Genetics of Cystic Fibrosis

• Cystic fibrosis


  • Defective CF gene
  • Deficient CFTR protein
  • Decreased chloride secretion
  • Altered ionic transport
  • Increased water absorption
  • Abnormal mucus composition
• Ions involved in cystic fibrosis

  • Sodium
  • Chloride
  • CFTR
  • ENaC
• From mutation to phenotype to therapy

  1. Bronchial obstruction

  2. Bacterial infections

  3. Inflammation

  4. Bronchiectasis and lung insufficiency
• Pleiotropic disease

  • Organ involvement: Lung, Gastrointestinal tract, Pancreas, Hepatobiliary tract, Sweat gland, Reproductive tract
  • Phenotype: Abnormal mucus leading to infection and damage, Meconium ileus (neonatal bowel obstruction), Intestinal obstruction, Pancreas dysfunction, Malabsorption, Biliary cirrhosis, Gallstones, Elevated chloride and sodium in sweat, Absence of vas deferens, Thick cervical secretions
• Cystic fibrosis is an autosomal recessive disorder
• Molecular testing is important for prognosis and therapy
• Cystic fibrosis gene


  CFTR gene, located on chromosome 7q31.2, 27 exons, 6 kb cDNA, 1,480 amino acids, over 2,000 pathogenic variants identified
• Cystic fibrosis has a UK prevalence of 1 in 2,500 newborns and a carrier frequency of 1 in 20-25 in North Europe
• F508delFAM test


  Fluorescent PCR amplification of a region of exon 11 of CFTR, an allele with p.F508del will produce a fragment 3bp smaller than a normal allele
• CF4 test


  Tests for 4 mutations, showing homo-/heterozygosity, based on ARMS (amplification refractory mutation system)
• CF-EU2 test


  Tests for 50 mutations, showing homo-/heterozygosity, based on ARMS, primers split into two mixes: "A mix" for all mutations & p.F508 normal allele, "B mix" for all normal alleles
• The most common CF mutation is c.1521_1523delCTT (p.Phe508del), accounting for ~70% of all CF mutations in NW European populations
• Neonatal screening for cystic fibrosis is based on elevated immunoreactive trypsin levels
• Genotype-phenotype correlation in cystic fibrosis

  There is significant clinical heterogeneity and variable phenotype due to different types of mutations in different domains of the gene/protein and gene modifiers
• CFTR protein


  Chloride channel in the apical membrane of secretory cells, has 5 major domains: 2 transmembrane domains, 2 nucleotide binding domains, 1 regulatory domain
• Mutation classes in cystic fibrosis

  • Class I: Defective or reduced protein production (e.g. nonsense, frameshift, splice site mutations)
  • Class II: Defective processing (e.g. p.Phe508del)
  • Class III: Defective gating (e.g. p.Gly551Asp)
  • Class IV: Defective conduction (e.g. p.Arg117His, p.Arg334Trp, p.Arg347Pro)
• Defective processing


  Majority of CF mutations, including p.Phe508del, give rise to a translation product unable to fold in the same way as normal. p.Phe508del CFTR does not escape the ER and trafficking is therefore disrupted, protein does not reach the cell membrane.
• Under certain conditions, p.Phe508del CFTR can reach the cell membrane where it has some residual chloride channel activity.
• Class III


  Defective gating - Many mutations of NBDs, including severe mutations, such as p.Gly551Asp, and less severe mutations, e.g. p.Ala455Glu and p.Pro574His, interfere with binding of ATP or with ATP stimulation, result in a decrease in chloride channel activity
• Class IV


  Defective conduction - Include mutations located in the pore, e.g. p.Arg117His, p.Arg334Trp, p.Arg347Pro, most mutations are in the MSD, normal phosphorylation and ATP dependent regulation, but reduced single channel currents – some mutations reduce the length of time the channel is open.
• Therapy approaches


  • Restore function
  • Restore airway surface liquid
  • Mucus alteration
  • Anti-inflammatory drugs
  • Anti-infective drugs
  • Transplantation
  • Nutrition
• Modulator therapies


  Restore CFTR function - Correctors and potentiator, Potentiator, Corrector and potentiator, Correctors and potentiator, Read-through modulator
• CFTR read-through's


  Protein repair - Ataluren (PTC124) promotes read-through of premature stop codons such as p.Gly542* (class I mutation), ELX-02 also promotes read-through of premature stop codons such as p.Gly542* (class I mutation)
• CTFR correctors


  Protein transport - Lumacaftor (VX809) acts as a chaperone to deliver defective CFTR to the cell membrane, for instance CFTR:p.Phe508del (class II mutation)
• CTFR potentiators


  Protein assistance - Ivacaftor (VX770) acts as a potentiator, shown to increase gating activity, studies carried out using p.Gly551Asp and p.Gly1349Asp (class Ill mutations)
• Combination drugs


  Symkevi ® /Symdeko ® : Tezacaftor and ivacaftor (Corrector & potentiator), Orkambi®: Lumacaftor and ivacaftor (Corrector & potentiator), Kaftrio ® /Trikafta™: Elexacaftor, tezacaftor and ivacaftor (2 correctors & 1 potentiator)
• Protein amplification


  Nesolicator (PTI-428) acts as an amplifier, shown to increase the amount of CFTR protein in the cell
• Other protein strategies


  • Gene supplementation therapy using a correct version of CFTR - transfected or transduced into the respective target cells
  • Transcript supplementation therapy using a correct version of CFTR-mRNA transfected into the respective target cells
  • Protein supplementation therapy using a correct version of CFTR - transfected or transduced into the respective target cells
• Cystic fibrosis is a pleiotropic disease with extensive allelic heterogeneity and some genotype/phenotype correlation.
• Distribution of mutations depends on populations: Only few different mutations account for more than 90% of CF chromosomes in N/W/C Europe.
• Mutations in CFTR can be assigned to distinct functional classes based on their pathomechanism.
• Causal pharmacological therapy is promising - Various drugs, including small molecules, are being developed for specific mutation classes.
• Sodium (Na+)


  An ion that helps regulate water movement in and out of cells. In cystic fibrosis, problems with sodium regulation can lead to thick, sticky mucus in the lungs and other organs.
• ENaC


  A protein that helps regulate sodium levels in the body. In people with cystic fibrosis, problems with ENaC function can contribute to the buildup of thick, sticky mucus in the lungs.
• CFTR


  A protein that acts as a channel for chloride ions to move across cell membranes. In people with cystic fibrosis, mutations in the gene that codes for CFTR can lead to a variety of symptoms, including lung disease, digestive problems, and infertility.
• Chloride (Cl-)


  An ion that plays a key role in regulating water balance in the body. In people with cystic fibrosis, a genetic mutation affects the function of the CFTR protein, which helps regulate the movement of chloride ions in and out of cells.
• CFTR

  A protein that acts as a channel for chloride ions to move across cell membranes. It is primarily found in the lungs, pancreas, and digestive system, where it helps regulate the balance of salt and water in these organs.
• CFTR function in the lungs

  The CFTR protein helps to keep the airways moist and clear of mucus. When it is functioning properly, the CFTR protein allows chloride ions to move out of cells and into the airways, which helps to thin and clear mucus. In people with cystic fibrosis, mutations in the gene that codes for CFTR can lead to a variety of symptoms, including lung disease, digestive problems, and infertility.
• F508delFAM test


  A genetic test used to detect the p.F508del mutation in the CFTR gene using fluorescent PCR amplification and fragment analysis
• Fluorescent PCR amplification


  A laboratory technique used to make many copies of a specific region of DNA, in this case, exon 11 of the CFTR gene