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Key Points • Linkage analysis provides evidence for the existence of a single CFTR locus on human chromosome 7 (region q31) 1 • There are more than 1900 known mutations of the CFTR gene 2 Of the mutations reported so far, 140 meet clinical and functional criteria and are categorized as causing CF 2 • The loss of CFTR protein activity is a result of mutations in the CFTR gene that lead to decreased quantity and/or function of CFTR • protein at the epithelial apical cell surface . This leads to defective Cl – transport through CFTR protein channels and is the underlying defect resulting in CF 3 The F508del mutation is the most common cause of CF, present in about 83% of patients worldwide 4 • • About 43% of the CF population is homozygous for the F508del mutation worldwide 4 References 1. Kerem JR et al. Science. 1989;245:1073-1080 2. Sosnay PR et al. Nat Genet . 2013;45(10):1160-1167. 3. Zeilinski J. Respiration . 2000;67:117-133 4. CFTR2.org. 3
Key Point CFTR protein is delivered to the apical epithelial membrane in a multi-step process • comprising CFTR synthesis, CFTR folding and processing, CFTR trafficking, CFTR turnover and CFTR function at the cell surface Additional Information CFTR Synthesis: CFTR gene is transcribed to mRNA in the nucleus. Introns are removed • from mRNA in a splicing process. mRNA is translated to immature CFTR protein in the endoplasmic reticulum with the aid of ribosomes CFTR Folding and Processing: Immature CFTR protein is folded in the endoplasmic • reticulum, thereby becoming mature CFTR protein. Any protein that is not properly folded is degraded (processed) by intracellular quality control system CFTR Trafficking: Mature CFTR protein is trafficked to the Golgi apparatus and then to • the cell surface CFTR Turnover: CFTR channels at the cell surface are eventually removed through the • process of turnover CFTR Function: CFTR at the cell surface functions as a transporter of chloride and other • ions References • Derichs N. Eur Respir Rev 2013;22:58–65 • Lommatzsch ST & Aris R. Semin Respir Crit Care Med 2009;30:531–8 Lukacs GL & Durie PR. N Eng J Med 2003;349:1401–4 • 4
Key Point • CFTR mutations can be grouped based on the type of molecular defect in the CFTR protein Additional Information • Different CFTR mutations cause disruptions at various stages of CFTR protein synthesis, resulting in a reduction in the amount of protein present at the apical membrane or affecting several aspects of the function of the CFTR protein • Mutations that cause the introduction of a premature stop codon result in defective CFTR protein biosynthesis, with no functional CFTR protein expressed at the apical cell membrane (Class I) • Other CFTR mutations, including the most common one, F508del , affect post-translational folding and transport of the CFTR protein to the cell surface, also known as a trafficking defect. These misfolded proteins fail to reach the Golgi apparatus, and the result is that only a small quantity of the misfolded CFTR protein reaches the cell surface (Class II) • Mutations that cause a splicing defect result in CFTR mRNA that is not properly processed. Although some functional protein is produced, the amount of CFTR at the cell surface is decreased in comparison with normal levels (Class V ) • Other mutations affect the function of the CFTR protein, resulting in a CFTR protein that reaches the apical membrane; however, either the channel does not open properly, which is known as a gating defect (class III), or it has impaired chloride movement, also known as conductance defect (Class IV). These mutations result in diminished chloride transport across the cell membrane Class VI mutations do not impair the biogenesis or function of CFTR, but often cause a 70-100 base pair C-terminus • truncation, leading to degradation of the mature CFTR protein at a rate 5-6 times faster than normal As previously described, overall these CF-associated mutations in the CFTR gene can result in the cell surface having • less, an absence of, or dysfunctional CFTR protein • Some mutations may have defects in more than 1 class • A single CFTR mutation can result in multiple defects in the CFTR protein. Any particular defect as a continuum of severity may lead to a range in Total CFTR Activity References • Boyle MP & De Boeck K. Lancet Respir Med 2013;1:158–63 • Wilschanski M et al. J Pediatr 1995;127:705–10 • Ratjen F. Curr Opin Pulm Med 2007;13:541–6 5
MacDonald KD et al. Paediatr Drugs 2007;9:1–10 • • Sheppard DN et al. Nature 1993;362:160–4 • Castellani C et al. J Cyst Fibros 2008;7:179–96 • Rowe SM et al. N Engl J Med 2005;352:1992–2001 • Zielenski J. Respiration 2000;67:117–33 • Rowntree RK & Harris A. Ann Hum Genet 2003;67:471–85 • Welsh MJ & Smith AE. Cell 1993;73:1251–4 5
• Some CFTR mutations produce multiple defects that place them into multiple classes. For example: – The R117H-CFTR mutation is classically thought of as a class IV mutation due to it having low levels of conductance; however, it also displays defective gating placing it into class III 1 – The most common CFTR mutation, F508del , has strong class II defects in processing and trafficking; however, some of the mutated protein is partially active at the cell membrane having defects in surface stability 2 placing it in class VI as well – The E831X mutation results in the introduction of a stop codon and, therefore, it might be categorized as a classic class I mutation. However, alternate splicing of its mRNA has been shown to result in some, albeit reduced, synthesis and therefore, it could also be categorized as a class V mutation 3 – The class system is further limited due to mutation in the same class exhibiting different degrees on the same defect. For example: § Three different class IV mutations show a wide range in channel open probability 1 § Three class II mutations show a wide range in protein levels 4 References 1. Sheppard DN et al. Nature . 1993;362(6416):160-164. 2. Gentzsch M et al. Mol Biol Cell. 2004;15(6):2684-2696. 3. Hinzpeter A et al. PLoS Genet . 2010;6(10):e1001153. 4. Sosnay PR et al. Nature Genetics 2013;45(10)1160-1167. 6
Key Points Total CFTR activity—the total chloride ion transport mediated by CFTR surface channels—is • determined by both CFTR quantity (the number of CFTR channels on the cell surface) and CFTR function (the functional ability of each channel to open and transport chloride) 1 The degree to which the CFTR mutation reduces CFTR quantity and/or function determines the • total CFTR activity 1 Additional Information CFTR quantity is determined by CFTR synthesis ( CFTR gene transcription and mRNA translation), • CFTR trafficking (delivery of normally processed, mature CFTR protein to the cell surface), and CFTR surface stability (normal CFTR channels are eventually removed from the cell surface) 2 • CFTR function is determined by channel open probability (the fraction of time that a specific channel is open and transporting chloride) and channel conductance 2 References 1. Sheppard DN et al. EMBO J. 1995;14(5):876-883. 2. Derichs N. Eur Respir Rev. 2013;22(127):58-65 . 3. Sheppard DN et al. Nature . 1993;362(6416):160-164 7
Key Points • The degree to which the CFTR mutation reduces CFTR quantity and/or function determines the total CFTR activity 1 • A single CFTR mutation can result in multiple defects in the CFTR protein Additional Information • Total CFTR activity—the total chloride ion transport mediated by CFTR surface channels—is determined by both CFTR quantity (the number of CFTR channels on the cell surface) and CFTR function (the functional ability of each channel to open and transport chloride) 1 • CFTR quantity is determined by CFTR synthesis ( CFTR gene transcription and mRNA translation), CFTR trafficking (delivery of normally processed, mature CFTR protein to the cell surface), and CFTR surface stability (normal CFTR channels are eventually removed from the cell surface) 2 • CFTR function is determined by channel open probability (the fraction of time that a specific channel is open and transporting chloride) and channel conductance 2 References 1. Sheppard DN et al. EMBO J. 1995;14(5):876-883. 2. Derichs N. Eur Respir Rev. 2013;22(127):58-65. 8
Key Points • Ussing chambers are an extensively used experimental procedure to measure ion transport across epithelia measured as a voltage 1 • The method consists of the measurement of current (or voltage) between two hemi- chambers separated by a section of epithelium or an epithelial cell monolayer grown on a permeable support 1 • The classic parameter measured in an epithelium mounted Ussing chamber is the “short-circuit”, I SC , that is the current across the epithelium when it is short circuited 1 Additional Information • Fischer rat thyroid (FRT) cells expressing different mutations of CFTR result in lower voltage due to decreased Cl - transport compared with cells expressing wildtype CFTR 2 References 1. Moran O, Zegarra-Moran O. J Cyst Fibros. 2008;7(6):483-494. 2. Li H et al. J Cyst Fibros . 2004;3(Suppl 2):123-126 9
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