Duncan - Cystic Fibrosis Flashcards
cystic fibrosis - the key facts?
Autosomal recessive
Affects epithelial tissue (not just in the lungs). Classified as a disease of electrolyte transport
1 in 2500 births, 1 in 25 is a carrier
50 yrs ago, most did not survive past one. Now life expectancy = 50
what organs are impacted and how?
give a time point for when this symptom typically becomes apparent
Airways - clogging, inc, in thick mucus, resulting in high risk of infection (one month)
Liver - blockage of small bile ducts, causes problems in liver function (5% of cases) (1-2 yrs)
Pancreas - pancreatic insufficiency - blockage of ducts prevents proper secretion of digestive enzymes, seen in 65% of cases. Inability to absorb all nutrients from diet, hard to maintain body weight (used to spot in babies before establishment of the Gunthrie test) (1 week - 1 month)
Small intestine - obstruction due to thick content, seen in 10% of cases of newborns - require surgery to survive. Known as meconium ileus (very quick)
Reproductive tract - don’t have a vas deferens (95 % of male cases) it doesn’t develop, so males have obstructive azoospermia (no sperm in semen). Very small % of females are also infertile potentially due to mucus in cervix too thick
Skin - excess secretion of Na+ and Cl- in sweat glands. Note there are other symptoms, these are the main ones
what is the function of the CFTR gene?
forms chloride channel in membrane of secretory epithelial cells
ENac is the Sodium ion channel
These cells produce mucus, sweat, saliva, tears, & digestive enzymes
CFTR Transports negatively chloride ions out of cells, Enac controls movement of Na+ into cells
Together control the movement of water from cells and the secretions (which together form mucus)
necessary for production of thin, freely flowing mucus,
which is normally good - lubricates & protects the lining of the airways, digestive system, reproductive system
in CF - focusing on the lungs - what is the issue caused by defective CFTR?
Cl- not being secreted means water doesn’t follow, so
Mucus is thicker and the PCL layer (periciliary) is of a smaller height
this…
Makes cilia bent, so they cannot effectively ‘beat’ mucus up to be swallowed, so bacteria/viruses etc… are stuck in the lungs (infections)
CFTR not functioning = it doesn’t interact with and inhibit ENaC so Na+ is moving into the cell more, so not only is water not entering the PCL, it is actually leaving it and going in the opposite direction, exacerbating said effects
what test can be done for CF that isn’t genetic?
Sweat chloride test
Induce sweating with pilocarpine iontophoresis, collect sweat using filter paper, measure Cl- concentration.
Diagnostic cut off is 60mM (above it = CF, tho some males with no vas deferens can be just below)
what is CF like in carriers?
Carriers have NO symptoms despite having 50% of the normal functional protein levels of CF gene products.
Studies have shown if you get around 15-20% WT function of the protein, you will relieve symptoms of CF
outline how quick results for CF can be, depending on the reason for referral/testing
5 different ones to get
reason for referral = Recurrent chest infection
Failure to Thrive
Chronic pancreatitis
then you get results in 2 weeks, 1 week for neonates
if foetal echogenic bowel (meconium ileus) is seen - 3 days for results
reason for testing is family history/want to find out if you’re a carrier - 2 weeks, but 1 week if a couple is already pregnant and want a predictive test (on them to see if they are carriers still)
its only 3 days for prenatal tests (i.e. on the foetus)
if infertility is the cause - 2 weeks
all new-borns are screened for CF once born.
explain how and how long it takes to get results form genetic testing if they get a positive result
they test for raised IRT on NBS - newborn blood spot.
Immunoreactive trypsinogen (IRT) is an enzyme precursor produced by the pancreas.
In CF, pancreatic ducts can be obstructed due to thick mucus, leading to leakage of trypsinogen into the blood
if positive, resultant genetic testing takes 5-6 days
the CFTR gene mutations involved in CF -
is it just a few mutations/one region of the channel?
what is the most common mutation?
no, there are more than 2000 mutations throughout it’s 24 exons associated with CF
Most common = p.Phe508del (70% of variants). So its a 3-nucleotide deletion
NOTE - MUTATION FREQUENCIES VARY BETWEEN POPULATIONS
(according to Duncan) what are the 5 classes of CFTR mutation?
I - impaired production - (NMD) mRNA is degraded in quality control
II - impaired trafficking - mRNA and protein made, quality control detects misfolding and sends channel for degradation instead of to the membrane. ΔF508 is class II
Reduced open probability (PO)
III - impaired gating (G551D is an example) so this means impaired open probability. If the channel cannot open in response to signalling molecules like cAMP, its open probability is low
Reduced conductance (G)
IV - decreased conductance - smaller number of ions can move through the pore per unit time. So they’re made and can open, but have reduced function
V - you get less/insufficient protein from the mRNA than normal
What’s the key distinction between classes I-III and classes IV-V CFTR mutations?
Classes I-III: classic CF (e.g. ∆F508) No functional protein
Classes IV-V: mild CF (e.g. R117H) Some functional protein
there are two other classes of mutation Louise taught us, what are they?
VI - decreased membrane stability - CFTR is made, trafficked, and functions, but leaves the membrane to quickly, so there is a smaller n number at any given time
VII - impaired production - mRNA produced but not the full length (so protein isn’t full length)
explain two possible reason for why CFTR mutations are seen in such high frequencies in heterozyogtes
- People are perfectly healthy/don’t have any negative symptoms
- Might have an advantage - Overstimulation of CFTR in intestinal epithelial cells by bacterial toxins leads to secretory diarrhoea, as Cl- leaves cells, water follows (i.e. too much CFTR activity also cause adverse effects)
…So less active CFTR = possible selective advantage, because CFTR variants cause thicker mucus which may provide some protection from secretory diarrhoea
Secretory diarrhoea much larger world health problem: 3 million deaths per year of children the age of 5
what genetic test is used to identify CF variants?
- name and what it tests for
CF-EU2V1 ARMS ASSAY, which uses fluorescent ARMS PCR (amplification refractory mutation system)
Tests for 50 common mutations in the CFTR gene associated with cystic fibrosis
how does ARMS-PCR work?
don’t include what the results look like
Primers: Mutation-specific AND wild-type primers target CFTR gene regions.
PCR Amplification: Only DNA with complementary primers (mutant or wild-type) is amplified. Primer sequence is specific for variant, if variant not present, the primer doesn’t bind and you get no PCR product
Fluorescent Probes:
PCR products are labelled with unique fluorescent signals. So each mutant (and WT) peak can be distinguished.
Detection:
Real-time fluorescence measurement identifies mutations due to unique fluorescent probe
Each mutation produces a specific signal, enabling identification…
