PKU Screening - Part 1 Flashcards
Outline the standards for Newborn BloodSpot Screening.
1) . Standard 1: Completeness of offer
- Notification of receipt of sample in lab, result or decline of test for all 5 conditions for 100% of babies with PCT.
2) . Standard 2: Enhanced tracking ability
- 100% of cards received with baby’s NHS number.
3) . Standard 3: Timely samples collection:
- 95% of samples taken 5-8 days of life.
4) . Standard 4: Timely sample dispatch:
- 100% of samples received within 4 working days of sample collection.
5) . Standard 5: Quality of blood spot sample.
- Avoidable repeat rate of less than or equal to 2%.
6) . Standard 6: Timely receipt of a repeat/second blood spot sample.
- 95% of samples received by laboratory within 72 hours of request.
7) . Standard 7: Timely processing of screen positive samples.
- 100% of screen positive results and clinical referral within 4 working days of sample receipt by laboratory.
8) . Standard 8: Timely identification of babies for whom child health records has not received notification of sample receipt, screening test results or decline.
- 100% of untested babies identified by 17 days of age.
9) . Standard 9: Completeness of uptake.
- Screening test results for all conditions for 100% of babies accepting test.
Describe Phenylketonuria (PKU).
- AR recessive disorder.
- Described by Folling in 1934 “Imbecillitas phenylpyruvica”.
- Reduction in phenylalanine hydroxylase activity.
- Phenylalanine from ingested protein or from muscle metabolism is converted to tyrosine in the circulation and liver and peripheral cells. When the phenylalanine is converted to tyrosine the tyrosine is then further used for metabolism and the manufacturing of neurotransmitters and other hormones. If the phenylalanine hydroxylase is reduced or has low activity then the phenylalanine will increase in circulation. The increased phenylalanine is converted through other pathways and that’s why the phenyl pyruvate appears in the urine. There are some cofactors involved in the process of converting phenylalanine to tyrosine including tetrahydrobiopterin (cofactor BH4) and dihydropteridine reductase (recycles BH4). If your DHPR is abnormal or you have BH4 deficiency then that would also lead to raised phenylalanine. In a screening test if you pick up a raised phenylalanine level then you would always screen for BH4 and DHPR abnormalities also.
- More than 400 different mutations resulting in reduction of between 0-25% of normal of normal enzyme activity.
- UK incidence is about 1 in 10,000 births.
- Carrier incidence is about 1 in 70.
- Of all the metabolic diseases PKU is probably the most common as a single entity - makes it a good disease to screen for at birth.
- In affected babies phenylalanine accumulates rapidly in the blood. From the moment the baby is born the maternal liver is not able to metabolise the phenyl alanine any longer. Babies shortly after birth are catabolic. They release protein into the circulation that is metabolised resulting in the production of phenylalanine. Because they don’t feed well in the first few hours the phenylalanine starts to increase. The normal level for phenylalanine in the neonate would be 50-60uM per litre. In PKU within 5 or 6 days it would increase to 1500-1600uM per litre. This phenylalanine leads to severe and irreversible learning difficulties if the problem is not rectified. The babies start out fine but after 6 to 8 months their development begins to deteriorate and then as they get older they have severe and irreversible learning difficulties. Once the damage has occurred it is irreversible. Babies with PKU may have a mousy odour, reduced hair and skin pigmentation (because tyrosine is a precursor to melanin), reduced growth, microcephaly, long term may develop Parkinsonian signs, abnormal gait, lack of concentration and significant learning difficulties in the majority of these patients if left untreated.
- Bloodspot phenylalanine levels of more than 240u/mol/L considered a positive screen.
- All labs now use Tandem MS to measure phenylalanine levels.
- Diagnostic confirmation is based on the plasma phenylalanine.
- May diagnose either classical PKU (phenylalanine > 1200umol/litre), hyperphenylalaninaemia (HPA; phenylalanine 600-1200 umol/litre), or mild hyperphenylalaninaemia (phenylalanine <600umol/litre).
- Often upon full amino acid quantitation after a positive bloodspot screen we see phenylalanine levels of >1000 or even 2000umol/L (20-40 times as high as it should be).
- Normal levels as follows:
1) . Adult = 58+/-15 umol/L
2) . Teenage 60+/-13 umol/L
3) . Child 63+/-18 umol/L
4) . Newborn <120 umol/L (2mg/dl)
What is the UK incidence of PKU?
UK incidence is about 1 in 10,000 births.
What is the UK carrier incidence of PKU?
Carrier incidence is about 1 in 70.
Describe what happens if you have a phenylalanine hydroxylase deficiency as may be seen in PKU.
- Reduction in phenylalanine hydroxylase activity.
- Phenylalanine from ingested protein or from muscle metabolism is converted to tyrosine in the circulation and liver and peripheral cells.
- When the phenylalanine is converted to tyrosine the tyrosine is then further used for metabolism and the manufacturing of neurotransmitters and other hormones.
- If the phenylalanine hydroxylase is reduced or has low activity then the phenylalanine will increase in circulation.
- The increased phenylalanine is converted through other pathways and that’s why the phenyl pyruvate appears in the urine.
- There are some cofactors involved in the process of converting phenylalanine to tyrosine including tetrahydrobiopterin (cofactor BH4) and dihydropteridine reductase (recycles BH4).
- If your DHPR is abnormal or you have BH4 deficiency then that would also lead to raised phenylalanine.
- In a screening test if you pick up a raised phenylalanine level then you would always screen for BH4 and DHPR abnormalities also.
What enzymes and cofactors are involved in the conversion of phenylalanine to tyrosine?
- Phenylalanine hydroxylase is the enzyme involved.
- Tetrahydrobiopterin (cofactor BH4) and Dihydropteridine Reductase (recycles BH4) are also involved in the conversion.
Describe the pathology and features that may be present in babies with PKU.
- In affected babies phenylalanine accumulates rapidly in the blood.
- From the moment the baby is born the maternal liver is not able to metabolise the phenyl alanine any longer.
- Babies shortly after birth are catabolic. They release protein into the circulation that is metabolised resulting in the production of phenylalanine.
- Because they don’t feed well in the first few hours the phenylalanine starts to increase.
- The normal level for phenylalanine in the neonate would be 50-60uM per litre.
- In PKU within 5 or 6 days it would increase to 1500-1600uM per litre.
- This phenylalanine leads to severe and irreversible learning difficulties if the problem is not rectified.
- The babies start out fine but after 6 to 8 months their development begins to deteriorate and then as they get older they have severe and irreversible learning difficulties.
- Once the damage has occurred it is irreversible. Babies with PKU may have a mousy odour, reduced hair and skin pigmentation (because tyrosine is a precursor to melanin), reduced growth, microcephaly, long term may develop Parkinsonian signs, abnormal gait, lack of concentration and significant learning difficulties in the majority of these patients if left untreated.
What is considered a positive PKU screen on the bloodspot test?
Bloodspot phenylalanine levels of more than 240u/mol/L considered a positive screen.
What PKU concentrations would result in the following diagnoses?
1) . Classic PKU
2) . Hyperphenylalaniaemia
3) . Mild Hyperphenylalaninaemia
1) . Classic PKU = >1200umol/L
2) . Hyperphenylalaninaemia = 600-1200umol/L
3) . Mild Hyperphenylalaniaemia = <600umol/L
Explain why classical PKU may be termed a ‘small molecule disease’. What are some of the features of PKU and what causes some of these features?
- Classical PKU is referred to as a small molecule disease because it gets transferred into the brain - the exact mechanism and neurotoxixity has not been described yet - if it is untreated you have severe mental retardation with an IQ of less than 40, usually the patients might have seizures and severe neurological signs.
- High phenylalanine levels - patients with hyperphenylalaninaemia might have some developmental delay and poor concentration.
- A low tyrosine contributes to the executive function and also tyrosine converts to neurotransmitters and that might mean that these patients have neurotransmitter defects.
What are considered to be the normal phenylalanine ranges in the following cases?
1) . Adult
2) . Teenage
3) . Child
4) . Newborn
Normal levels as follows:
1) . Adult = 58+/-15 umol/L
2) . Teenage 60+/-13 umol/L
3) . Child 63+/-18 umol/L
4) . Newborn <120 umol/L (2mg/dl)
Describe the genetics of PKU.
- Chromosome 12q24.1
- Gene cloned 1983 90kB, 13 exons
- > 350 mutations described - some common
- Little genotype to phenotype correlation
- Combined/compound heterozygosity
- mutations in modifying genes
- variability of therapies and outcome measures used - IQ, MRI, neuropsychiatric testing
- Environmental factors
What is the location of the chromosomal abnormality involved in PKU?
- Chromosome 12q24.1
Describe genotype/phenotype correlation in PKU.
- Genotype correlates well with biochemical phenotype (this is actually very arguable and it may be said that the correlation is not actually that good).
- In most patients with classical PKU there is not a good genotype-phenotype relationship in most patients.
- Complete or near complete enzyme deficiency leads to classical PKU.
- Atypical/benign forms: disease severity in most is determined by the least severe of the 2 PAH mutations (i.e. determined by the enzyme activity of the least severe mutation - if the enzyme activity is 10% then you convert some phenylalanine to tyrosine and the disease will therefore not be that severe).
- 2 mutations with similar severity may confer a milder phenotype than either would do alone.
Describe how PKU screening was originally carried out. What technology is now used?
- Guthrie 1961.
- Prevention maternal PKU syndrome.
- Blood spot (filter paper) samples using the Guthrie bacterial inhibition assay. Would collect blood on the card, punch the card out and then put it on agar gel. In the agar gel was Bacillus subtilis and an alanine derivative called B-2-thienylalanine (inhibitor to the bacteria). For the bloodspot from a patient with PKU who has lots of phenylalanine in the blood the phenylalanine will leak into the agar gel and the inhibition will be overcome. The bacteria will then start growing because the baby’s phenyl alanine overcomes the inhibitor. By measuring the growth of the bacteria you could then tell what the phenylalanine level would be. This previous manual microbiology test took a long time.
- They next began to do thin layer chromatography where you spot out the blood spot onto a chromatogram and just run it in a one dimensional manner. You would pick up increased phenylalanine levels. Still very time consuming. Could also do it using 2D chromatography letting solvents run both ways.
- Now use tandem mass spec - every blood spot is eluted into a solvent, this liquid is then injected into the front of the tandem mass spec and it goes through very strong magnets. Inject the liquid and it gets ionised - there is a vacuum at the back and the vacuum will suck the ions through the quadrapoles/magnets - the magnets will split off the magnets according to weight and the speed that they travel at. At the end there is a detector and it will tell you what has come through the detector. You can set the tandem mass spec to search for specific molecules. They will quantitate it through an internal standard and by quantitating it you can tell what it is. This is all automated.
