Phenylketonuria Screening

Question 1

Give four main examples of inborn errors of metabolism (IEMs)

Answer 1

• PKU
• MCADD
• Galactosaemia
• Smith Lemli Opitz Syndrome

Question 2

What mode of inheritance does PKU show?

Answer 2

Autosomal recessive

Question 3

What is the main feature of PKU?

Answer 3

• Reduction in phenylalanine hydroxylase activity
• causes accumulation of phenylalanine in affected babies following commencement of milk feeding
• if untreated leads to severe LD and neurological disability

Question 4

What are the main clinical symptoms of PKU?

Answer 4

• Mousy odour
• Reduced skin and hair pigmentation
• Reduced growth and microcephaly
• Parkinsonian signs
• Abnormal gait
• lack of concentration

Question 5

What is the incidence of PKU?

Answer 5

• globally = 1 in 20,000
• Europeans = 1 in 10,000
• population variations within U.K: 1 in 12,000 for England vs 1 in 4,500 for Ireland
• extremely rare in Finland and Japan (1 in 200,000)

Question 6

How is enzyme activity affected in PKU?

Answer 6

More than 400 different mutations an result in a reduction of between 0-25% of normal enzyme activity

Question 7

What is considered a positive screen for PKU bloodspot?

Answer 7

Phenylalanine level greater than 240 umol/L

Question 8

What is diagnostic confirmation based on for PKU screening?

Answer 8

Plasma phenylalanine

• Classical PKU = more than 1200 umol/L
• Hyperphenylalaninaemia (HPA) = 600-1200 umol/L
• mild HPA = less than 600 umol/L - query treatment required?

Question 9

What are normal phenylalanine levels across age groups?

Answer 9

• Adult = 58 umol/L (plus or minus 15)
• Teenage = 60 (plus or minus 13)
• Child = 63 (plus or minus 18)
• Newborn = less than 120 (hence abnormal cut off of more than 240)

Question 10

How do genotype and phenotype correlate in PKU?

Answer 10

• Genotype correlates well with biochemical phenotype
• some mutations (e.g. Arg408Trp) significantly reduced PAH activity and result in a severe phenotype
• others (e.g. Glu390Gly) leave residual activity so there is a greater tolerance to dietary Phe and therefore a milder phenotype

Question 11

What are the advantages of tandem mass spectroscopy (MS/MS)?

Answer 11

• Profile approach
• Screening for wider group of disorders
• Shorter analytical time and high throughput
• increased analytical sensitivity and specificity
• earlier and more accurate screening in the post natal period

Question 12

What are the cut-offs for follow up of HPA using tandem MS?

Answer 12

• less than 200 umol/L = neg PKU test result
• 200-400 bracket get a Gal, Tyr1, DHPR and biopterin screen on the bloodspot (if pos then immediate follow up needed, if neg then is equivocal result and need to repeat blood test and if still more than 200 then needs immediate follow up)
• more than 400 bracket = PKU positive - immediate clinic appointment needed and have repeat blood test and other marker tests as above for confirmatory purposes

Question 13

What does treatment involve for PKU?

Answer 13

• Dietary: low phenylalanine content - Supplemented with a protein substitute free of phenylalanine
• Small and controlled amount of PHA is required for normal growth and development and in neonates this is provided by breast or formula milk
• in early infancy plasma PHA should be maintained between 120-360 umol/L
• diet must be strictly controlled during pregnancy to avoid severe foetal damage

Question 14

What does the enzyme phenylalanine hydroxylase (PAH) do?

Answer 14

• Catalyses the irreversible hydroxylation of phenylalanine to tyrosine
• it’s activity requires the cofactor tetrahydrobiopterin (BH4)

Question 15

PKU has a multifactorial cause based on what two main things?

Answer 15

• Genetic mutations of the PAH gene

- Environmental exposure to dietary phenylalanine

Question 16

The PAH enzyme is primarily expressed in the liver but PKU pathology is almost entirely restricted to the brain. Why?

Answer 16

• Aetiology of cognitive problems due to HPA is unclear but blood brain barrier thought to be involved
• elevated plasma Phe impairs brain uptake of other large neutral amino acids in PKU patients
• direct effects of elevated brain Phe and depleted large neutral amino acids may be major cause for disturbed brain development and function in PKU

Question 17

BH4-deficient HPA occurs when PAH activity is normal but there is a defect in the biosynthesis or recycling of the cofactors BH4. Give more details

Answer 17

• this cofactor is essential for activity of PAH enzyme
• BH4 deficiency can be caused by defects in four genes (GCH1, PCDP1, PTS and QDPR)
• usually present as severe phenotype affecting both hepatic PAH metabolism and CNS neurotransmitter biosynthesis
• can be treated with BH4 supplements

Question 18

Provide some details on PAH structure

Answer 18

• Comprised of 4 subunits that form 2 dimers
• dimers then pair to form a tetramer
• location of mutations can be used as a predictor of the phenotype

Question 19

What is the carrier frequency for PKU?

Answer 19

1 in 50

Question 20

Opinions differ on whether we should genetically test for PKU. What are some of the reasons for testing?

Answer 20

• Provides a molecular confirmation of the diagnosis (if two muts identified)
• Allows for cascade screening/carrier testing
• Helps in prediction of disease severity (well documented phenotype:genotype correlations for many muts)
• helps in prediction of treatment response (BH4 supplementation)

Question 21

Describe an alternative treatment to dietary restriction for PKU patients

Answer 21

• BH4 supplementation alongside dietary restriction
• administration of exogenous BH4 supplements in some PKU patients can increase PAH activity thus reducing Phe levels to a clinically significant extent
• not responsive in all patients and depends on type of PAH mutation the patient carries
• responsive mutations tend to be those associated with significant residual PAH activity and are generally milder mutations associated with a less severe PKU phenotype

Question 22

What type of mutation make up the majority of PAH gene mutations in PKU?

Answer 22

Missense mutations (approx 60%)

Question 23

What are the three common PAH mutations across the UK?

Answer 23

• Exon 12: c.1315+1 G to A
• Exon 12: p.Arg408Trp
• Exon 3: p.Ile65Thr

Question 24

What are the four types of PAH mutation and what affect do they have (if any) on protein function?

Answer 24

• Null alleles/deletions: no activity
• Vmax alleles: reduced activity
• Kinetic alleles: reduced affinity for substrate or cofactor
• Unstable alleles: increased turnover and loss of PAH protein

Question 25

Is the PKU gene amenable to Sanger sequencing?

Answer 25

Yes: fairly small exons (between 190 and 500bp) and isn’t GC rich

Question 26

Sanger sequencing for PKU will not detect large dels or dups. What methods should be used instead?

Answer 26

Quantitative methods such as MLPA

Question 27

It is important to exhibit caution when interpreting genotype in context of phenotype for PKU. Why?

Answer 27

• patients with same genotype will often have different phenotypes, even between sibling pairs
• suggests environmental or other genetic factors involved such as how well Phe intake is managed or native BH4 expression levels

Question 28

How does BH4 supplementation work?

Answer 28

• May stabilise mutant PAH (molecular chaperoning)
• Increased affinity for Phe by PAH
• Reduced rate of deactivation of PAH

Question 29

What are the untreated risks for maternal PKU?

Answer 29

• placenta concentrates PHA 2-4x
• 92% mental retardation
• 73% microcephalic
• 40% growth retardation
• 12% congenital anomalies
• recommend that maternal levels stay between 100-300 umol/L for preconception and during pregnancy

Question 30

What method do labs use to measure phenylalanine for PKU testing?

Answer 30

Tandem MS