Cystic Fibrosis Past Paper PDF

Cystic fibrosis Clinical manifestations Pathophysiology Genetics Targeted therapy Cystic fibrosis (CF) AR inheritance Most common lethal AR disease in Europe, affecting 1 in 3000-6000 live births Multisystem disease affecting exocrine glands; production of viscous mucu...

Cystic fibrosis Clinical manifestations Pathophysiology Genetics Targeted therapy Cystic fibrosis (CF) AR inheritance Most common lethal AR disease in Europe, affecting 1 in 3000-6000 live births Multisystem disease affecting exocrine glands; production of viscous mucus → obstruction, chronic inflammation Main features: progressive lung disease, pancreatic insufficiency, malnutrition, susceptibility to infections Phenotypic variability (affected organs, severity, age of onset) Genetic features: heterozygote advantage allelic heterogeneity variable expressivity allelic disorders genetic modifiers Ratjen et al Nat Rev Dis Primers. 2015;14;1:15010 Clinical manifestations Classical CF: severe phenotype multiple organs affected Nonclassical /atypical CF milder phenotype affects 1 or 2 organs normal pancreatic function Ratjen et al Nat Rev Dis Primers. 2015;14;1:15010 Clinical manifestations Chronic sinusitis: long-lasting sinus inflammation and infection Pancreatic insufficiency: deficiency in the production of pancreatic enzymes → compromises ability to digest food → malnutrition and impaired growth CF-related diabetes: reduced insulin release and elevated blood sugar (40-50% of adult patients) Obstructive lung disease: airflow blockage causing difficulty breathing; deterioration of pulmonary capacity → cause of death Liver disease: obstruction of bile ducts, inflammation, fibrosis Meconium ileus: blockage of terminal ileum by dense meconium (first feces of newborn) → life-threatening Congenital bilateral absence of vas deferens (CBAVD): vas deferens fail to form before birth → sterility (97-98% of male patients) Pancreatitis: inflammation of pancreas Ratjen et al Nat Rev Dis Primers. 2015;14;1:15010 Neonatal Screening Worldwide implementation of cystic fibrosis newborn screening as of 2020 Mandated by law in many countries: Italy - Ministero della Salute: Articolo 6, legge quadro 5 febbraio 1992, n.104 Radioimmunologic test serum immunoreactive trypsin (IRT) CF → high IRT levels Scotet et al. Int J Neonatal Screen. 2020 Mar 4;6(1):18. In case of positive IRT test: sweat test DNA test Clinical diagnosis of CF using the sweat test Sweat glands Sweat test: measurement of Cl- and Na+ in sweat following stimulation with pilocarpine (muscarinic agonist). CF: [Cl-] > 60 mM Na, Cl concentration (mM) CF normale  Cystic fibrosis conductance regulator (CFTR) Gene: CFTR, locus 7q31.2 Protein: CFTR anion channel for Cl- and HCO3- expressed on apical surface of epithelial cells of exocrine organs: respiratory tract, pancreatic duct, GI tract, sweat glands, salivary gland, reproductive tract, bile duct member of ABC transporter family (ATP-binding cassette protein), consisting of: 2 membrane spanning domains (MSD1 and MSD2), each containing 6 transmembrane segments, forming the channel pore 2 intracellular nucleotide-binding domains (NBD 1 and NBD2) that bind ATP 1 intracellular regulatory domain (R domain) with phosphorylation sites Ratjen et al Nat Rev Dis Primers. 2015;14;1:15010 CFTR is a Cl- channel activated by phosphorylation a. Closed channel b. Open channel Cl- out out in in dominio R R domain N ATP ATP ATP ATP N O O ADP ADP O P ADP ADP O- O- O P O O cAMP-dependent C protein kinase A dominio R R domain O C -O O O P P O O -O O CFTR is normally closed CFTR opens following activation via cAMP signaling pathway: ligand binding to membrane receptor (e.g., muscarinic or purinergic receptor) adenylate cyclase activation elevates cAMP → activates protein kinase A (PKA) PKA phosphorylates CFTR regulatory domain through ATP hydrolysis → conformational change leading to pore opening and chloride flux CFTR in the respiratory tract Bronchial epithelium Normal Cystic fibrosis Normal CFTR Mutant CFTR: localized on apical membrane absent or dysfunctional CFTR mediates Cl- efflux no Cl- efflux inhibitory effect on Na+ channels → enhanced Na+ and H2O reabsorption limits Na+ reabsorption dehydration and acidification of airway Green = cilia hydration of airway surface liquid surface liquid Orange = CFTR Blue = nuclei mucociliary clearance impaired mucociliary clearance  Pathophysiological cascade of respiratory disorder in cystic fibrosis Lopes-Pacheco Front. Pharmacol., 05 September 2016  CFTR variants >2000 variants, of which ~380 CF-causing variants Most frequent variant = F508del (a.k.a. ΔF508) deletion of Phe at aa 508 accounts for 50-70% of all CF alleles >80% of CF patients carry at least one copy CFTR variants CF genotypes Lopes-Pacheco. Front Pharmacol. 2020;10:1662  Global distribution of CF patients - prevalence per 100,000 habitants. Global distribution of F508del allele - % CF patients bearing at least 1 allele. Lopes-Pacheco. Front Pharmacol. 2020;10:1662 Italian CFTR mutations Mutations Campania Basilicata Puglia Northern Italy DF508 55.6 55.8 46.8 47.6 N1303K, G542X, W1282X, 1717-1G>A, R553X 18.6 13.4 18.1 10.1 4382delA, 1259insA, I502T, G1349D 0.3 0 10.5 0 4016insT, G1244E, R1158X, 711+1G>T, 7.2 3.8 1.7 0 L1065P 852del22 0 5.8 0.6 0 2183AA>G, R1162X 2.3 1.9 1.1 19.1 Other 6.6 7.6 6.0 13.6 Carrier frequency Genotype frequencies according to Hardy-Weinberg equilibrium f(AA) = p2 f(Aa) = 2pq f(aa) = q2 Cystic fibrosis occurs in 1 in 2500 live births f(aa) = q2 = 1/2500 f(a) = q = √(1/2500) = 1/50 We know that p+q = 1, therefore p=1-q f(A) = p = 1-1/50 = 49/50 ≈ 1 f(Aa) = 2pq = 2 x 1 x 1/50 = 1/25 → frequency of CF carriers = 1/25 Heterozygote advantage: protection against infectious diseases Resistance to cholera Resistance to typhoid fever Bacterium Vibrio Cholerae Bacterium salmonella typhi → fever, → severe diarrhoea → dehydration headache stomach pain Cholera toxin stimulates cAMP signalling → Bacterium invades gastrointestinal cells by CFTR activation → Cl- and water loss attaching to normal CFTR protein Mutant CFTR allele limits Cl- and water loss Mutant CFTR allele protects against infection Classification of pathogenic CFTR variants CFTR function Minimal or none Residual Phenotype SEVERE MILD Classical CF Nonclassical/atypical CF Pancreatic insufficieny Pancreatic sufficiency Modified from Quintana-Gallego et al., Arch Bronconeumol.2014;50:146-50 Genotype-phenotype correlation Pancreatic function: good correlation presence of 1 or 2 mild alleles (class 4, 5, 6) → pancreatic sufficiency presence of 2 severe alleles (class 1, 2. 3) → pancreatic insufficiency Pulmonary disease: poor correlation variability in severity and age of onset among individuals with same CFTR genotype, even within same family Genetic factors contibuting to phenotypic variability Phe508del Phe508del Combination of CFTR variants in trans Phe508del Ala455Glu more severe less severe Arg117His 5T Arg117His 7T Combination of CFTR variants in cis Phe508del Phe508del  CFTR common variants Allele Poly(T) tract frequencies Poly(T) tract located in intron 8; length affects splicing of exon 9 3 common poly(T) variants in the general population: T9, T7, T5 5T variant → loss of exon 9 → reduced quatity of funtional CFTR Clautres, RBMOnline 10; 14-41 (2015) CFTR-related disorders Clinical entities associated with CFTR dysfunction that do not fulfill diagnostic criteria for CF (allelic disorders): Congenital bilateral absence of the vas deferens (CBAVD) male infertility, alone or with mild CF features (e.g., slightly elevated sweat Cl- levels) accounts for 1-2% of all male infertility 80% of affected individuals carry CFTR variants 1 or 2 mild CF alleles + T5 variant Recurrent idiopathic pancreatitis: 10-20% present CFTR variants distinct CFTR variants affecting HCO3- transport in pancreatic ducts, but not Cl- transport Bronchiectasis: rare presentation in carriers unclear correlation with CFTR genotype Correlation of CFTR function with sweat chloride levels and clinical phenotype Bradbury (2016) https://www.researchgate.net/publication/301265898 CF modifier genes Variants in modifier genes contribute to variable severity of clinical manifestations in patients with same CFTR genotype Paranjapye. J Cyst Fibros. 2020;19(Suppl 1):S10-S14 Therapeutic strategies Symptomatic drug therapy antibiotics (infections) glucocorticoids (bronchial inflammation) bronchodilators and mucolytics (bronchial obstruction) pancreatic enzymes (malnutrition) Genetic gene therapy - direct delivery of CFTR gene to airways by inhalation; limited effectiveness in clinical trials; none currently approved genome editing to correct defective gene; still at in vitro research stage (e.g. CRISPR/cas9) Targeted/personalized drug therapy CFTR modulators to improve expression/function of defective protein based on molecular defect Strategies to tailor CF therapy to the underlying molecular defect Lopes-Pacheco Front. Pharmacol., 05 September 2016 https://doi.org/10.3389/fphar.2016.00275 CFTR modulators in development POTENTIATOR Potentiators increase the opening time of the CFTR channel resulting in higher ion flow. Correctors facilitate the processing of mutated CORRECTOR CFTR protein leading to improved delivery to cell membrane AMPLIFIER Amplifiers increase the amount of functional CFTR e.g., read-through agents to promote read-through of premature stop codons in CFTR mRNA Brodlie et al., Genome Medicine 2015 Vertex Pharmaceuticals (https://www.vrtx.com) CFTR potentiators: to potentiate channel activity Ivacaftor (VX-770) CFTR correctors: to correct defective folding and trafficking Lumacaftor (VX-809) Tezacaftor (VX-661) Elaxacaftor (VX-445) Ivacaftor (VX-770): in vitro studies in CF cell models Cell-based assays transepithelial -currents (Ussing chamber) CFTR gating (patch clamp) airway surface liquid volume ciliary beating (microscopy) CF cell models VX-770 identified by high throughput cells expressing recombinant CFTR screening of 228000 chemicals human bronchial epithelial cells from CF patients CFTR mutations G551D: class III mutation, defective channel gating DF508: class II mutation, defective trafficking Van Goor et al. Proc Natl Acad Sci USA. 2009; 106(44): 18825–18830.  Ivacaftor (VX-770) acts as a CFTR potentiator to increase Cl- transport Ussing chamber Fig 1: Transepithelial chloride transport cells expressing recombinant G551D or F508del CFTR note: F508del-CFTR cells incubated at 27oC O/N to improve cell surface density of mutant channels cells stimulated with forskolin (adenylate cyclase activator) to activate CFTR G551D-FRT cells G551D-FRT cells → Ivacaftor does not directly activate CFTR but potentiates activation by forskolin → greater effect against G551D versus F508del CFTR Van Goor et al. Proc Natl Acad Sci USA. 2009; 106(44): 18825–18830. Ivacaftor (VX-770) acts as a CFTR potentiator to increase Cl- transport Ussing chamber Fig 3: Transepithelial chloride transport bronchial epithelial cells from CF patients bearing G551D/F508del CFTR or homozygous F508del CFTR amiloride to block Na currents forskolin to activate CFTR → Ivacaftor is more effective in bronchial epithelium from patients bearing G551D/ F508del CFTR compared to homozygous F508del CFTR Van Goor et al. Proc Natl Acad Sci USA. 2009; 106(44): 18825–18830.  Ivacaftor (VX-770) improves hydration of airway surface and ciliary beating Figure 5 bronchial epithelial cells from CF 100 ml fuid patients patients bearing G551D/F508del CFTR stimulation of cAMP pathway by VIP to activate CFTR VIP (A-B) Volume of airway surface liquid (ASL) 100ul fluid deposited on surface; at regular interval, fluid is absorbed onto filter paper, and filter paper weighed microscope objective (D-E) Ciliary beating frequency light intensity of ciliary surface is monitored by microscopy VIP Van Goor et al. Proc Natl Acad Sci USA. 2009; 106(44): 18825–18830. Ivacaftor improves lung function in CF patients bearing G551D CFTR Clinical trial in CF patients aged >12 yrs with at least 1 copy of G551D mutation 48 wk treatment with placebo or ivacaftor Measurement of sweat chloride, lung function (FEV1) and body weight Ramsey et al. N Engl J Med 2011; 365:1663-1672 → significant reduction in sweat Cl- levels, and improvement in both pulmonary function and body weight compared with placebo Lumacaftor (VX-809): in vitro studies in F508del CF cell models CF cell models bronchial epithelium from CF patients homozygous for F508del CFTR cells expressing recombinant F508del CFTR Cell-based assays Immunoblotting to evaluate protein maturation Ussing chambers to measure transepithelial chroide transport Van Goor et al., Proc Natl Acad Sci U S A. 2011;108(46):18843-8. Lumacaftor increases F508del CFTR maturation and chloride secretion A-B Glycosylation pattern of CFTR (Western blotting) C-D Transepithelial currents due to chloride secretion Van Goor et al., Proc Natl Acad Sci U S A. 2011;108(46):18843-8. Lumacaftor (VX-809) is additive with the CFTR potentiator VX-770, and is more effective than other CFTR correctors Figure 3 G-H Transepithelial currents due to chloride secretion (Ussing chamber) Lumacaftor (VX-809) is selective for correction of CFTR processing Figure 4 VX-809 does not improve the maturation of non-CFTR misfolded proteins A. Normal hERG (top), mutant hERG (bottom) B. Normal Pgp (top), mutant Pgp (bottom) In contrast, VRT-325 and Corr-4a are non- selective correctors Lumacaftor-ivacaftor clinical trial in CF patients CF patients homozygous for F508del CFTR 24 wk treatment with combined lumacaftor- → Combined treatment with lumacaftor and ivacaftor or placebo ivacaftor improves lung function in CF patients LUM (600 mg/day)-IVA bearing homozygous F508del CFTR LUM(400 mg/12h)-IVA Wainwright et al. N Engl J Med. 2015 Jul 16;373(3):220-31. Trikafta: triple combination therapy for CF patients with at least one copy of F508del mutation Triple combination therapy: 2 correctors (Tezacaftor and Elexacaftor) to improve CFTR maturation 1 potentiator (Ivacaftor) to enhance CFTR channel opening Suitable for >90% of patients with CF Bear. Cell. 2020 Jan 23;180(2):211 Trikafta combination therapy improves CFTR maturation and chloride transport in vitro Bronchial epithelial cells from CF patients bearing F508del CFTR left: heterozygous F508del + minimal function allele right: homozygous F508del Treatment: tezacaftor (corrector), ivacaftor (potentiator) and VX-445 (corrector) Results: increased protein maturation and chloride transport, with greatest effect in cells bearing homozygous F508del CFTR double corrector treatment → greatest effect on protein maturation triple combination treatment → greatest effect on chloride transport Keating et al., N Engl J Med 2018; 379:1612-1620 Trikafta combination therapy improves lung function and decreases sweat chloride levels in vivo Clinical trial in CF patients aged > 18years left: heterozygous F508del + minimal function allele right: homozygous F508del CFTR Keating et al., N Engl J Med 2018; 379:1612-1620 FDA-approved CFTR modulators Table modified from Ridley & Condren. J Pediatr Pharmacol Ther. 2020;25(3):192-197. Trikafta: 2020 approved by EMA, marketed in Europe as Kaftrio; 2022 approved by AIFA for prescription under the Italian public health service Simulation model to predict log-term health outcomes of Trikafta treatment: “ … the median projected survival for patients with CF initiating ELX/TEZ/IVA between the ages of 12 and 17 years was 82.5 years, an increase of 45.4 years compared with best standard care alone.”  2024 Breakthrough Prize awarded to Sabine Hadida, Paul Negulescu and Fredrick Van Goor for the development of Trikafta Further reading Available on Virtuale Ratjen et al., 2015. Cystic fibrosis. Nat Rev Dis Primers;1:15010. Cutting, 2015. Cystic fibrosis genetics: from molecular understanding to clinical application. Nature Reviews Genetics 16: 45–56. Lopes-Pacheco. CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis. Front Pharmacol. 2016 Sep 5;7:275. Other sources: Gene Reviews Cystic Fibrosis and Congenital Absence of the Vas Deferens. https://www.ncbi.nlm.nih.gov/books/NBK1250/