20.03.08 Cystic Fibrosis

Question 1

Mode of inheritance in CF

Answer 1

Autosomal recessive

Question 2

Prevalence

Answer 2

1 in 2600 affected individuals in UK population

Question 3

Carrier frequency

Answer 3

1 in 25 in UK. Lower in other populations

Question 4

What is CFTR

Answer 4

• Cystic fibrosis transmembrane conductance regulator
• Cyclic AMP-acticated chloride ion channel
• Located in plasma membrane of secretory epithelial cells in sweat ducts, vas deferens, intestine, lungs, kidney, pancreas

Question 5

What phenotypes arise from CFTR mutations

Answer 5

• Classical CF
• Mild CF
• CFTR-related disease

Question 6

What is the most common UK mutation

Answer 6

• p.(Phe508del)

- 75% of mutations in UK

Question 7

What causes CFTR channel to open

Answer 7

R (regulatory) domain is phosphorylated by protein kinase A and when ATP is bound to NBD (nuclear binding domain)

Question 8

Phenotype of CF (remember as CF PANCREAS)

Answer 8

C= chronic cough
F= failure to thrive
P= pancreatic insufficiency
A= alkalosis and hypotonic dehydration
N= neonatal intestinal obstruction (meconium ileus)
C= clubbing of fingers
R= rectal prolapse
E= electrolyte elevation in sweat (salty skin)
A= Absence of vas deferens
S= sputum with staph or pseudomonas

Question 9

What is fetal echogenic bowel

Answer 9

-Bright bowel on antenatal ultrasound imaging

Question 10

What proportion of echogenic bowels are due to CF

Answer 10

3%

Question 11

What are other causes of fetal echogenic bowel

Answer 11

• Fetal aneuploidy
• Intra-amniotic hemorrhage
• infection (Cytomegalovirus CMV)

Question 12

How can mild CF manifest

Answer 12

Pancreatic sufficiency, variable lung disease, lower salt chloride (still higher than normal)

Question 13

How can CFTR-related disease manifest

Answer 13

• Mild dysfunction of one organ system.

- Disseminated bronchiectasis, CBAVD, chronic idiopathic pancreatitis

Question 14

Methods used to detect CF mutations

Answer 14

• ARMS (amplification refactory mutation system)
• OLA (oligonculeotide ligation assay)
• Sanger
• MLPA
• NGS methods

Question 15

What proportion of CFTR mutations are copy number changes

Answer 15

10%

Question 16

What could be a possible reason for finding a homozygous CFTR variant in a non-consanguineous couple

Answer 16

• UPD7

- Deletions

Question 17

What 9 diseases are tested in the newborn screening program in UK

Answer 17

• CF
• Congenital hypothyroidism
• Glutaric aciduria type 1
• Homocystinuria
• Isovaleric acidaemia
• phenylketonuria
• MCADD (medium-chain acyl-CoA dehydrogenasae deficiency)
• Maple syrup disease
• Sickle cell disease

Question 18

How old are babies when blood spots are taken

Answer 18

1) 5 days old

2) 28 days (if first is raised)

Question 19

What is the indicator used in CF newborn screening

Answer 19

• Immunoreactive trypsinogen (IRT)

- Elevated (above 99.5th centile)

Question 20

What is IRT

Answer 20

Precursor to proteolytic enzyme trypsin.

Question 21

Where is IRT produced

Answer 21

Pancreas

Question 22

Where is IRT converted to trypsin

Answer 22

Small intestine

Question 23

Why is IRT elevated in newborns with CF

Answer 23

Mucous plugs block pancreatic ducts, preventing IRT reaching intestine.

Question 24

Other reasons for elevated IRT in newborns

Answer 24

Stressful delivery, premature

Question 25

Which common 4 CFTR mutations are examined in newborn screening

Answer 25

Phe508del, Gly551Asp, Gly542Ter, c.489+1G>T

Question 26

Other biochemical tests for CF

Answer 26

- Sweat test (quantitative pilocarpine iontophoresis). People with CF lose more salt in sweat that unaffected people. Two abnormal readings above 60mmol/L are indicative of CF (90% of CF patients have positive sweat test). - Transepithelial nasal potential difference. Assess ion conductance in upper respiratory epithelium. Measures chloride and sodium ions.

Question 27

Are there genotype phenotype correlations

Answer 27

- Yes - Severe mutations (leading to no CFTR protein, class 1-2) are associated with pancreatic insufficient CF. - Mild mutations (some functional CFTR, class 3-5) result in pancreatic sufficiency, lower sweat chloride levels, less severe respiratory disease.

Question 28

Give example of partial penetrant alleles

Answer 28

-5T allele of intron 8 -c.3850-2477C>T Both alleles lead to leaky splice sites and the proportion of normal and truncated transcripts correlates with disease severity.

Question 29

What is the significance of the intron 8 polyT and poly TG tract

Answer 29

- Length of polyT at the splice acceptor site of intron 8 has an effect on the splicing of exon 9. - 5T allele causes exon 9 to be skipped in 90% of transcripts - 5T with TG12 or TG13 causes increased exon 9 skipping.

Question 30

Which poly T allele is associated with CBAVD when found in trans with a path variant

Answer 30

5T12TG (5T13TG more associated with mild CF)

Question 31

Which allele is modified by the presence of 5T in cis

Answer 31

p.Arg117His

Question 32

What is the significance of R117H/7T when seen in trans with a pathogenic allele

Answer 32

Associated with a variable presentation or can even be benign.

Question 33

What is the significance of 5T/5T

Answer 33

Phenotype is highly variable and more likely to be associated with CFTR-RD

Question 34

When is 5T reflex testing recommended

Answer 34

- Males with infertility caused by obstructive azoospermia - Patients with bronchiectasis/ pancreatitis, where 1 pathogenic mutation is detected - Patients with R117H

Question 35

Which polyT allele is exclusively seen in cis with p.Phe508del

Answer 35

9T

Question 36

What are possible treatments in CF

Answer 36

- Daily airway clearance - Mucolytic enzymes - Antibiotics - Lung transplantation - Pancreatic enzyme replacement therapy (in PI CF) - CFTR modulators - DNA editing (CRISPR-Cas9, to remove mutated CFTR, then homologous recombination with WT) - RNA editing (antisense oligonucleotides to replace deleted segments of RNA)

Question 37

3 types of CFTR modulator drugs

Answer 37

- Read through therapy | - Corrector therapy

Question 38

An example of read through therapy

Answer 38

- Gentamicin. - An aminoglycoside antibiotic that can correct for nonsense mediated decay of truncated CFTR. - However issues with ototoxicity (toxic to ear).

Question 39

An example of corrector therapy

Answer 39

- Lumacaftor - Facilitates maturation of CFTR and delivery to the membrane - Corrects proteins that cause misfolding (e.g. F5008del)

Question 40

An example of potentiator therapy

Answer 40

- Ivacaftor - Improves CFTR channel function - Corrects for proteins that reduce gating efficiency (e.g. G551D)

Question 41

How do aminoglycoside antibiotics reduce the fidelity of translation

Answer 41

By inhibiting ribosomal “proofreading", allows transcription past stop codon to yield full length protein

Question 42

What is orkambi

Answer 42

- A combination of Lumacaftor and Ivacaftor. | - Used for hom F508del patients.