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Cystic Fibrosis (CF) is an autosomal recessive genetic disorder (Knowles & Durie, 2002) associated with mutations in the ATP-binding cassette (ABC) superfamily on chromosome 7 (Guggino & Stanton, 2006). The ABC proteins play a crucial role in transporting molecules across the plasma membrane (PM) by binding to ATP (Amaral & Kunzelmann, 2007). One such related ABC membrane is the cystic fibrosis transmembrane conductance regulator (CFTR) which is a cyclic-AMP dependant protein kinase that functions as a selective Cl- ion channel responsible for the proper regulation of tissues in the pancreas, intestine, lungs and kidneys (Guggino & Stanton, 2006). Individuals homozygous for the CFTR missense mutation demonstrate classical symptoms of CF such as inflammation, infection of mucosal areas (sinuses and respiratory airways), increase concentrations of NaCl sweat, male infertility, and indigestion as a result of pancreatic insufficiency (PI) (Knowles & Durie, 2002). Although these clinical symptoms are applicable to the majority of individuals (due to a common form of mutation in Î”F508-CFTR), a second rare and non-classical form of CFTR leads to poor prognosis as this class of mutation results in silent and late-onset of pulmonary dysfunction, lower Cl- sweat concentration, and absence of epithelial vas deferens (Knowles & Durie, 2002) . Due to the vast array of silent symptoms, such complications often lead to early mortality (Knowles & Durie, 2002). However promising therapeutic options (such as miglustat) have greatly increased the quality of life and life expectancy of affected individuals over the past years (Noel, Wilke, Bot, De Jonge & Becq, 2008).
Source for figure 1: Zeitlin, P. (2004). Can curcumin cure cystic fibrosis? The New England Journal of Medicine, 351(6), 606-608.
Despite the lack of a definite cure for both the classical and non-classical forms of CF, promising treatment options can be expected with the numerous discoveries of the roles of CFTR. Many of such advancements have lead researchers to discover CFTR's ability to suppress Na+ activity in epithelial cells (ENaC), mediate bicarbonate ions, regulate anion exchange, and regulate bicarbonate-chloride ion exchangers in epithelial cells (Guggino & Stanton, 2006). Furthermore, it has been found that the presence or absence of CFTR mediates the secretion of Cl- and water into the lumen of the kidney, the intestine, and pancreas (Guggino & Stanton, 2006). Hence, in normal individuals (see figure 1A), the presence of CTFR causes hydration of the mucosal layer of the lungs (Guggino & Stanton, 2006) as the osmosis of water out of the epithelial cells mimics the movement of ions leading to the secretion of bacteria (by cilia in our respiratory tract) out of the cells (Zeitlin, 2004). However, in CF patients (see figure 1B), the absence of the CFTR gene causes abnormal displacement of ions to flow in to the epithelial cells leading to the dehydration of the mucosal layer and build up of bacteria and mucus causing clinical symptoms mentioned previously (Zeitlin, 2004). Even though the trafficking of ions is affected as a result of mutations in the CFTR gene, the effects of mutational variations and how they impede the functioning of the CFTR gene itself becomes the imperative question.
Amaral and Kunzlmann (2007) help shed light on to this matter as they approximate 1500 CFTR mutations are responsible for causing CF. Of these mutations, the Î”F508 (which lacks as single phenylalanine codon) in the CFTR is the most prevalent form as 90% and 70% of patients with CF are either heterozygous or homozygous (respectively) for this allele (Guggino & Stanton, 2006). In addition, five separate mechanisms (see figure 2) are used as a framework for the various modes of CFTR mutations (Guggino & Stanton, 2006). Accordingly, Class I mutations lead to prematurely truncated CFTR transcripts (consequently resulting in a lack of expression) while Class II (Î”F508-CFTR) are caused by missense mutations that result in improper folding of proteins which are inhibited by the endoplasmic reticulum (ER) quality control process and tagged by chaperone molecules for degradation by proteosomes (Rowntree & Harris, 2003). Mutations of Class III are caused by low regulation and secretion of Cl- as a result of aberrant ion channel activation, while Class IV mutations are a product of defective Cl- conduction across the PM (Guggino & Stanton, 2006). Lastly, Class V mutations are a result of alternative splicing which ultimately dissipates the number of functional CFTR proteins in the cell (Guggino & Stanton, 2006).
Source for figure 2: Rowntree, R. K., & Harris, A. (2003). The phenotypic consequences of CFTR mutations. Annals of Human Genetics, 67, 471-485.
The enumerated classes of mutations and insight in to the Î”F508-CFTR mechanism are the basis of effective therapeutic regiments used today (Rowntree & Harris, 2003). Since the majority of CF cases are due to the confinement of Î”F508 mutations in the ER by chaperone molecules, effective drug therapies focus primarily on allowing Î”F508-CFTR to leave the ER for functional operation in the PM (Zeitlin, 2004). The disruption of the clanexin chaperone in miglustat (N-butyldeoxynojirimycin) and cuminin (a natural constituent of the spice turmeric) are plausible yet controversial as only a few results have been confirmed (Amaral & Kunzelmann, 2007). However, Noel et al. (2008) confirm that miglustat down-regulates the hyper-absorption of Na+ and allows for proper functioning of ion transport by CFTR in the PM. Curcumin on the other hand, has been found to impede calcium pump activity in the ER and since chaperone proteins demonstrate strong calcium dependency, it is hypothesized that by inhibiting calcium levels the chaperone molecules will be unable to function as effective retention molecules (Zeitlin, 2004). However, further studies are needed to confirm the effectiveness of this treatment (Zeitlin, 2004). In addition, Genistein (a bioflavonoid found in soy and tofu) stimulates proper regulation of c-AMP mediated Cl- transport by activating the G551DCFTR channel (Guggino & Stanton, 2006). Furthermore, aminoglycosides can be used to inhibit nonfunctional premature transcripts of CFTR (Class I) as the selectively force ribosomes to omit the mutated codon to produce a functioning CFTR protein transcript (Zeitlin, 2004). Another commonly used therapy includes GSNO (bronchodilator) that improves function of cilia by augmenting surface airway volume and increasing Î”F508-CFTR in the PM (Guggino & Stanton, 2006). Despite these advances in therapeutic options, one must question if there are alternative options if faced with drug resistance. Allen and Visner (2007) indicate lung transplantation as an alternative route to increase quality of life as research demonstrates no particular advantage in overall survival of CF patients. These results may also be a consequence of the waiting time for the lung itself (Allen & Visner, 2007).
The obvious interdependence of the CFTR gene in the function of many distal organelles such as the lung, kidneys, and pancreas has major implications at the cellular and molecular level (Knowles & Durie, 2002). Current therapeutic options include chaperone targeting drugs that cause proper folding of Î”F508-CFTR genes. However with the exhaustion of all drug therapies, patients may chose to maintain their quality of life with lung transplants.
I approached this problem summary by looking at my lecture notes and briefly skimming the text book for any Cystic Fibrosis related articles. This provided a good overview for what constituted CF and which components were important for this problem summary (for instance key words such as chaperone molecules, CFTR, Î”F508, therapy such as miglustat, and how they interact with the plasma membrane). After reading articles in the textbook, and looking at the problem summary question posted on Blackboard, I began to research these on the PubMed database and Google Scholar. I then narrowed down my search by reading the abstracts that best suited this problem summary and began to compose this research paper following the general outline of questions posted. I also did not run into any problems when researching this problem summary. Finally, after working laboriously and sweating over my computer for a long time, this problem summary was the end product. (By the way- thank you very much for the extension Dr. Mayi -I didn't need to sweat too profusely!)
Allen, J. F., & Visner, G. (2007). Lung transplantation in cystic fibrosis--primum non nocere? New England Journal of Medicine, 357(21), 2186-2188.
This source provided a good overview of alternative treatment options (using lung transplantations in children) that can increase the quality of life in CF patients. This therapy is an alternative to classical targeting of the chaperone molecules.
Amaral, M. D., & Kunzelmann, K. (2007). Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends in Pharmacological Sciences, 28(7), 334-341.
This source provided insight on the chaperone targeting therapeutic treatments used presently such as miglustat, aminoglycosides, and cuminin. It also provided a good detailed mechanism of how some of these treatment methods work on a molecular level.
Guggino, W. B., & Stanton, B. A. (2006). New insights into cystic fibrosis: Molecular switches that regulate CFTR. Nature Reviews. Molecular Cell Biology, 7(6), 426-436.
This source provided a very clear overview of what constitutes CF along with past and recent therapeutic advances (cuminin, genistein, and G551DCFTR channel). It also provided clear diagrams that enhanced understanding of the role of CFTR gene and CF in the PM.
Knowles, M.R., & Durie, P. R. (2002). What is cystic fibrosis? New England Journal of Medicine, 347(6), 439-442.
This source was helpful in introducing the problem summary and clearly demonstrating the clinical symptoms of CF in both normal and affected individuals.
Noel, S. F., Wilke M., Bot A.G., De Jonge, H. R., & Becq, F. (2008). Parallel improvement of sodium and chloride transport defects by miglustat (n-butyldeoxynojyrimicin) in cystic fibrosis epithelial cells. Pharmacology and Experimental Therapeutics, 325(3), 1016-1023.
This source showed the therapeutic relevance of miglustat through clinical results at a cellular and molecular level.
Rowntree, R. K., & Harris, A. (2003). The phenotypic consequences of CFTR mutations. Annals of Human Genetics, 67, 471-485.
This source provided a clear and concise explanation on the various types (classes I-V) of mutations that result due to mutated CFTR and what mechanism if the cause behind its improper functioning. It also provided a clear diagram that was used in this problem summary to help the reader follow the various classes of mutations.
Zeitlin, P. (2004). Can curcumin cure cystic fibrosis? The New England Journal of Medicine, 351(6), 606-608.