Maggie's Paper

Maggie Fast
Genetics Paper
Rough Draft
Cystic Fibrosis
Cystic fibrosis, commonly referred as CF, is an autosomal recessive disorder. Clinical symptoms such as chronic pulmonary disease, pancreatic exocrine insufficiency and an increase in the concentration of sweat electrolytes affecting organs such as the liver and salivary glands vary in the effects and severity they have on a patient (7, 9). Despite the complications of the numerous organs affected, the leading cause of death in CF patients, 95%, is respiratory failure (9). The genetic mutation causing these symptoms is found on the cystic fibrosis transmembrane regulator (CFTR) gene found on human chromosome 7 (1, 3, 7). Several different types of mutations have been found to be the cause and through various diagnostic techniques, treatments can be formulated that are geared toward the effects created by the mutation.

Mutations of Cystic Fibrosis Transmembrane Regulator (CFTR)
CFTR is a cAMP and protein kinase A (PKA) regulated chloride channel that is found in the apical membranes of secretory epithelia (3, 6). Mutations in CFTR disrupt the transport of epithelial salt and water, causing tissue dehydration and an accumulation of thick mucus in the pancreatic, bronchial and vas deferens ducts. The mucus leads to obstruction and inflammation of the ducts (4). Implications in many other processes have been noted as well, including; regulation of other ion channels, membrane trafficking, pH regulation and apoptosis (10). The gene itself consists of 2 membrane spanning domains each containing; 6 transmembrane segments, 2 nucleotide binding domains (NBDs) and a regulatory region with multiple sites for phosphorylation by protein kinases A and C (4). In a gene that spans approximately 250,000 base pairs of DNA, more than 850 mutations have been identified and cover all types of mutations, including; missense, frameshift, nonsense, small and large in-frame deletions or insertions, and splice (7, 10).
With so many mutations possible and each capable of defining the severity of the disease, a classification system has been developed. The classification system is divided into 6 classes based on their known or predicted molecular mechanism or dysfunction and their functional consequence for the CFTR protein. Class I: Defective Protein Synthesis is a lack of the CFTR protein at the apical membrane. Mutations of this caliber produce severely altered structures that tend to be unstable and deficiently cleared from the cell. This gives little to no functional CFTR in the apical membrane, typically resulting in a severe phenotypic effects.
Class II: Abnormal Processing and Trafficking is another mutation where no CFTR is at the apical membrane; however this particular mutation involves a failure of heterologous systems to be properly processed to a mature glycosylated form. Generally this results in severe phenotypic effects (10). However, the most common mutation of this class ΔF508 when processed correctly still maintains residual chlorine channel activity. The specific mutation occurring at this position is the deletion of the phenylalanine (5). Patients suffering from this particular mutation have a reduced sensitivity of cAMP-sensitive chlorine conductance in epithelial cells, but still maintain some function. This leads many to believe that cellular mislocalization, and not impaired ion transport, is the cause of the abnormal behavior of the gene (ΔF508CFTR) (5). Contrast to the wild-type CFTR, which becomes fully glycosylated and is generally at the plasma membrane, the mutant is only core-glycosylated and accumulates in intracellular compartments. This “mis-location” in intracellular compartments has been attributed to misfolding of the protein, which is supported by a temperature sensitivity of the defect. Mutations of this sort are referred to as down regulation of the protein, meaning the protein still has limited function is thus less severe to the patient.
Class III: Defective Regulation is a mutation that affects the regulation of CFTR function by preventing ATP binding and hydrolysis at the nucleotide binding domains (NBD1, NBD2) required for the channel activation (10). Other channels, such as chloride channels or ROMK2 potassium channels, may be affected through missense mutations in G551D.
Class IV: Decreased Conductance is a class of mutations that affect the properties of CFTR single-channel conductance. Mutations of this sort are located within the MSD1 which is implicated in forming the channel pore. This allows corresponding CFTR variants to retain residual function. Alleles of this class are typically associated with milder pancreatic phenotypes.
Class V: Reduced Synthesis/Trafficking results in a reduced amount of functional CFTR at the apical membrane. Several mutations may be associated with reduction of fully active CFTR due to partially aberrant splicing, promoter mutations or ineffiecient trafficking. The CF phenotype is generally mild.
Class VI: Decreased Stability is generally a nonsense or frame shift mutation that results in the truncation of the C-terminus of CFTR. This leads to instability of, what is otherwise, a fully processed and functional variant. The mutations cause anywhere from 70-100 base pair truncation of the C-terminus and is usually associated with sever CF mutation.

Symptoms and Complications of Cystic Fibrosis
The most prevalent complication of cystic fibrosis is that involving the lungs. Lung disease generally develops due to thick, dehydrated mucous which impairs airway mucociliary clearance, making the patient more susceptible to recurrent bronchial infections (9). The most prevalent of these infections is that of the bacteria Pseudomonas aeruginosa. This pneumonia causing bacteria progressively destroy the lungs, leading to respiratory failure.

Diagnostic Techniques
Currently, diagnosis for CF is reliant on measuring the amount of sodium and chloride ions in sweat (2). This is a reflection of the defective re-absorptive section of the sweat gland ducts (2). Testing is usually done on both arms simultaneously and quantification of potassium is generally included. To re-enforce the results, the test is usually completed on two separate occasions. This is in hopes of providing double-proof results to avoid misdiagnosis. The classical Gibson Cooke quantitative pilocarpine iontohoresis test requires a sufficient amount of sweat (>30µL) and a set of very involved steps that require considerable training. The complexity of the Gibson Cooke method has led to other tests being devised that are more simplistic, but provide accurate results. Two tests using a specifically designed macroduct sweat collection system followed either by ionic quantification of the sweat or putting the sweat through a sweat check conductivity analyzer. “Cut off” values have been determined in terms of ionic composition in 98% of CF patients; a chloride concentration of >60mmol/l and a sodium concentration of >70mmol/l (2). The majority of patients generally have much higher concentrations. However, 1-2% of patients have normal range or borderline values making a definite diagnosis difficult.
For the 1-2% of patients on the borderline of the sweat test, additional investigation is necessary. To make a specific diagnosis, however, sequencing of the entire CFTR genome must be completed and this is currently not realistic in clinical settings. Diagnostics then turn to the exocrine pancreatic function and upper and lower respiratory tract status, with the aim of avoiding a false-negative which would delay treatment.
Prenatal and Neonatal screening have allowed treatments to begin earlier. Pilot studies are screening for the carrier state of ΔF508 in early pregnancy. Diagnosis of the fetus is completed by early chorionic villus biopsy. For neonates, the detection of increased blood levels of immunoreactive trypsin is evaluated. This is followed by a mutational analysis.

Treatment Modalities

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-ShareAlike 3.0 License