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…
since I refuse to get pro, describe what the results from an ARMS assay looks like (hetero vs homozygous…)
a load of green peaks show, these are from the WT primers and indicate WT sequences in the sample
red peaks are simply controls added
any blue peaks indicate pathogenic variants. A delta508 WT probe is located on the pathogenic variant plot as a reference/control
heterozygous = there should be a green and blue peak at the same position
homozygous = just a blue peak.
treatment for CF -
it has massively improved because of…
treatments aimed at respiratory issues include…
Physiotherapy
– Oral, inhaled or IV antibiotics
– Bronchodilators
– Mucolytic agents to reduce
mucus viscosity
– Anti-inflammatory agents
– Home oxygen therapy
– Lung or heart-lung transplantation
– Lifestyle (exercise)
CF -
name some treatments aimed at GI and infertility symptoms
GI -
high calorie and fat diet
supplemental feeding
pancreatic enzyme replacement therapy
infertility -
MESA (microscopic epididymal sperm aspiration) and ICSI
in terms of molecular therapies for CF, what are the three categories of drug (with an example for each one)?
Read through agents - force production of full length CFTR when the mRNA has a premature stop codon/ nonsense mutation
e.g. aminoglycoside antibiotics (gentamicin)
Correctors - force mutant CFTR protein to the cell membrane, if the mutant is functional then Cl- secretion is restored (Lumacaftor)
Potentiators - increase the Po (open probability) of the CFTR channels (which must be trafficked normally). The G551D mutation is a target for the potentiator VX-770, or Ivacaftor
Cystic Fibrosis treatment - what is Orakambi?
Combination drug made up from ivacaftor and lumacaftor –
Effective for people homozygous for F508del (50% of people with CF in the UK).
It is licensed for people over the age of 2.
Slows decline of lung function by 42%
give a statistic that shows that CF treatment has improved
Adult CF population surpassed paediatric population in roughly 2012
what is bronchiectasis caused by in CF?
Persistent and progressive dilation of bronchi or bronchioles in lungs
result of thick mucus blocking airways leading to chronic obstructive airway disease
who is doing pretty fucking amazing?
you bby gorl
karyotyping - briefly how does it work?
what do you do with the karyotype?
how good is it?
How it works - Trypsin produces dark and light bands. Dark - AT rich, late replicating, gene poor, heterochromatic regions. Light - GC rich, early replicating, gene rich, euchromatic regions
What you do - Analysis Count and pair up chromosomes, match up bands, look for missing or extra chromosome / bands
How good it is - 5-20 Megabase resolution which is pretty low, and 5 is still hard to spot. It does show the whole genome but low resolution means its best for spotting aneuploidy (or very large changes in CN)
overview of FISH -
what do you do method-wise?
How good is it?
Overview - uses ssDNA probes that are complementary to target sequences, labelled with fluorescent protein (fluorochrome)
What you do - denature your sample (to part strands enough to allow probe to get access to complementary sequence). Hybridise sample with probes at 37 degrees. Wash to remove background signal due to non-specific binding.
Apply counterstain to visualise the actual chromosomes, so you can see where the fluorescence is when analysed with fluorescent microscope
Higher resolution than karyotyping - large abnormalities >3-5Mb, and can detect balanced changes. 10% mosaicism detection. ~5% of patients diagnosed
what types of probes (5) can be used in FISH?
Whole Chr paints - a whole bunch of probes all across the chromosome, can be helpful in karyotyping abnormalities, like translocations where a bit of one chromosome shows up attached to another
Aneuvysion = used in prenatal testing, centromeric probes used in order to count how many copies there are of a chromosome, to detect aneuploidies like DS
Locus specific - probes used to detect specific syndromes, e,g, williams syndrome caused by deletion of ELN gene. Centromeric probe shows two CHr 7s, but only one of them is showing the ELN gene = William’s syndrome
Cancer - amplification - Looks really messy like background fluorescence. Can be used to identify a certain gene that’s been amplified - duplicated again and again to form a ‘homogenous staining region’, a continuous stretch of amplified DNA integrated into a chromosome, lacking normal banding pattern. Also shows double minutes - dots of DNA fragments all over the cell
Fusion - probes designed to hybridise together in a normal situation or an abnormal situation, e.g. see if a gene is broken a part or if genes are fused together, like BCR/ABL
MICROARRAYS -
what are they used to detect?
what is there resolution like?
what is their biggest downfall?
Used to detect CNVs (CNV can refer to different number of chromosomes, genes, exons etc…)
Higher resolution than karyotyping and FISH, so detects a lot more genetic abnormalities. (if referred due to developmental delay -)Karyotype = 5% diagnosis, array = 20%
Biggest con - cannot detect balanced rearrangements/translocations
microarrays are used in cases of…?
Pretty much replaced karyotyping, one of the first tests for children with developmental delay (Autism, Intellectual disability, facial dysmorphism, congenital abnormalities (e.g., heart defects).
Prenatal Analysis:
fetal abnormalities detected on ultrasound, e.g. structural heart defects, skeletal abnormalities.
Effective for large genomic changes containing multiple genes; less effective for single-gene disorders.
Applications in Cancer -
Limited Use, Specific to certain conditions like Myelodysplastic Syndrome caused by common copy number changes
Acute Myeloid Leukaemia: Less effective as arrays cannot detect gene fusions (balanced translocations)
how does an oligonucleotide array work?
Patient and control DNA is labelled with different fluorescence
The DNA’s are fragmented to short DNA sequences and together they are hybridised to the oligonucleotide array.
The amount of patient DNA versus control DNA bound to an oligonucleotide array is measured by the relative intensity of fluorescence.
If the patient DNA is present in the same quantity as the control DNA at a given oligonucleotide, relative fluorescence is the same, 1:1. indicates (at the location the oligo probe is specific for) the patient is normal disomic.
More patient DNA relative to the control DNA, as in the fluorescence for the patient DNA is stronger by half, ratio of 1 to 2, indicates patient has a gain of genetic material.
Less patient DNA relative to the control, 2:1, patient has a loss of genetic material
SNP microarray - explain the terms involved.
How does the actual array work?
SNP = harmless, 10 million in the human genome, points with single diff. base
You’ve got SNP A (WT, most commonly seen) or B (variant/polymorphism)
You are homozygous, AA or BB, or heterozygous AB
The actual array -
Similar process but its the probes that fluoresce and you don’t need control DNA
Use ~25 nucleotide-long probes, 900,000 for different SNPs all attached to the array chip. These are probes for both alleles A and B. they are labelled with different fluorescence
Amplify & digest/fragment patient DNA, hybridise to array
Image scanning, identify which SNP fluoresces, A, B or both
in a SNP microarray, what do the results look like?
loads of tiny dots
Each dot = a SNP
For each SNP - you can get fluorescence from both the A and B alleles, heterozygous AB genotype with a 1:1 ratio of fluorescence, seen along the middle band
Or the SNP may be homozygous for A or B, and therefore the relative fluorescence will be skewed in favour of A or B. The log ratio of relative fluorescence is therefore at the extremes, 0 or 1
Plotted in order they occur on chromosome so you can look at neighbouring SNPs together
what does a deletion look like in a SNP microarray?
what does a duplication look like?
deletion - you lose one of your alleles along an entire region, so no heterozygous calls, only homozygous
duplication - (in the region of the duplication) you’ve got an extra allele, so you can still have either extreme (ratio of 1 or zero, all A or all B) BUT never just AB, you get these FOUR regions of calls all with different relative fluorescence (AAA, BBB, AAB, BBA)
give me 5 questions you would be trying to answer to determine how ‘bad’ a CNV is
Are genes included in gain or loss? (could be intronic) - how many? The more that are = more likely pathogenic
Is it in a known syndromic location? (makes sense, also use of arrays has identified a lot more syndromic locations)
Do genes fit with referral reasons? (can be hard to answer if the variant involves 100s of genes, but if it’s that many it’s very unlikely it’s not pathogenic)
Is it seen in the normal population? (If the CNV is found in more than 1% of the population we can dismiss it as disease causing)
Do parents have the same change? (if yes and parents have same phenotype - likely to be causative, if parent has it but no phenotype = less likely to be pathogenic, if de novo - likely pathogenic)
does a parent having the same CNV as the child mean the parent must have the same symptoms as the child?
No, its not always this clear cut. this is due to ‘penetrance’.
Penetrance tends to be expressed as a percentage of times a variant would be expected to cause disease. For example, 100% penetrance means that all carriers would have the disease. Whilst 50% penetrance would mean that only 50% would be expected to have the disease.
For rare genetic variants we cannot predict the percentage penetrance, but we must be aware of the possibility of low penetrance when considering whether an inherited variant is pathogenic
QF-PCR
what is the purpose?
give an overview of how the process works?
Detects aneuploidy in prenatal testing, specifically used for Down syndrome as an urgent 24-hour test for DNA from amniotic fluid or chorionic villus samples
Amplification & Quantification:
Uses fluorescently labeled primers, targeting Small Tandem Repeats (STRs, DNA sequences with repeats of 2 or more nucleotides e.g. 10 repeats of “GAC”).
Number of repeats varies between individuals (alleles).
Maternal allele = 5 repeats.
Paternal allele = 9 repeats.
Measures the relative copy number of each allele via fluorescence.
Visualized as peaks on a graph (alleles have different sized STRs so give different peaks)
explain how to interpret the results of QF-PCR
Normal/Disomy (2 copies):
Two alleles (peaks) in a 1:1 ratio.
Equal amplification of both alleles.
Trisomy (3 copies):
1:1:1 ratio: Three peaks (three alleles with different STR sizes).
2:1 ratio: Two peaks (one allele amplified twice gives a large peak, one allele amplified once).
1 peak - Uninformative Results:
A single peak can indicate identical alleles but cannot confirm the number of chromosomes.
Efficient and rapid, ideal for prenatal diagnostic use
how does digital droplet PCR work?
DNA from a patient sample divided into thousands of tiny droplets using oil, each droplet contains DNA from an individual cell, then do thousands of separate PCR reactions. Is in effect single cell analysis
digital drop PCR - explain the probes used and the following PCR reaction
Two probes are designed:
One specific to the wild-type DNA sequence, one specific to the variant DNA sequence.
Each probe is tagged with a fluorescent marker and a quencher to prevent fluorescence (different tag for WT vs variant)
PCR Reaction in Droplets:
Specific primers flank the target DNA sequence in each droplet.
If a probe binds to its target DNA, the PCR reaction proceeds.
During the reaction, polymerase activity separates the quencher and fluorescent marker, activating fluorescence. if the e.g. WT sequence is not present, the WT probe cannot bind to the amplicon, and the fluorphore and quencher will not be separated
digital droplet PCR - once you’ve carried out the PCR reactions, describe and explain the process of fluorescence detection, and result interpretation
Fluorescence Detection:
The number of fluorescent-positive droplets is counted:
e.g. Heterozygous individual: ~50% of droplets fluoresce for each probe.
Low-level mosaicism (this is what it’s good for): Detects even rare variants (e.g., present in 1 in 20,000 cells)
Blue dots = droplets emitted variant fluorescence,
Green dots = droplets emitted WT fluorescence
Orange = emitted both.
Depending on the concentration of the original DNA sample there will be a proportion of droplets that contain no DNA.
So if the variant of interest is present there will be blue and orange calls, if it is not present there will only be the green wildtype calls.
The number of green vs blue and orange calls can tell us the proportion of cells that have the variant.
ddPCR is an excellent technique for the exclusion and measurement of mosaicism
