Cystic Fibrosis Newborn Screening Flashcards
Is immunoreactive trypsonogen (IRT) a good CF marker?§
Crossley et al. first discovered elevated blood immunoreactive trypsinogen (IRT) in babies with CF in 1979.
An immuno assay for IRT was the first serious introduction of newborn screening for CF.
It is actually not a very good test on its own for CF. IRT is a poor marker for CF during the first few days of life (3-10% positive predictive value). Better at 2-4 weeks (approximately 50% PPV).
In normal individual IRT levels drop off in the first few weeks of life but in CF babies it remains elevated for longer.
What are the key factors to consider for a screening test?
1) . Sensitivity - want to pick up as many people who have got the disease as you can.
2) . Specificity - don’t want to identify anyone who hasn’t got the disease as high risk.
3) . Positive predictive value - if you get a positive test result you want as many as possible of those results to be true and actually have the condition.
4) . Technology (+ cost) - gold standard testing may be too expensive for large scale screening.
What strategies have been used for CF newborn screening?
1) . IRT-IRT - those with a raised IRT at day 5 then get a second sample taken at day 28 when the test is better. Those that still have an elevated IRT would go on to have the sweat test. Still get quite a few IRT false positives so will end up sweat testing a lot of negatives.
2) . IRT-DNA - do initial IRT and do initial testing on that first blood spot sample. Don’t need to take any second samples which takes up resources and increased anxiety in families. Definitive diagnosis relatively quickly. DNA testing relatively expensive. When 1 mutation is found we don’t know for certain whether they are carriers or affected individuals where we just haven’t identified the second mutation.
3) . IRT-DNA-IRT - IRT DNA testing on the first sample and will get some where the DNA mutation is identified and you get the diagnosis and then where no mutations are identified they make the decision that it is low risk of CF. If 1 mutation is identified then you do a day 28 IRT - if IRT is still raised and have 1 mutation then they are seen as high risk of having CF, if the IRT has gone back down to normal they are seen as probable unaffected carriers.
4) . IRT-PAP? - pilot studies in other countries has looked at using pancreatitis associated protein as an alternative to DNA testing. It is cheaper an it doesn’t identify carriers as we don’t want to identify carriers in screening programmes. No evidence that it is particularly better than other strategies just yet.
What is the UK newborn screening centre trying to do by CF screening? i.e. what is the benefit of newborn CF screening?
The UK newborn screening centre is trying to maximise the diagnosis of CFTR defects producing preventable or treatable disease (respiratory, digestive) in infancy.
It is using preventable or treatable disease and focussing on the severe side of things where it is worth detecting early so you can do something about it.
It is trying to minimise the need for second samples which take a lot of time, coordination and increases anxiety.
It aims to minimise diagnostic delay - want to get a diagnosis and begin treatment as quickly as possible.
Want to minimise detection of unaffected heterozygotes - don’t want to find carriers if we can avoid it as it complicates things.
Want to avoid diagnosis of very mild forms of CFTR defect producing late onset, essentially unpreventable disease.
The strategy they use at time of lecture is an IRT-DNA/DNA-IRT method.
Describe the IRT-DNA/DNA-IRT method of screening for CF.
- Initial IRT then 2 stages of DNA testing, if there is uncertainty after that then second IRT is performed at day 28.
- Combination of biochemical and genetic testing.
- Measurement of Immunoreactive Trypsonogen (IRT) at day 5 (and day 28).
- 2 stage testing for CFTR gene.
Describe the CF newborn screening protocol.
1) . Initial day 5 blood spot sample - single measurement of the IRT performed with IRT cut-off values. Rough and ready initial test.
2) . Most will be in normal range below the cut-off value. Those that are above that initial cut-off will have an extra 2 assays to get an accurate IRT figure to which they will apply the actual cut off value that they want between low risk and high risk. Usually the top 0.5% of population IRT values that are targeted for extra screening (usually around 70ng/ml).
3) . If the IRT value is above the actual cut-off value then it will go for DNA testing. There are 2 stages to DNA testing. It is firstly tested for 4 mutations which may detect 2 CF mutations, 1 CF mutation, or no CF mutations.
i) . If you find 2 mutations you have confirmed the diagnosis.
ii) . If you find 1 mutation then you will screen for an extra panel of mutations on which you may find a 2nd mutation. If you still only have 1 mutation after the larger panel then you don’t know whether they are a carrier or affected and so you would go on to perform a second IRT test at 28 days. If the IRT has gone back down to normal then conclude that they are probably an unaffected carrier. If the IRT is still high then CF suspected and will be referred for sweat testing and appropriate follow up.
iii) . If no mutations detected on 4 mutation genetic screen then for most signed off as CF not suspected. However, if the IRT is really high would take a second sample anyway - this is mainly to reflect the diversity of populations - not a good test if not of western european descent.
How is IRT measured?
- IRT measurement is usually performed using a specific kit.
- Most UK labs use products from PerkinElmer (AutoDELFIA (TM) Neonatal IRT kit and AutoDELFIA (TM) immunoassay system) - may be new systems in place now.
- A lot of time is spent on the quality control of testing - the levels produced are not exactly consistent. Can get variation from batch to batch of reagents of 5-7%. To try and minimise variation and get some form of countrywide consistency the newborn screening centre spend a lot of time talking to PerkinElmer and arranging to have huge batches made up specifically for the UK screening labs. 2 big batches at a time will get sent out to the labs so at any one time only 2 batches of reagents are being used in the country. With new batches of kit the cut-off values might need to be changed.
What mutations are analysed in stage 1 of DNA analysis for CF newborn screening? How is it performed?
- 4 most common, severe mutations:
1) . deltaF508
2) . 621+1G>T (c.489+1G>T)
3) . G542X
4) . G551D mutations
- Should pick up approximately 85% of UK mutation alleles.
- Genotyping performed using methods such as fluorescent multiplex ARMS (e.g. Elucigene CF4 kit), pyrosequencing, and other methods.
Describe the basics of pyrosequencing.
- Get PCR product, the you’ve go sequencing primer again but you couple the DNA polymerase reaction to 2 reactions including a reaction involving luciferase which generates light when a base is incorporated.
- Instead of having a soup of all the nucleotides you are adding 1 base at a time and seeing if it extends the chain or not.
- If it extends the chain you get a flash of light and if it doesn’t you get nothing.
- You use a reaction to mop up the nucleotide and add another one and see if you get a flash of light or not and so on.
- You add a base at a time usually in an order determined by the software that reflects the sequence you are looking at.
- It is quantitative so if you have a run of 2 G’s you will get twice the height of peak.
- If you tell it what base you want to look at and what change you are expecting then the software will design an assay for you.
Describe stage 2 of DNA sequencing used in CF newborn screening.
- Detects approximately 90% of mutation alleles in the UK.
- 2 main kits used include the CFEU2 Elucigene kit and the CF OLA kit from Abbott.
- CFEU2 kit is a fluorescent multiplex ARMS PCR that includes a 50 mutation panel.
- CF OLA is a multiplex PCR and oligonucleotide ligation assay that includes a 32 mutation panel.
Have a look at interpreting pyrosequencing and elucigene results in infertility module.
Look at lectures on CF and flashcards possibly.
Are we looking to find the p.Arg117His(R117H) mutation in a newborn screen for CF? Justify your answer.
- R117H is not what we are looking for in a newborn screening programme as it is not going to cause classic CF. It is more of an unwanted byproduct of the testing we do.
- R117H with a 5T variant in intron 8 is more sever than the 7T and together with a severe mutation would typically lead to pancreatic sufficient CF. However, there is variability.
- The deltaF508:R117H(7T) genotype is more wooly - can be associated with mild lung disease, CBAVD, or no symptoms (especially in females).
- Various studies have been done looking from the newborn screening angle - Scotet et al. followed up 9 individuals with deltaF508:R117H(7T) that was identified by newborn screening - no clinical symptoms were seen in any at a mean age of 7. Approximately 1% or less were actually giving symptoms.
