Overview of cystic fibrosis. Cystic Fibrosis is an autosomal recessive disorder associated with an abnormal expression of a chloride ion transporter known as cystic fibrosis transmembrane regulator (CFTR). (Rowe) CFTR channels are known to exist throughout the body making a cystic fibrosis a multisystem condition affecting all major organs. Nonetheless, the majority of conditions associated with cystic fibrosis are the direct or indirect consequence of damage to one of two systems; the pulmonary or the gastrointestinal. Respiratory defects are known to result in a persistent cough that produces thick sputum and mucus, wheezing, breathlessness, a decreased ability to exercise, inflamed or stuffy nasal passages and a repeated incidence of lung infections. Gastrointestinal defects are known to cause foul-smelling greasy stools, poor weight gain and growth, intestinal blockage and severe constipation. (ADAM CF) While signs and symptoms vary on the severity of the condition, a diagnosis for cystic fibrosis is usually achieved by the age of 2. Nonetheless, there are cases where patients with a milder form of the disease have gone undiagnosed till the age of 18. (Zielenski 2000, Davies)
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Figure . Global Incidence of Cystic Fibrosis (WHO)2.0 Epidemiology. Cystic fibrosis has traditionally been classified as a European disease that only affects either Europeans or those with European heritage. With an allele frequency upwards of 2%, cystic fibrosis is highly prevalent in western societies. (Bertranpetit) In the United States alone, a defective CFTR gene is carried by an estimated 1 in 29 Caucasian Americans with 30,000 individuals having two copies and thus consequently expressing the cystic fibrosis phenotype. (CF Foundation) Over the past decade, a more concentrated effort on the part of the World Health Organization (WHO) has seen fruitful results in attempts to provide a more in-depth classification of cystic fibrosis cases worldwide. While these efforts do reinforce the known fact that cystic fibrosis is predominantly a European disease, it nonetheless provides new insight that cystic fibrosis in African populations is more prevalent than is commonly believed. (WHO) Furthermore, large scale data classification indicates that at least one abnormal CFTR allele is still carried by approximately 1 in 48 Hispanics, 1 in 61 Africans and 1 in 88 Asians. (Hamosh) Figure 1 depicts the incidence of cystic fibrosis globally. (WHO)
2.1 Europe. With the prevalence of modern technology and classification methods, the incidence of cystic fibrosis is well documented throughout Europe. On average, 1 in 2000-3000 newborns is affected with cystic fibrosis. However, a closer look at CF incidence rates indicates that there are local and regional variations as seen by a higher incidence rate in northwestern France when compared to statistics in southern France.
While CF can be attributed to a wide array of possible mutations, variant Î”F508 is by far the most common and on average is present in 66% of the European population. Frequencies for the Î”F508 mutation vary from a maximum of a 100% in the isolated Faroe Islands of Denmark to about 20% in Turkey. Central, northern, western and north-eastern Europe sees a frequency of 70% which is closer to the overall average. Mapping out the allelic frequencies across Europe allows one to visualize the presence of a north-south cline that appears to have formed due to differing local evolutionary pressures.
Apart from Î”F508, mutations such as G542X, N1303K and G551D mutations contribute to about 10-15% of all the CF-causing CFTR mutations. These mutations predominate in the southern nations bordering the Mediterranean countries that have significant genetic influences from the African gene pool. Lastly, there are ethnic specific mutations as well as those that are either private or limited to a small number of individuals; the classic example of which is the 97% CF incidence rate seen in Ashakenazi Jews. (WHO)
2.2 Africa. Historically, Africa as a continent has been subject to immense political unrest and economic fragility, a fact that has directly contributed to a lack of infrastructure and technology that is at par with the most western nations. This unfortunate reality has made it difficult to accurately obtain data from African populations and thus far, most of the detection studies conducted have been small and infrequent. The limited data nonetheless show largely European mutations such as Î”F508, G542X and N1303K, albeit at different frequencies. The identification of CFTR mutations in the African population presents evidence of cystic fibrosis as being a relatively common disease in Africa, but one that is grossly under-diagnosed. In addition, studies indicate a uniquely African mutation, 3120+1G-->A, which is found in about 46% of the CFTR alleles derived from cystic fibrosis patients. Combining data from across the continent results in a carrier frequency of 1 in 42, with an expected incidence of CF in 1 in 7056 healthy Africans. (WHO)
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2.3 North America. Given that the majority of North American inhabitants possess some geographic origin to the European continent, the incidence of CF is second only to Europe at 1 in 3500 with Î”F508 being the most prevalent allele. 3120+1G-->A, the African mutation, is the second most prevalent allele in the African American CF patients after Î”F508 which is hypothesized to have emerged via ethnic admixture with Caucasians. (WHO)
2.4 Asia. Cystic fibrosis is a rare disorder in Asian populations and has an incidence of about 1 in 40,000 in South Asian populations and 1 in 200,000 in East Asian populations. In addition, no single mutation has been found to have a frequency higher than 15% and most cystic fibrosis cases are the result of rare and unique mutation anomalies. (WHO)
2.5 Oceania. Given the historical emigration of Europeans to oceanic territories, the incidence rates found in Oceania parallel those found in European populations. (WHO)
2.6 Historical theories for defective allele persistence. Over time, many hypotheses have been floated in attempts to understand the primary reason for the high allelic frequency seen in cystic fibrosis as well as its persistence over time. These hypotheses include: high spontaneous mutation rates, a founder effect that is followed by a genetic drift, and a heterozygote advantage. There is no doubt that each of these hypotheses has some merit to itself, however, given the population history of Europe, for the recessive lethal frequency to be as high as 2%, there has to be a significant heterozygote advantage occurring due to the defective CF allele.
The concept of Heterozygote advantage confers a relatively higher fitness to individuals who have one but not two alleles for a given gene. In the case of cystic fibrosis, having two alleles of the mutated CFTR gene leads to systemic loss of function for CFTR, however, having one allele reduces but not entirely diminishes CFTR function, a scenario that has been thought to be exploited by the body in order to combat conditions such as lactose induced diarrhea, typhoid fever, cholera as well as tuberculosis. (Poolman, Modiano, Pier, Cuthbert) While each of these conditions, at some point in history, has caused widespread death in Europe, cholera and typhoid fever can be ruled out as possible evolutionary pressures for the persistence of cystic fibrosis. This is because cholera, a disease endemic in Southeast Asia and Africa did not make an appearance on European soil until the early 19th century, thereby making it a highly unlikely causative agent to achieve an allele frequency as high as 2% in just 5 generations. (Bertranpetit, Sack) Similarly, recent clinical studies in Indonesia, an area endemic for typhoid fever, has shown a localized allele frequency of a meager 0.13%, a number well below the 2% seen in European populations. The study makes use of a statistically significant sample size of 775 individuals leading one to conclude that evidence for typhoid fever as a causative agent is highly lacking. (vandevosse) On the other hand, lactose induced diarrhea and tuberculosis have both caused localized mortality in Europe for hundreds of years, an event that could have resulted in a large enough selective pressure to increase the heterozygote frequency as high as 2%. (Bocquet-Appel, Kapur)
3.0 Genetic basis for disease. The CFTR gene is located on the long arm of chromosome 7. (Poolman) Encompassing approximately 180,000 base pairs, the resultant protein is composed of 1480 amino acids with more than 1000 disease-associated mutations that result in errors in the coding sequence, messenger RNA splice signals, and other regions. (Zielenksi 2000, Gibson, Tsui) Defects arising from CFTR mutations can be classified according to the mechanism via which they are believed to cause disease. Scientists thus far have categorized six major classes of defects, class I and II of which encompass approximately 80% of all CFTR cases. (Rowe, Gibson)
3.1 CFTR Structure and Function. CFTR is a member of the ATP-binding cassette transporter family of membrane proteins. (Hunt, Rowe, Sheppard) As is characteristic of a majority of ATP-binding cassette transport family members, the final CFTR protein product contains two nucleotide-binding domains (NBD) and two 12 membrane spanning alpha-helixes, along with a unique regulatory R domain with multiple phosphorylation sites. (Welsh 1994, Hunt, Sheppard)
Figure . CFTR activation via PKA and 2ATP (Hunt)The R domain serves as a target for phosphorylation by cAMP induced protein kinase A (PKA). The domain has been shown to integrate PKA signaling and has been hypothesized to receive input from other physiological systems as well. (Rowe, Hunt, Sheppard) Studies indicate that deleting the R domain results in an activity that is 50% of maximal efficacy in an unphosphorylated state suggesting that the R domain plays a role in inhibiting the constitutive activity of the chloride transporter. (Welsh 1994, Hunt) As seen in Figure 2, the R domain is thought to carry out its inhibition by interacting with NBD1 in the CFTR thereby preventing the interaction between NBD1 and NBD2 that would be required to open the chloride channel. When PKA is activated by cAMP, the PKA molecule is transported to the R domain phosphorylating it. The phosphorylation of the R domain releases the inhibition on NBD1 thereby allowing it to interact with NBD2. However, this interaction is not complete until the arrival of 2 ATP molecules which are necessary to lock NBD1/NBD2 interaction into place consequently opening the channel for chloride flow. (Welsh 1994, Hunt, Sheppard)
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CFTR channels, located primarily on the apical plasma membrane, are part of a multi-protein assembly that works to keep all parts in close proximity to each other. The carboxyl terminal of the CFTR protein product, composed of threonine, arginine and leucine, anchors the protein to PDZ-type receptors forcing the CFTR to be present in close proximity to a number of membrane receptors, ion channels and the cytoskeleton. (Rowe) The diversity among proteins surrounding CFTR helps to explain the role of CFTR in a number of cellular functions distinct from chloride permeability; studies have shown that CFTR possesses the ability to down regulate transepithelial sodium transport by down-regulating the sodium channel in itself; CFTR regulation of calcium-activated chloride channels and potassium channels has been shown to impact exocytosis and the formation of molecular complexes in the plasma membrane. (Rowe)
3.2 Class II defect Î”F508, a class II defect, is the most common CFTR mutation and accounts for 70% of all CFTR cases worldwide and up to 90% of all cases in the United States. (Welsh 1993, Zielenski 1995, Rowe) A class II defect is categorized by a mutation that results in defective protein maturation and eventual premature degradation. CFTR with Î”F508 mutation results in a missing phenylalanine residue at the 508 position causing it fold inappropriately in the cytosol. This fact is rapidly recognized by the normal cellular machinery resulting in the degradation of the misfolded protein and consequently preventing the CFTR to be inserted into the membrane. (Welsh 1994, Rowe) Specifically, Î”F508 mutation interferes with the folding of NBD1. Without any mutations, NBD1 exists in equilibrium with a relatively low energy "molten globule" formation. Increasing the temperature decreases the stability of NBD1 to resemble the "molten globule" formation. On the other hand, ATP binding to NBD1 increases the stability of NBD1 allowing it to interact with NBD2 to form an intact and functioning chloride channel. In the Î”F508 mutated state, NBD1 is destabilized to resemble a molten globule state at physiology temperatures and during the presence of ATP. The molten globule state is prone to aggregation and thus is degraded resulting pathologies are associated with a chloride channel loss. (Hunt)
While Î”F508 is the most common class II mutation, there are several other clinically important mutations such as N1303K, G85E, and G91R which are prevalent in non-European populations and lead to a misfolded CFTR protein that is prematurely degraded. (WHO)
3.3 Class I defect Class I defect accounts for about 5 to 10 percent of all CFTR mutations and is caused by premature truncation or a nonsense allele. G542X, the most common class I defect, is particularly prevalent amount the Ashkenazi Jews and their descendants. (Welsh 1993, Rowe, WHO)
3.4 Class III-VI defect Classes III-VI defects present mutations that result in a phenotype indistinguishable from either class I or class II, however their prevalence is significantly lower than either one of them and accounts for a combined 10-15 percent of all CFTR cases. Class III mutations encode a full length CFTR protein, however they a defect in the two nuclear binding domains which due to abnormal adenosine triphosphate (ATP) gating results in little or no ion channel activity. An example of such a mutation is G551D. Class IV defects, seen in A455E mutations, results in a full length CFTR that only possesses partial ion-channel activity. Class V defects cause abnormal or alternative splicing that result in a reduced number of functional CFTR transcripts. Class VI defects results in a defective CFTR stability at the cell surface. It is important to note that specific mutations may have characteristics that can be categorized in multiple classes. (Welsh 1993, Rowe, Gibson)
4.0 CFTR Physiology. Depending on the location in the body, the CFTR protein can be located on either secretory or absorptive cells. (Kunzelmann) As stated previously, the primarily role of CFTR is to conduct chloride ions across the apical surface of the epithelial cells. In order to do so, PKA phosphorylates the regulatory domain, and ATP binds to the two nucleotide binding domains. Most clinical symptoms observed in cystic fibrosis as associated with defective chloride conductance, however, some transport defects observed in cystic fibrosis are not as simply explained because CFTR in itself is regulated by several secondary messengers, cytosolic factors and membrane receptors, while at the same time regulating other membrane conductances. (Kunzelmann)
Figure . Secretion and absorption of chloride ions in the sweat duct. (BB-sweatduct)4.1 Sweat ducts. The sweat glands, as seen in Figure 3, are composed of two distinct cell types; the coil cells and the duct cells. The coil cells are secretory in nature are localized to exclusively the dermis. In contrast, the duct cells are absorptive and are located in both the dermis and epidermis. (Segal) In normal coil cells, stimulation by Acetylcholine activates the muscarinic G-protein coupled receptor that leads to the activation of phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Diffusion of IP3 to the endoplasmic reticulum prompts the ER to release its stores of calcium into the cytosol raising the intracellular calcium concentration. Increases in calcium, via an unknown mechanism, stimulates chloride channels located on the apical membrane to secrete chloride ions into the lumen. The hyperpolarization of the lumen due to the chloride ions provides the gradient for paracellular transport of sodium ions from the interstitital space into the lumen. The movement of sodium and chloride ions into the lumen generates as osmotic gradient that allow for the water to follow, thereby making the net secretion taking place in the coil cells isotonic. The isotonic fluid moves upwards from the distal end of the dermis towards the proximal end and then onto the epidermis layer. (Quinton, Segal)
In the proximal dermis and epidermis layer, the fluid interacts with the duct cells which are absorptive in nature. Epithelial ENaC channels located on the apical membrane allow for the entry of sodium ions into duct cells. Simultaneously CFTR channels, also located on the apical membrane, absorb chloride ions. The duct cells have a lower water permeability compared to the coil cells hence the net effect is absorption of sodium and chloride ions without absorbing water. Consequently, the final solution reaching the sweat pores is a hypotonic fluid with low amounts of sodium and chloride ions. (Quinton, Segal)
In cystic fibrosis patients, a defective or non-existent CFTR channel impairs the reabsorption of chloride ions and consequently with it sodium ions. Due to this, little post-coil cell alteration takes place thereby making the fluid exiting the sweat pores rich in sodium chloride. The resulting salty taste of the skin makes for the gold standard for diagnosis today and is the reason why cystic fibrosis has been historically associated with the phrase "a child that taste salty when kissed will soon die." (Quinton)
4.2 Lungs and Sinuses. In the airways, CFTR is predominantly located on the luminal membrane of cells forming serous end pieces of submucosal gland. In addition, the CFTR protein, to a lesser degree, is also expressed in the superficial epithelium.
CFTR, in the submucosal gland, is located on the basolateral membrane with an associated role to secrete chloride ions. The Na/2Cl/K (NKCC1) cotranporter located on the basolateral membrane transports the necessary chloride ions to the cell generating the pools from which chloride ions are secreted. The incoming potassium ions are recycled back into the lumen via potassium pumps located on the basolateral membrane. In contrast to the submucosal gland, the CFTR channels in the superficial epithelium have an absorptive function. CFTR, in this case located on the apical membrane, colocalizes with the epithelial sodium channels (ENaC) and the depolarizing effect of sodium reabsorption is able to drive the chloride ions into interior of the cell. CFTR displays self-regulation by inhibition the epithelial Na channels upon CFTR activation. In doing so, CFTR limits sodium transport across apical membrane, consequently limiting the gradient that allows for chloride transport. (Kunzelmman)
In the airways, located directly adjacent to the epithelial cells is the Airway Surface Liquid (ASL). The ASL consists of two distinct layers - a mucus layer and periciliary liquid layer. Cilium, originating from the periciliary layer, extends to a height of 7uM and generates a sweeping motion in order to clear mucus out of the lungs. The volume in the periciliary layer is tightly regulated to maintain a low-viscosity condition favorable for the cilium to beat. In addition, the periciliary layer also lubricates the gel-forming mucins secreted from the cell surface, a fact that is integral for the successful clearance of mucus by the cilium. The mucus layer on the other hand consists of high molecular weight mucins that originated from the cell surface but have had their properties altered by the surrounding water content, ion concentrations and pH. In a normal epithelial cell, the presence of the periciliary layer of normal volume promotes efficient mucus clearance. In addition, oxygen consumption by a normal epithelial cell leaves behind oxygen partial pressure gradient within the ASL. (Gibson, Puchelle, Kunzelmann)
Two competing hypotheses have been proposed to explain the frequent lung infection associated with cystic fibrosis. The first hypothesis, termed the "low volume" hypothesis, proposes that a lack of functional CFTR in the epithelial cell leads to a diminished inhibition of the apical ENaC channels. A lack of inhibition leads to an abnormally high sodium absorption, which via the osmotic gradient is followed by water. The elevated isotonic fluid absorption depletes the ASL leading to reduced mucociliary clearance. However, while mucociliary clearance is reduced, mucus secretion from the cell surface continues. Over time, a lack of clearance causes the mucus layer to be abnormally thickened. Elevated oxygen consumption in the epithelial cells generates hypoxic gradients in the mucus layer which is exploited by bacterial pathogens that are able navigate through the mucus layer and penetrate into the hypoxic zones. Pathogens are able to adapt to the hypoxic environment with increased alginate expression and the formation of microcolonies that over time develop into biofilms. The extracellular matrix of the biofilm protects the pathogens from host lung defenses in chronic airway infection. (Gibson, Ratjen)
The second hypothesis, termed the "high salt" hypothesis, proposes that the ASL under normal conditions is a low salt environment; excess salt and chloride is absorbed via the ENaC and CFTR channels in comparison to water. In CF patients, a malfunctioning/non-existent CFTR leads to decreased salt absorption across the apical membrane resulting a saltier than normal ASL fluid. The high salt environment is thought to inactivate endogenous, salt-sensitive antimicrobial peptides resulting in an impaired defense against pathogens and result in chronic airway infection. (Gibson)
4.3 Pancreas. The pancreas is a lobulated structure with its head contained within the curve of the duodenum. The role of the pancreas is to secrete a bicarbonate rich fluid that is rich in digestive enzymes. These secretions drain through the pancreatic duct which merges with the common bile duct to flow into the duodenum via the ampulla of vater. (Cohn, Taylor) There are two major cell types in the pancreas; the acinar and ductal cells. Composing of 75-80% of all cells, the acinar cells are predominant cell type in the pancreas. The acinar cells store digestive enzymes in the form zymogens granules that upon stimulation are discharged from the cell via exocytosis. The act of eating induces a combination of hormonal secretions and neural mechanisms, both of which act upon the acinar cells to release the zymogen granules. In contrast, the ductal cells, upon stimulation by the hormone secretin, release a bicarbonate rich fluid that has a two-fold effect; to dilute the secretions made the acinar cells and to maintain the flow of the digestive enzymes into the duodenum. Bicarbonate flow from the ductal cells takes place using the Cl-/HCO3- exchanger located on the apical membrane thereby making the secretion of chloride ions into the lumen via the CFTR a matter of utmost importance. Over a span of 24 hours, the ductal cells secrete about 2.5 L of bicarbonate which plays an integral role in neutralizing the acidity in the gastric tissue as well as mediating the digestion of carbohydrates, fats and proteins. (Cohn, Taylor)
CFTR in gastrointestinal epithelial cells are located on the apical membrane and undergo the same aforementioned regulation via cAMP activated PKA. In normal epithelial cells, the secretin receptor is a G-protein located on the basolateral membrane. The binding of secretin to the secretin receptor initiates a signaling cascade that raises intracellular cAMP and stimulates PKA. PKA goes on to phosphorylate the R-domain on the CFTR activating it. The result of CFTR activation is the translocation of chloride ions across the apical membrane into the lumen of the duct. The chloride ions are then utilized to translocate bicarbonate into the lumen of the duct. The transport of bicarbonate into the lumen has a hyperpolarizing effect, establishing a downward voltage gradient that is utilized by the sodium to move from the interstitium to the lumen paracellularly. The flow of ions into the lumen generates an osmotic gradient that pulls water into the lumen effectively hydrating it. The water that flows into the lumen is necessary for the formation of the "juice" that facilitates the movement of enzymes into the duodenum. (BB)
Cystic fibrosis patients possess either a non-existent or malfunctioning CFTR channel, both of which impair the transport of chloride ions in the lumen duct regardless of stimulation. Impairment of chloride transport has a direct effect on bicarbonate secretion as the presence of chloride ions in the duct is necessary for bicarbonate to be exchanged across the Cl-/HCO3 exchanger. Deficiency in bicarbonate secretion consequently fails to provide an osmotic gradient for paracellular water transport resulting in a pancreatic exocrine secretion that has low bicarbonate, low pH and a lower than normal volume. The inspissated secretion is unable to effectively flow into the duodenum and over time gets builds up in the lumen blocking the secretion ducts. (Taylor) Furthermore, the blockage prevents future enzyme secretion from the acinar cells resulting in acinar cell atrophy, pancreatic fibrosis and eventual auto-digestion of the pancreas resulting in pancreatic insufficiency. Compounding the issue is that without the necessary digestive enzymes in the small intestine, the body is unable to breakdown the complex food substrates taken in as part of a meal. This in turn prevents the absorption of essential nutrients from fats, proteins and carbohydrates. The stunted growth and mental deficiency associated with cystic fibrosis is a direct consequence of malabsorption of essential nutrients. (Cohn, Taylor)
In addition to a defective CFTR channel, studies indicate that cystic fibrosis patients have increased levels of Arachadonic acid (AA) and lowered levels of docosahexanoic acid (DHA). AA has been implicated as a potent mediator of inflammation and a stimulant of mucus production. Furthermore, excess AA has the potential to compete with DHA for incorporation into the membrane. Arachadonic acid insertion into the membrane has the potential to alter membrane fluidity and with it the chloride movement across the membrane. (Taylor)
4.4 Endocrine. The pancreas, along with acinar and ductal cells, is also home to the beta cells in the islets of Langerhans. The primary function of the beta cells is to synthesize and secrete insulin, a molecule that is required for glucose uptake into the muscle and adipocyte tissue. After a meal, glucose levels in the blood rise for a temporary period. The rise in glucose levels is buffered by the passive uptake of glucose into the liver, kidney and pancreas by the GLUT2 transporter; the uptake of glucose into pancreas is a stimulus for insulin release from the pancreatic beta cells. In addition, insulin dependent GLUT4 transporters also allow for a portion of the glucose to be taken up and stored into the striated skeletal muscle and adipose tissue. (Bell)
In cystic fibrosis patients, beta cell destruction reduces the post-glucose ingestion insulin levels by up to 41%. (Alves) Consequently, glucose uptake into the skeletal muscle and adipocytes is severely impaired resulting in fasting, non-fasting and intermittent hyperglycemia as is seen in type I and type II diabetic patients. Nonetheless, according to the American Academy of Diabetes, the resulting Cystic fibrosis related diabetes (CFRD) is distinct from either class I and or class II diabetes and is classified as 'other types of diabetes', listed as being 'caused by exocrine pancreatic lesion.' (Alves)
4.5 Intestine. Meconium ileus is one of the earliest clinical manifestations of cystic fibrosis and is present in about 20% of all CF cases. (Eggermont, Williams) In utero, CFTR channels are fully developed by week 18 week post-gestation. A defective CFTR prevents the transport of chloride ions that normally would have established as osmotic gradient for water movement into the lumen. The dehydrated lumen results in an inspissated meconium that is unable to pass through the colon and gets stuck. The end result is a colon that is small and unused with an ileum that is dilated and filled with sticky meconium. The meconium is highly viscous, rich in serum albumin, and has increased levels of disaccharides, all of which can be lack of pancreatic proteolytic enzymes. (Eggermont)
A condition that accompanies the increase in fecal volume and is compounded by malnutrition and increased intra-abdominal pressure due to coughing is rectal prolapse. The protrusion of the internal rectal membranes is known to occur in about 20% of all children diagnosed with cystic fibrosis. (Haworth, Robertson)
5.0 Diagnosis. While there are multiple methods to diagnose cystic fibrosis, the current gold standard utilized by all 50 states is the sweat chloride test. (Laguna) During the test, an electrode is placed over gauze containing pilocarpine and electrolyte solution. A second electrode not containing pilocarpine is placed at a different site and mild current stimulates the sweat glands to release sweat. A piece of preweighed filter paper is placed on the test site and is utilized to collect the sweat for a length of 30 mins. After the test, the filter paper is weighed to measure the amount of sweat collected. (LeGrys) Theoretically, the sweat chloride test exploits the inability of the CFTR to conduct chloride ions therefore patients with cystic fibrosis will have higher than normal levels of chloride in their sweat.
A value of 60 mM/L is the threshold for diagnosis and is easily detected in the majority patients with cystic fibrosis. Nonetheless, in about 5-10% of cases, an intermediate value of 40-60 mM/L is measured, a fact that often leads to misdiagnosis as patients with a milder phenotype present with chloride levels in the intermediate range. A value less than 40 mM/L is usually considered normal. (Laguna, Ratjen)
In addition, as part of a newborn screening process, a blood or saliva sample is usually obtained from new born babies. The samples undergo genetic testing where the DNA obtained is screened for mutations known to cause cystic fibrosis. (Davies) Often, blood samples are also tested for elevated levels of the immunoreactive trypsinogen. While testing for trypsinogen is highly effective, trypsinogen has been known to be elevated in patients with only one mutated CFTR allele, or in some cases in patients with two normal CFTR alleles implying that there is a possibility of a false positive (Davies)
Owing to the time sensitive nature of cystic fibrosis treatment, another excellent option is prenatal testing for parents carrying at least one mutated CFTR allele. Prenatal testing allows a developing fetus to be diagnosed with cystic fibrosis at the earliest point thereby allowing medical personnel to alter their care and treatment accordingly. As part of the prenatal screening, a woman can opt for either an amniocentesis or a chorionic villus biopsy. (Davies) In an amniocentesis, a needle is inserted through the abdominal wall into the amniotic fluids where fetal cells are obtained. The extracted cells are then tested to assess the functionality of both CFTR genes in the fetus. In the case of chorionic villus biopsy, a needle is inserted through the vagina and cervix into the uterus where a small piece of placenta is extracted. The extracted placenta is then biopsied for test for CF in the developing fetus. One of the major complications surrounding prenatal screening is that amniocentesis has fetal death risk of 1 in 200 and the number for chorionic villus sampling is 1 in 100, thereby making prenatal testing an invasive option. (Tabor)
An early diagnosis for cystic fibrosis is integral for the care of the newborn as it allows for parents to be notified on the alterations that they must make to the infant's diet and to his/her lifestyle changes. In addition, patients receive specialized care from an early age which goes a long way in maintaining both the quality and longevity of a patient's life. (Moorcroft) Nonetheless, most countries around the world do not conduct newborn or prenatal screenings thereby delaying diagnosis until the onset of physiological symptoms.
6.0 Prognosis and Management. The historical prognosis of cystic fibrosis has been extremely poor with most patients failing to live past the first year of their life. Improved screening, earlier diagnosis and access to better health care in the past twenty years has positively impacted the prognosis for cystic fibrosis patients. The median age of survival for CF patients was 2007 was 37.4 with the majority of students graduating high school and college. (Median Age)
While a cure for cystic fibrosis has yet to be discovered, the improvement in prognosis is seen largely due to a strict management plan that includes the use of antibiotics, a regimented nutrition plan and routine tests to assess organ functionality.
Given the various gastrointestinal manifestations seen in CF, most of which are primarily due to pancreatic insufficiency, the importance of a regimented nutritional plan cannot be understated. Studies indicate that lung function and survival is directly proportional to BMI hence the nutritional goal is to keep the BMI for CF patients above the 50th percentile. (Shoff) While CF patients do not necessarily have major modifications to their diets, their meals are often supplemented with pills that contain the necessary degradative enzymes that would have ordinarily been obtained from the pancreas. (Kalnins) In addition to the enzyme secretion, the pancreas also secretes large amounts of bicarbonate which function to neutralize the acidic chyme coming in from the gastric tissue. The lack of bicarbonate in pancreatic insufficiency fails to neutralize the acidic chyme in the duodenum hence enzymes pills are often enteric coated to protect them from the acidic environment or are sometimes supplemented with bicarbonate pills. (Kalnins) The majority of supplemental enzymes are packaged as pellets that contain lipases, amylases and proteases all of which are predominantly derived from pigs. (Walters) In cases where fat malabsorption persists even with supplemental enzymes, one can consider adding a proton-pump inhibitor or histamine-2 blocker both of which decrease gastric acid secretion. The rationale behind doing so is that decreasing the acid will allow the capsulated enzymes to function more effectively and in most cases is a highly effective treatment. (Kalnins) In addition to enzyme supplementation, CF patients must also be given additional salts in their diet to compensate for the excessive salt loss due to a defective CFTR channel. (Davies) Furthermore, multivitamins containing vitamin A, D, E and K are given to prevent against the plethora of defects that result from vitamin deficiencies. (Davies)
Pulmonary infections arising on the built up dry mucus in the lungs is one of the leading causes or morbidity and mortality in CF patients. (Cohen) P. aeruginosa, the most common infectious agent in CF patients is naturally found in many domestic environments including plants, soils and warm moist environments that contain organic waste. In addition, P.aeruginosa has been shown to be suspended with aerosols for long periods of time indicating the threat of infection via an airborne route. (Cohen) While the route of infection is not necessarily clear, one of the major keys to ensuring a good quality of life in CF patients is by preventing infection by P.aeruginosa. P.aeruginosa has been shown to be responsive to antibiotic treatments during the initial stages of infection. (Cohen) However, as the time of the infection progresses, the synthesis of biofilms makes bacterial infection more impervious to treatment both by antibiotics and by components of the innate and adaptive immune system. (Cohen) Hence, it is essential that bacterial infections be treated as early as possible thereby once again reflecting the importance of an early diagnosis for a given patient.
The first line defense against bacterial infections is to prevent the occurrence of infections in the first place. Over the past decade, several mechanical devices and airway clearance techniques have been developed that are able to artificially clear mucus from the airway thereby removing the source environment for the bacterial infection. These techniques include the active cycle of breathing techniques, autogenic drainage, positive expiratory pressure masks, Flutter and Acapella with no clearance techniques being shown to be superior over the others. (Cohen) Nonetheless, if the patient were to develop an infection, effective treatment includes the use of oral or intravenous antibiotics that are appropriate of the specific culture. Antibiotics primarily used for treatment include ciprofloxacin and azithromycin, both of which are effective against preventing and controlling an infection. (Davies)
In addition to nutrition and antibiotics, CF patients undergo a series of routine checkups to assess the overall health of the body. These tests include imaging tests, lung function tests, sputum culture and organ function tests. Imaging tests are carried by a series of X-rays, CT scans and MRIs, all of which allow the presiding physician to determine the extent of damage to the patient's lungs, gastrointestinal tissues and any other possible organs. (MayoClinic) Lung function tests measure the size of the lungs, how much air a patient can breathe in and out, how fast the patient can breathe in and out, and effectiveness at which the lungs deliver oxygen to blood. FEV1, designated as the volume that has been exhaled at the end of the first second of forced expiration, is one measure of lung function that is commonly utilized and is measure by a test called spirometry. (MayoClinic) Sputum cultures are regularly obtained which are then analyzed for bacteria. (MayoClinic) Lastly, regular blood tests are carried out to assess the functionality of organs such as pancreas and liver, as well as test for cystic fibrosis related diabetes. (Mayoclinic, Alves)
While nutrition, antibiotics and routine checkups are effective preventative measures against cystic fibrosis induced defects, they are not always successful and a patient often reaches a point where a given organ is no longer viable. At that point, a last ditch effort for treatment is organ transplantation. The most common organ transplanted in cystic fibrosis patients is the lung which often declines to the point where assistance from mechanical devices is required for survival. (Belkin)
Recent studies indicate that regular exercise has the potential to help clear mucus built up in the airways and consequently help lung function. Thereby, regular exercise has been integrated into the aforementioned management and has shown to increase overall quality of life for a patient. (Moorcroft)
6.1 Quality of life. With the advent of modern technology, new drugs and a better understanding of the disease, the prognosis for cystic fibrosis patients is better than ever. However, improved prognosis has not necessarily translated into a higher quality of life. According to a 2006 study assessing the quality of life for cystic fibrosis patients "[cystic fibrosis] imposes considerable emotional stress on the individual and requires intensive and time-consuming daily therapy, which may have adverse effects on the quality of life." (Schmitz)
In addition, as CF patients' age, they face a set of medical issues that would have not presented themselves otherwise. As most cystic fibrosis patients are achieving sexual maturity, the previously irrelevant issue of reproductive health has come to the forefront. Studies over the past two decades have made it clear that ~95% of all males with cystic fibrosis are infertile. (Lyon) Dehydrated secretions have been implicated in the formation of intrauterine obstructions that can affect a spectrum of abnormalities. About 2% of male infertility suffer from the congenital absence of the vas deferens, however even males who do have an intact vas deferens are almost always azoospermic. The seminal vesicles have been shown to display various abnormalities including aplasia, hypoplasia or cystic dilation. In addition, the body and tail of epididymides are frequently absent or not fully mature. Lastly, the testes may be atrophic or normal. (Lyon) On the other hand, women are fortunate to not suffer from any reproductive tract abnormalities. Nonetheless, a reduction in cervical mucus water content has been thought to result in a mucus plug that is resistant to the passage sperm indicating that a level of sub-fertility may occur. (Lyon)
7.0 Current research Cystic fibrosis, at its core, is a genetic condition, and while no cure for it has been found thus far, scientists believe that the only manner cystic fibrosis can truly be cured is by altering the defective gene. With that being said, gene therapy has been at the forefront of cystic fibrosis research with attempts being made to substitute a normal of copy of the CFTR gene in all affected cells. The thought is that is the gene transfer would correct the basic condition without inducing an immune response; attempting to neutralize the effects of a defective gene by the introduction of a foreign entity is expected to induce a retaliatory immune response. (Ramaldho) While delivery of the therapeutic gene to the affected cell via a method of viral vectors has been largely successful, the number of affected cells expressing the transgene is relatively low. This frustrating phenomenon has largely been attributed to the remarkable natural defenses of the human body. (Koehler) Consequently, it has been hypothesized that a vector in combination with immunosuppressant drugs could potentially solve the issue. However, the addition of an immunosuppressant drug in a patient with an increased risk of infection for long periods of time could potentially end up doing more harm than good. (Koehler) Research attempts to circumvent the immune response are currently underway with one major viable option being to modify the vector backbone to prevent the expression of viral genes in the transduced cells thereby minimizing the chances of an immune response. (Koehler)
8.0 Conclusion. Cystic Fibrosis is a complex genetic disorder that currently has no known cure. Having its evolutionary origins in early Europe, cystic fibrosis has quickly become a condition that, to varying degrees, is found across the globe. The primary defect associated with cystic fibrosis is a malfunctioning or deficient chloride ion transporter named CFTR. The presence of CFTR channels in the majority of tissues makes cystic fibrosis a multifaceted lethal disease. A treatment for such a condition would have to include diverse and far ranging strategies in order to combat the specific disorders associated with each organ. Unfortunately, search for a cystic fibrosis treatment is still in infancy and a possible cure does not appear visible in the near future. Our current strategy of preventing pulmonary and gastrointestinal associated mortality for as long as possible hinges on the careful management of the disease. An early diagnosis, coupled with balanced nutritional, enzyme supplements and routine checkups has allowed patients to live up to an average of 37 years, a considerable improvement over the six month timeline that was seen before. Nonetheless, a prolonged life does not always equate with an improved quality of life. Patients living to 37 rarely live what is considered a "normal" lifestyle; hence it is imperative future research focus on not just managing the condition but rather to fully understand and cure cystic fibrosis.