function of CFTR protein (Figure 1). The most common mutation is the F508del (also known as ΔF508) mutation, with prevalence varying depending on the geographical region.
- Class I mutations result in no production of CFTR protein.
- Class II mutations, the most common mutations among patients, are associated with impaired post-translational modification, such as, misfolding, inadequate glycosylation leading to the defective trafficking of CFTR protein to the cell surface.
- Despite the presence of full-length CFTR protein to the cell surface, class III mutations alter channel gating and result in reduced Cl- channel activity.
- Class IV mutations lead to the regular production of CFTR protein with reduced Cl- permeability.
- Class V mutations lead to the production of minimal copies of functional CFTR.
- Class VI mutations negatively affect the protein stability causing an accelerated turnover of CFTR protein at cell surfaces thereby reduce the quantity.
Recently, 11 new rare CFTR mutations have been reported among patients from the Indian subcontinent that increased the spectrum of CFTR mutations worldwide.
Figure 1: Classes of CFTR mutations. Adopted from Lopes-Pacheco et al.
The conventional treatment options for CF patients aim to slow lung disease and its progression, to improve airway clearance. Since chronic infection in the lungs and exaggerated respiratory inflammation are the hallmark features of CF, antibiotics and anti-inflammatory drugs are frequently chosen for CF patients. Antibiotics are mainly given to the CF patients to eradicate the bacterial infections. Tobramycin is highly recommended for CF patients in US to delay colonization of bacteria in the lungs. Significant improvement of lung functions, decreased bacterial and neutrophil counts, reduced pulmonary inflammation and weight gain had been observed following tobramycin administration. In addition, tobramycin had been shown to promote translational read-through of the premature stop codon (class I CFTR mutation) and generate the full-length functional CFTR protein in AECs from CF patients. However, long-term usage of tobramycin increases the risk of renal failure and promote antibiotic resistance.
Azithromycin (AZM) is a macrolide class antibiotic that are known to reduce acute respiratory exacerbations, to improve lung function weight gain and quality of life in CF patients without correcting CFTR mutation. Macrolides are known to show anti-inflammatory actions by reducing neutrophilic inflammation. The antibiotic activity of macrolides resides in the “sugar” moiety attached to the macrolide ring, however removing this may not alter the anti-inflammatory actions. Non-antibiotic macrolides (NAMs) had been shown to restore the phagocytic function of airway macrophages. Therefore, whether NAMs would be beneficial in CF is under consideration. Ibuprofen as an anti-inflammatory drug is recommended for long-term treatment for CF patients. Inhaled dornase-α is recommended for CF patients with moderate to severe CF to reduce the viscosity of sputum. Hypertonic saline and inhaled β-agonists are recommended for CF patients over 6 years of age.
Although conventional treatment options showed significant improvement of the lung function, however, they are unable to repair the deleterious effects of the mutations in the CFTR gene. Therefore, mutation-targeted drugs that can restore normal CFTR function are in demand. A number of small molecules known as CFTR corrector and CFTR potentiators had been identified by high throughput screening (HTS). CFTR correctors increase the delivery of functional CFTR to the cell surface (effective for class II mutations), while CFTR potentiators increase channel gating (effective for class III mutations). Ivacaftor has been approved by FDA as CFTR potentiator for the patients carrying class II G551D allele and class IV mutations. Ivacaftor had been shown to enhance channel activity, improve lung function, lowers sweat chloride level and thereby improve the clinical outcomes in patients carrying class III mutation. Lumacaftor is a promising CFTR corrector that has been shown to selectively correct class II mutations with improved CFTR post-translational modification, trafficking to the cell surface and enhanced Cl- ion secretion. The maximal effect has been observed after 24hr of treatment. Unfortunately, monotherapy of lumacaftor in patients carrying homozygous class II mutations failed to show significant improvement of CFTR function in AECs and significant changes in lung functions. Furthermore, patients with heterozygous (class II/III) mutations require treatment with both Ivacaftor and Lumacaftor to achieve clinical benefit. Combinational therapy of Ivacaftor and Lumacaftor showed enhanced channel gating, improved lung function and lowered frequency of hospitalization compared to monotherapy or control group.
Gene therapy is an attractive tool to deliver the correct copy of CFTR gene via viral vector into the individual cells of the lungs. Since almost all structural and immune cells are affected in CF lung diseases, delivery of the correct copy of CFTR gene remained challenging. Clinical trial of gene therapy did not show substantial improvement of lung function. Adverse effects following gene therapy set this tool as hope more than a reality. CRISPR/Cas9 is a very recent and advanced gene delivery tool. Ongoing research is being conducted for development of novel CRISPR/Cas9-based CFTR gene delivery system.
CF is one of the most common life threatening recessively inherited disorders in the world. Those most affected are children, typically diagnosed before the age of two. The disease predominantly affects Caucasian populations compared with other ethnic groups. It is difficult to estimate CF prevalence, especially in non-Caucasian populations. Additionally, less severe cases may be diagnosed later in life. In predominantly Caucasian populations, the birth prevalence of CF is around 1 in 2500 live births. These figures may vary, with Scandinavian countries such as Sweden and Norway reporting a much lower rate. Finland has a CF birth prevalence as low as 1 in 40000 births. As of 2015, around 30000 people were living with CF in the United States and over 90% of individuals were Caucasian.
The life expectancy of CF has increased in recent years with advancements in diagnosis and care. The median survival time of patients with CF is around 37 years worldwide. This can vary depending on severity of disease and a key factor is the type of CFTR mutation. The F508del mutation is present in over 60% of CF cases worldwide, although prevalence varies depending on geographical region and ethnicity. Among the broad range of mutations present in CF cases globally, F508del is by far the most common, followed by G451X and G551D mutations.
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