Week 8 Part 1 - Cystic Fibrosis Flashcards
Cystic Fibrosis - Overview
Fatal monogenic recessive disease
A congenital disorder
Due to mutations in the gene for the CF transmembrane conductance regulator (CFTR) gene, Cl- channel
Phenotype of Cystic Fibrosis
Classic triad of manifestations includes:
- increased sweat Na and Cl
- pancreatic insufficiency
- pulmonary infections
Thick mucus production an important part of pathophysiology
90% of males are infertile due to congenital bilateral absence of the vas deferens
>90% of deaths due to recurrent infection, most commonly with Pseudomonas aeruginosa
Pathophysiology of Cystic Fibrosis in Lung and GIT
Decreased Cl channel activity
=> change to regulation of ENaC, an epithelial sodium channel -> increased Na and therefore water absorption
=> decreased water content of secretions, mucus secretions becoming thicker and impaired flow, plugs and obstructions
In lung:
=> decreased pericilliary volume
=> thick mucus leads to poor clerance of pathogens and recurrent infections
In gut:
=> blockage of pancreatic ducts
=> loss of water from GIT
=> meconium ileus
=> constipation
=> rectal prolapse
Pathophysiology of Cystic Fibrosis in Sweat Glands
Decreased Cl channel activity
=> change to regulation of ENaC, an epithelial sodium channel -> DECREASED Na and therefore water absorption (vs increased in airways/GIT)
=> increased sweat NaCl concentration
Cystic Fibrosis - Molecular Genetics
CFTR Gene
- in a 250 Kb region on chromosome 7 (region q31-q32)
- 27 exons
- 6.5 Kb transcript
- regulated by a cAMP sensitive protein kinase
CFTR Protein
- 1480 aa membrane protein
- Cl- ion channel expressed in the apical (usually) membrane of exocrine epithelial cells
- member of the ATP binding cassette (ABC) family of transporters
Mutation spectrum
- >1,000 mutations described
- one major mutation in Caucasians, ΔF508, accounts for ~ 70%
Most Common Mutations in Caucasians
ΔF508 - 70%
G542X - 2%
G551D - 2%
621 + 1G>T - 2%
W1282X - 1%
A large % of those with CF will be compound heterozygotes (e.g. ΔF508/ΔF508, ΔF508/W1282X, ΔF508/G542X)
Most Common Mutations in Caucasians - Defects
ΔF508 - Deletion -> misfolded-trafficking defect; not expressed on cell membrane, class II
G542X - truncation, class I
G551D - poor Cl- conduction, class II
621 + 1G>T - abnormal splicing; severe phenotype
W1282X - truncation, class I
Genotype/Phenotypic Correlations
The classic CF clinical phenotype (85-90% of patients) is associated with complete loss of CFTR function as a Cl- channel; and
- pancreatic exocrine insufficiency (birth/early life)
- chronic obstructive pulmonary disease
- abnormal concentrations of sweat electrolytes
- absence of the vas deferens in males
- 20% of cases have an obstructed bowel (meconium ileus)
However, depending on the CFTR mutation inherited, some patients may present with atypical, often mono-symptomatic (mono-organ) forms of CF
The degree of correlation between CFTR genotype and CF phenotype is highest for pancreatic involvement and lowest for pulmonary manifestations of the disease
Genotype/Phenotypic Correlations - Pulmonary Manifestations
Most common cause of morbidity and mortality
But there is a poor correlation with severity of genotype and the age of onset, severity and progression
For example, even among homozygotes for the most common mutation (ΔF508), lung function may vary from normal to severe dysfunction
Several factors may influence the severity of lung pathology including exposure to infectious pathogens, air quality/pollutants/cigarette smoke; nutritional status, and therapeutic regimens
Heterozygote Advantage - Resistance to Diarhoea
By acting on CFTR, toxins released by cholera (common killer throughout history) and other microorganisms, (e.g. Escherichia coli) can cause increased fluid flow in the intestine resulting in diarrhoea
- CF homozygotes fail to secrete chloride ions in response to a variety of stimulants, including bacterial toxins
Animal models
- mice heterozygous for CFTR null alleles show reduced intestinal fluid secretion in response to cholera toxin
Why is DNA Analysis not used as a Front Line Test
Because of the large number of CF mutations
The exact strategy used depends on the clinical context
Clinical and family history is important
Newborn Screening
Measure immunoreactive trypsinogen (IRT), 48-72h after birth
- levels raised in CF
- measured on blood spots (collected from heel prick)
Genotype for common mutations
If have 2 mutations =CF; if no mutations, CF unlikely
If have one mutation, carry out sweat test (test of sweat electrolytes)
CF patients secrete nearly normal volumes of sweat in the sweat acinus
CF patients not able to reabsorb NaCl from sweat, across the ductal epithelial cells, as sweat moves through the sweat duct
Sweat Test Interpretation
Cl 60 mmol/l, cystic fibrosis
Cl 30–59 mmol/l, borderline
Cl 29 mmol/l, normal
Molecular Methods of Testing
Sequencing
- NGS, Sanger sequencing
Kit Methods
- Elucigene CF29v2 multiplex ARMS PCR - detects 29 common CF variants as a first screen
- p.Phe508del confirmed by heteroduplex analysis and other variants by sequencing.
- second level testing involves screening by multiplex ligation-dependent probe amplification (MLPA) for large deletions and duplications of one or more exons, and genomic DNA sequencing of entire gene
Elucigene Method
Based on ARMS
The very basis of allele discrimination is based on the PCR reaction using allele specific PCR
- this is unusual: normally PCR amplification is NOT the basis of the allele discrimination
- normally PCR amplify part of the gene + allele discrimination with an added or complementary technology
Multiplexed-products of different sizes
- discerned by: electrophoresis separation in a EtBr containing agarose gel
Control bands for each of the multiplex reactions
