18.03.08 Cystic Fibrosis

Question 1

Give a brief overview of CF including its incidence, carrier frequency and clinical features.

Answer 1

Cystic fibrosis (OMIM #219700) is the most common autosomal recessive inherited disease affecting 1:2600 individuals in UK population

Carrier frequency for CF ~1/25 in the UK population, but is lower in Latin American, African-American and Asian populations

Complex multisystem disorder affecting, pancreatic, pulmonary, gastrointestinal and reproductive systems primarily.

Pathological process arises from mutations in CFTR expressed in the apical membranes of secretory epithelial cells and allows flow of chloride ions across the cell membranes (process also called conductance).

CFTR mutations can cause classical CF, mild CF, or CFTR-related disorders.

Question 2

What is the genomic position and structure of the CFTR gene?

Answer 2

7q31.2

27 exons, 66.1kb transcript and the protein is 1480 amino acids long.

CFTR protein domains: 2 transmembrane domains (TM1/MSD1, TM2/MSD2), 2 nucleotide binding domains (NBD1, NBD2), a regulatory domain (R) that is phosphorylated by protein kinase A.

Question 3

What is the function of the CFTR protein?

Answer 3

Cyclic AMP-activated chloride channel located in the plasma membrane of secretory epithelial cells in the kidney, pancreas, intestine, heart, lungs, vas deferens and sweat ducts.

Regulates several other transport proteins (including inhibition of the epithelial sodium channel, ENaC) and forms large macromolecular signalling complexes that are regulated by molecular switches. These complexes mediate transepithelial salt and water secretion into kidney tubules, pancreatic ducts and the intestine.

The rate of Cl secretion is determined by the number of functional CFTR channels – more channels = higher rate of Cl- secretion.

Channel opening is dependent on binding of protein kinase A to the R domain

Cl- secretion: Cl- ions bind to positively charged residues within transmembrane region to coat the pore. This is followed by repulsion of further Cl- ions by the now negatively charged channel to accelerate movement of Cl- out of the cell.

Question 4

When recalling the clinical features of CF, the mnemonic ‘CF PANCREAS’ is useful. What does this refer to?

Answer 4

Chronic cough and wheezing
Failure to thrive
Pancreatic insufficiency (symptoms of malabsorption like steatorrhea)
Alkalosis and hypotonic dehydration
Neonatal intestinal obstruction (meconium ileus)/ Nasal polyps
Clubbing of fingers/ Chest radiograph with characteristic changes
Rectal prolapse
Electrolyte elevation in sweat, salty skin
Absence or congenital atresia of vas deferens
Sputum with Staph or Pseudomonas (mucoid)

Question 5

What are the features of classical CF?

Answer 5

1. Severe and chronic lung disease: cough and sputum production, colonisation with pathogens associated with CF,
2. Pancreatic insufficiency (PI), recurrent pancreatitis,
3. gastrointestinal complications: up to 20% of neonates meconium ileus at birth, malabsorption, steatorrhoea (fatty stools), deficiencies of fat-soluble vitamins
4. high concentrations of chloride in sweat (≥60 mmol/L), congenital absence of the vas deferens (CBAVD).

Question 6

What is foetal echogenic bowel, when is it identified? What genetic abnormalities could this be caused by? What proportion are attributable to CF?

Answer 6

Speckling in the bowel on 20 week ultrasound can be indicative of CF: 3% of grade 2/3 FEB is due to CF. (FEB is graded from 1 to 3).

CF ascertained by hyperechogenicity have severe PI mutations and most commonly p.Phe508del mutation.

T21 is a differential diagnosis

Question 7

What is the phenotype of mild CF?

Answer 7

Pancreatic sufficiency
Variable lung disease
Lower sweat chloride (still higher than unaffected)

Question 8

What are the features of CFTR-related disease? Give some examples of CFTR-RD.

Answer 8

Might only have mild dysfunction in 1 organ system and might not have elevated sweat chloride levels

e.g. disseminated bronchiectasis, CBAVD, chronic idiopathic pancreatitis and other less common presentations such as allergic bronchopulmonary aspergillosis.

Question 9

What is the most common CFTR mutation in the UK population. What class of CFTR mutation is this?

Answer 9

p.(Phe508del) accounts for 75% of mutations in the UK

Question 10

How many CFTR mutations have been detected?

Answer 10

> 1800

Question 11

What proportion of CFTR mutations do CNVs account for?

Answer 11

10%

Question 12

Which CFTR mutations are included in the newborn screening programme?

Answer 12

1. p.Phe508del
2. p.Gly542X
3. p.Gly551Asp
4. c.489+1G>T

Question 13

Give a brief overview of the pathway for CFTR mutation detection as part of the newborn screening programme.

Answer 13

1. Day 5 blood spot taken - IRT assay
2. IRT > 60ng/ml = repeat in duplicate
3. If >99.5th centile - DNA analysis for four common mutations
4. Two mutations - report as CF suspected
   One mutation - 50 mutation test. Report as carrier or
   suspected if >99.9th centile
   No mutations - If IRT >99th centile, repeat on second
   blood spot, if still >99.9th centile report as CF
   suspected

Question 14

Which biochemical tests can be used in the diagnosis of CF?

Answer 14

1. Analysis of immunoreactive trypsinogen (IRT) at the day 5 Guthrie spot test
2. Sweat test –Gold standard test for confirmation of diagnosis– CFTR is a chloride ion (Cl-) transporter. Individuals with CF will lose more Cl- in sweat than unaffected people. Two abnormal readings above 60 mmol/L are indicative of CF and more than 90% of CF patients will produce abnormal sweat chloride readings.
3. Transepithelial nasal potential difference measurements to assess ion conductance in the upper respiratory epithelium: separately measures transport of sodium (Na+) and chloride ions (Cl-). The status of Na+ and Cl- movement in the upper respiratory tract is thought to reflect that of the lower airways in CF patients. Characteristic traces are observed when different buffered solutions are applied are characteristic of normal and abnormal CFTR function.

Question 15

Describe the genotype-phenotype correlation of pancreatic function in CF.

Answer 15

There is a good genotype/phenotype correlation for pancreatic sufficiency. PI is associated with two class 1, 2 or 3 mutations, and levels of pancreatic function correlate well with CFTR genotype.

Severe mutations associated with pancreatic insufficiency (>95%), diagnosis in infancy, liver disease, high sweat chloride (>60mmol/L) and meconium ileus (~20%)

‘Mild’ mutations (some functional CFTR protein) result in pancreatic sufficiency (70-80% of cases), onset usually after 10 years, no meconium ileus, lower sweat chloride levels and less severe respiratory disease.

An individual with pancreatic sufficiency will have 1 or 2 pancreatic sufficient mutations.

CFTR mutations have also been identified in patients with idiopathic chronic pancreatitis but no respiratory symptoms.

Question 16

What is the clinical significance of the intron 8 poly(T) and poly(TG) tract?

Answer 16

The length of the poly(T) tract at the splice acceptor site of intron 8 has an effect on the splicing of exon 9, which is 90% skipped when the length of the tract is reduced to 5T.

The pathogenicity of the poly(T) tract is also mediated by the length of the adjacent poly(TG) tract, where long TG tracts are more likely associated with disease phenotype than shorter tracts (higher penetrance).

Question 17

Which genes have been shown to modify the phenotype in CF? What is the evidence that modifier genes exist in CF?

Answer 17

Variation in phenotype between monozygotic twins raised in a shared environment suggests the existence of modifier genes

E.g. MBL2 (mannose binding lectin) deficiency is associated with predisposition to early infection with Pseudomonas aeruginosa and subsequently more severe lung disease.

At least 9 genes have been identified from GWAS: ADIPOR2, ENDRA, IFRD1, IL8, MBL2, MRSA, SERPINA1, TCF7L2 and TGFB1. However, not all studies give the same results and the associations are not entirely proven yet.

Question 18

Describe the association between CF mutations and congenital bilateral absence of the vas deferens (CBAVD).

Answer 18

CBAVD frequently carry mutations in CFTR but show no sign of classic CF phenotype.

CBAVD which results in infertility, has one CFTR mutation in >70% of cases, 2 CFTR mutations ~10% of cases.

5T allele is 4-6 fold higher in CBAVD cases than normal or CF cases. Frequently seen as 5T hets with another CFTR mutation. CFTR mutations are also found with (CUAVD) but mutations are found lower frequency than those with CBAVD.

Question 19

What are the current and potential treatments available for patients with CF?

Answer 19

1. Daily airway clearance – physiotherapy and devices for percussion/vibration/ventilation
2. Hypertonic saline: hydrates mucus
3. Mucolytic enzymes therapy to remove mucus from the chest
4. Inhaled antibiotics: antibacterial
5. Oral steroids/ibuprofen/antibiotics: anti-inflammatory
6. Lung transplantation
7. Pancreatic enzyme replacement therapy for exocrine pancreatic insufficiency, e.g. CREON
8. Gene therapy
9. CFTR modulators
10. DNA editing CRISPR-Cas9 - remove mutated CFTR followed by homologous recombination with wild-type
11. RNA editing - antisense oligonucleotides to replace deleted segments

Question 20

Describe the use of mutation-specific therapy in CF patients (CFTR modulators).

Answer 20

Readthrough therapy to correct for nonsense mediated decay of truncated CFTR: Aminoglycosides e.g. gentamicin can readthrough PTCs by inhibiting ribosomal ‘proofreading’: yields full length protein. Problems with ototoxicity from prolonged use. Ataluren readthrough therapy has less side effects.

Corrector therapy e.g. Lumacaftor: Facilitate proper maturation of CFTR and delivery to the membrane. Corrects for mutations that cause misfolding and degradation e.g. F508del

Potentiator therapy e.g. Ivacafor: Improving CFTR channel function to correct for mutations that reduce gating efficiency e.g. G551D

Can be taken in combination

Question 21

Describe class I CFTR mutations, give an example of a mutation in this class and which drug therapy may be appropriate.

Answer 21

Gly542X

No Synthesis (50% mutations). Premature transcription termination leading to unstable truncated transcript or no CFTR expression (no protein). = severe phenotype
Includes: Nonsense mutations (14%), frameshift (22%) or splice mutations (8%) or Abnormal or alternative splicing that result in reduction in the amount of functional CFTR expressed in the membrane

Drugs that permit read through of nonsense mutations:

1. Ataluren (PTC124)- in phase 3 trials although there are doubts regarding the mechanism of action of this drug.
2. Agent geneticin (G418) / Gentamicin- aminoglycoside antibiotic

For splicing:

1. epigallocatechin gallate (EGCG)/tocotrienol increase the level of correctly spliced CFTR transcripts
2. Antisense oligonucleotides

Question 22

Describe class II CFTR mutations, give an example of a mutation in this class and which drug therapy may be appropriate.

Answer 22

p.Phe508del

Block in processing: Protein misfolding and defective maturation leads to retention in the endoplasmic reticulum, which results in protein degradation thereby reducing the amount of CFTR present at the cell surface.

“Correctors” – promote folding leading to expression
Lumacaftor (VX809) (small molecule therapy to target Phe508del mutation) in combination with Ivacaftor being used in phase 3 trials. Results so far show increased lung function.

Question 23

Describe class III CFTR mutations and give an example

Answer 23

G551D

Block in regulation: Reduced capacity to secrete Cl due to defects in channel activation, either affecting ATP binding or phosphorylation of the R domain (severely reduced function of protein).

Potentiators” increase the functional CFTR present at the cell membrane

Ivacaftor- phase 3 trial results showed markedly improved patient lung function, lowered amounts of salt in sweat and patient weight gain. Approved for use in US for patients >6y and trialled in those <6y & 8 additional gating defect variants.

Question 24

Describe class IV CFTR mutations and give an example

Answer 24

p. Arg117His-5T
p. Asp1152His

Altered conductance: Reduced capacity for Cl- conductance across membranes due to mutations within the transmembrane domains (partial function of protein) = milder phenotypes.

Potentiators needed”

1. Flavonoids compounds, e.g., genistein, which act directly on the channel to increase open probability.
2. Ivacaftor now being trialled for patients with Arg117His

Question 25

Describe class V CFTR mutations and give an example

Answer 25

Deletion of the initiation codon (the protein lacks the N-tail required for cytoskeleton anchoring)

Regulation of other ion channels: Mutations that affect the regulation of other ion channels such as ENaC sodium channel and the outwardly rectifying chloride channel (ORCC).

Compounds that enhance CFTR retention / anchoring
1. Hepatocyte growth factor (HGF) an activator of Rac1 Signalling promoters anchoring to the actin cytoskeleton via NHERF1.

Question 26

What is the potential issue with using OLA for mutation detection?

Answer 26

33 mutations including reflex testing for polyT tract. Problems: p.Phe508del & p.Phe508Cys in trans can result in the failure of the normal probe for p.Phe508del to hybridise and ligate giving false p.Phe508del homozygote. p.Ile507Val & p.Ile507del in trans can interfere with the hybridisation of the normal probe for p.Ile507 (I507) leading to a false +ve p.Ile507del (I507del) homozygote.

Question 27

What is the basis for the Devyser CF Kit?

Answer 27

Wild-type and mutant alleles in separate fluorescent multiplexes

CF Core Kit tests for 33 mutations. There are European regional kits e.g. Iberian, Italian and UK. The UK kit includes Chinese and Pakistani mutations.

Question 28

Describe the genotype-phenotype correlation of lung function in CF.

Answer 28

There is a poor genotype/phenotype correlation for pulmonary disease indicating that there might be genetic modifiers in addition to CFTR, affecting clinical outlook.

Pulmonary disease is variable, even amongst patients with the same genotype, but a patient with two class 1 or 2 mutations is likely to have worse respiratory disease and a lower probability of survival than a patient with milder mutations (class 3-5).

Patients with a class 4 or 5 mutation have a later onset of respiratory symptoms as there is some function of CFTR (although less than wild-type CFTR).

Question 29

Describe the genotype-phenotype correlation of digestive function in CF.

Answer 29

Reduced Cl- conductance mutations are associated with digestive symptoms (e.g. diarrhoea).

Question 30

Describe the genotype-phenotype correlation of CBAVD in CF.

Answer 30

CBAVD patients are less likely to carry two severe mutations and will often have one severe and one mild mutation, or two mild mutations.

Question 31

Describe the common TG tract length. Which length is associated with increased skipping in the presence of 5T?

Answer 31

Common TG lengths = 11, 12 or 13.

The TG tract commonly has 11 repeat units (TG11), less commonly TG12 or TG13. Increased skipping of exon 9 is caused by TG12, TG13 and 5T.

Question 32

What phenotypes are associated with TG13/5T and TG12/T5

Answer 32

TG13/T5 associated with atypical CF

TG12/T5 with CBAVD.

Question 33

What is the effect of 5T seen in cis with p.(Arg117His)?

Answer 33

R117H is a class IV mutation; the mutations alters the conductance of the chloride channel resulting in a milder form of CF.

5T is known to alter the expression of the mild CFTR mutation, R117H and is commonly seen in patients with CBAVD.

5T/R117H seen in trans with a severe mutations give a presentation of classical CF with variable severity.

Question 34

What is the effect of 7T seen in cis with p.(Arg117His)?

Answer 34

7T/R117H seen in trans with a severe mutation gives a more variable phenotype and can even be benign.

Question 35

What is the effect of 9T seen in cis with p.(Arg117His)?

Answer 35

9T/Arg117His are very rare and thought to be benign.

Question 36

What is the effect of 5T in trans with another CFTR mutation of 5T/5T?

Answer 36

The phenotype is highly variable and more likely CFTR related disease.

Question 37

What is the relationship between the 9T allele and the p.(Phe508del) patahogenic variant?

Answer 37

Always in cis

Question 38

When is reflex testing for the 5T allele recommended?

Answer 38

1. All males with infertility caused by obstructive azoospermia
2. Patients with bronchiectasis/pancreatitis with only one pathogenic mutation
3. Patients with R117H mutation

Question 39

What is disseminated bronchiectasis? What alleles are seen more frequently in these patients?

Answer 39

Disseminated bronchiectasis is an obstructive pulmonary disorder associated with childhood lung infections also showed more frequent detection of the 5T allele and CFTR mutation.

Question 40

What proportion of pancreatitis patients carry a CFTR mutation?

Answer 40

Pancreatitis which involves atrophy of pancreatic acinar tissue, fibrosis, and inflammation. ~14% carry a CFTR mutation on one allele and ~10% have the T5 allele. Some studies dispute the T5 findings so the role of T5 in pancreatitis is unclear

Question 41

What proportion of chronic rhinosinusitis patients carry a CFTR mutation?

Answer 41

Chronic rhinosinusitis, the inflammation of the sinus epithelium show CFTR mutations in ~7% cases.

Question 42

Briefly describe the use of gene therapy in CF.

Answer 42

Gene therapy: introducing a normal copy of CFTR

Trials of using adenoviral, lentiviral and liposomal vectors containing plasmid DNA encoding CFTR: