Genetics of PKU

Question 1

Describe PKU.

Answer 1

• PKU is an autosomal recessive inborn error in metabolism.
• PKU is caused by the deficiency of the hepatic enzyme phenylalanine hydorxylase (PAH). This enzyme is encoded by the PAH gene on chromosome 12.
• PKU has a multifactorial cause:
  1) . Genetic mutations of the PAH gene = deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH)
  2) . The PAH enzyme catalyses the irreversible hydroxylation of phenylalanine to tyrosine leading to environmental exposure to increased dietary phenylalanine or hyperphenylalaninaemia (HPA).
• PAH activity requires the co-factor tetrahydrobiopterin (BH4).

Question 2

What is the phenotype of phekylketonurea (PKU) based on?

Answer 2

• PKU is a highly heterogeneous disorder and the phenotype classification is based on serum phenylalanine levels.
• Classical >1200umol/L
• Moderate = 900-1200
• Mild = 600-900
• MHP (mild hyperphe) <600
• This data is based on the UK PKU expert review 2010. The levels are based on diagnosis through newborn screening at day 5-8 of life.

Question 3

Describe the clinical features of phenylketonurea (PKU). How can some of the clinical features of PKU be managed?

Answer 3

• If PKU is undetected by neonatal screening chronic, untreated severe HPA in infants and children leads to:
• Albinism
• Musty odour (phenylacetate)
• Severe intellectual disability
• Seizures
• Ataxia
• Motor deficits
• Behavioural problems (aggression, self harm)
• Autistic features
• Correction of HPA in the first few weeks of life by dietary restriction prevents the severe mental retardation associated with PKU.
• Dietary control should start ASAP (UK guidelines recommend before 21 days of life) and continue throughout life.
• Even with early diagnosis and lifelong treatment most PKU patients suffer from some degree of neurocognitive deficit.
• Hyperphenylalaninaemia in adults is often associated with attention problems, mood instability and poor job performance.
• Dietary restriction in affected females is particularly important especially when they consider starting a family due to the risk of maternal PKU. It is recommended that dietary control should be in place before conception.

Question 4

Describe the pathology in PKU.

Answer 4

• The PAH enzyme is primarily expressed in the liver.
• PKU pathology almost entirely restricted to the brain.
• The aetiology of cognitive problems due to HPA is unclear, but the blood brain barrier is thought to be involved.
• Evidence suggests that elevated plasma Phe impairs brain uptake of other large neutral amino acids (LNAAs) in patients with PKU.
• Direct effects of elevated brain Phe and depleted LNAAs may be the major cause of impaired brain development and function in PKU.
• HPA can have effects on brain proteins and other neurotransmitters, oxidative damage, white matter damage, impaired LNAA uptake into the brain and other unknown effects.

Question 5

Describe how HPA may occur when PAH activity is normal.

Answer 5

• A rarer form of HPA occurs when PAH activity is normal but there is a defect in the biosynthesis or recycling of the cofactor tetrahydrobiopterin (BH4).
• This cofactor is essential for the activity of the PAH enzyme.
• There are two types of BH4 deficiency. The first type is where there is associated HPA and the second type is where there is no associated HPA. Associated HPA type involves GTPCH, PTPS, DHPR and PCD. No associated HPA type involves DRD and SR.
• BH4 deficiency usually presents as a severe phenotype affecting both hepatic PAH metabolism and CNS neurotransmitter biosynthesis.
• Can be treated with BH4 supplements.

Question 6

Outline the structure of the PAH enzyme.

Answer 6

• The PAH gene encodes a 452 aa monomer which has a regulatory, catalytic (which contains an iron molecule) and tetramerisation domain.
• The active PAH enzyme exists in a dimeric or tetrameric form.
• The tetrameric form is preferred.
• Location of mutations can be used as a predictor of phenotype.

Question 7

Describe the epidemiology of PKU.

Answer 7

• Globally PKU has an incidence of 1 in 20,000.
• Incidence in European populations is 1 in 10,000; with a carrier frequency of 1 in 50.
• However, there are population variations:
• 1 in 12,000 in England
• 1 in 4,500 in Ireland
• 1 in 7,600 in Scotland
• 1 in 13,000 in Wales
• Extremely rare in Finland and Japan - approximately 1 in 200,000 births.
• Latin America 1 in 25,000 - 1 in 50,000.
• Yemenite Jews 1 in 5,000.

Question 8

Describe referrals for PKU?

Answer 8

• Receive about 30 referrals for PKU annually.
• Bristol Genetics Lab is recognised as a specialist centre and receive referrals from UK and internationally.
• Referrals mainly from Dublin, Scotland and Liverpool.
• Types of referrals:
  1) . Diagnostic/Confirmation: 4% of cases where either one or no mutations are identified.
  2) . Cascade testing: Parental samples to confirm inheritance of mutations; followed by other family members.
  3) . Prenatal referrals: Not encouraged as PKU is a treatable disorder.
  4) . Population risk: Partners of PKU affected patients or mutation carriers. Biochemial testing (Phe/Tyr ratio) can be undertaken, but this can be unreliable and cannot be undertaken in females who are pregnant or who are taking the contraceptive pill. Test for common mutations in the population vs full gene screen.
• Target TATs as set by UKGTN.

Question 9

Why bother genetic testing for PKU given that we can make a biochemical diagnosis? Should we genetically test for PKU?

Answer 9

The main reasons for genetic testing for PKU:

• provides a molecular confirmation of the diagnosis - if two mutations identified.
• Allows for cascade screening/carrier testing.
• Helps in prediction of disease severity - well documented phenotype/genotype correlations for many mutations.
• Helps in prediction for treatment response - BH4 supplementation.

Should we genetically test for PKU?
Differing opinions from centre to centre:
- Locally: No value in testing
- Nationally: Testing is useful to confirm diagnosis and provide information for family.
- Europe: It is important that genetic testing is carried out on all positive PKU cases to help predict disease severity and predict treatment response.

Question 10

Describe BH4 supplementation as a treatment for PKU.

Answer 10

• Treatment for PKU is mainly dietary restriction of Phe.
• Another treatment is the use of Tetrahydrobiopterin (BH4) supplementation - Kuvan or saproterin dihydrochloride.
• This treatment is used alongside dietary restrictions.
• It has been found that administration of exogenous BH4 supplements in some PKU patients can increase PAH activity. This reduces Phe levels to a clinically significant extent.
• This treatment is not responsive in all patients.
• BH4 responsiveness depends on the type of PAH mutations carried by the patient.
• Mutations which are associated with residual PAH activity are generally reported to be responsive to BH4 supplementation.
• The majority of these are milder mutations, associated with a less severe phenotype.

How does this treatment work?

• BH4 may stabilise mutant PAH (molecular chaperoning).
• The mutant PAH protein has an increased affinity for Phe.
• Reduced rate of deactivation of PAH.
• Many papers available describing BH4 responsive mutations, at Bristol lab generally use two sources to determine the BAH responsiveness of a mutation. This includes a paper and the biopku database of real patient data.

Question 11

Describe the PAH gene and the mutation spectrum.

Answer 11

• Autosomal recessive inheritance.
• Human PAH gene located on 12q23.2
• Spans 171kb.
• 13 exons, cDNA 2.4kb.
• Encodes a 452 aa polypeptide.
• Undergoes very little post translational modification.
• To date there are more than 560 pathogenic mutations reported in the PAH gene. Most reported to the PAH knowledgebase.
• Mutations are spread throughout the gene and are of many different types.
• Missense mutations = 62%
• Small or large deletions = 13%
• Splicing defects = 11%
• Non-pathogenic polymorphisms = 6%
• Nonsense mutations = 5%
• Insertions = 2%
• As with other genes not all mutations in the PAH gene have the same effect on protein function.
• Null alleles/deletions = no activity.
• Vmax alleles = reduced activity.
• Kinetic allele - reduced affinity for substrate or cofactor.
• Unstable alleles = increased turnover and loss of PAH protein.
• Depends on type of change and location in the gene/protein (functional domains).

Genotype/Phenotype Correlations:

• The PAH gene has a broad spectrum of mutation severity from complete loss of PAH activity, causing classical PKU to high residual activity associated with mild HPA.
• Use many sources to determine severity of identified mutations.
• Prediction of phenotype is not always possible due to novel mutations, unclassified phenotype or variable phenotype (e.g. p.Ile65Thr is associated with mild to moderate PKU phenotype).
• As with other autosomal recessibe disorder the general rule with phenotype prediction is:
  1) . Severe + Severe Mutation = Classical PKU
  2) . Severe + Mild Mutation = milder PKU

The less severe mutation of the two determines the phenotype.

• If dietary restriction is implemented from birth the physical phenotype may be mild so the PKU phenotype means the biochemical phenotype - which can be complicated by a patient’s Phe loading (physical intake) tolerance.

Question 12

Describe Genotype/Phenotype correlations in the case of the PAH gene.

Answer 12

• The PAH gene has a broad spectrum of mutation severity from complete loss of PAH activity, causing classical PKU to high residual activity associated with mild HPA.
• Use many sources to determine severity of identified mutations.
• Prediction of phenotype is not always possible due to novel mutations, mutation may have been reported by may have an unclassified phenotype or many of the mutations have a variable phenotype (e.g. p.Ile65Thr is associated with mild to moderate PKU phenotype).

Question 13

Outline the different PAH mutations that have been identified by Bristol Genetics Laboratory.

Answer 13

• More than 119 different PAH mutations have been detected at BGL.
• Of these only 16 have a frequency >1%, 56 only seen once.
• 12 of these are novel.
• Certain mutations are more common in specific populations. c.1066-1G>A in Mediterranean and Turkish populations. Could target specific exons based on ethnicity of patient.
• Most PKU patients have 2 different PKU mutations in trans.
• Not uncommon for 3 pathogenic PKU mutations to be identified in a patient. In these cases it is important to establish the phase of the mutations so that the phenotype can be predicted.

Question 14

What are the 3 common PAH mutations in the population local to the Bristol lab?

Answer 14

Three common mutations in the local population:

1) . c.1315+1G>A in exon 12
2) . p.Arg408Trp in exon 12
3) . p.Ile65The in exon 3

The incidence of these mutations actually varies quite significantly across different regions of the UK.

For a patient at population risk of carrying a PAH mutation testing for exons 12 and 3 alone can exclude the above common mutations, however the consensus is that a full gene screen is more appropriate.

A fourth mutation in exon 12, p.Tyr414Cys, accounts for 2% in SW England, so screening exons 3 and 12 would cover 46% of PAH mutations in the local population.

Question 15

How is genetic testing for PKU usually performed?

Answer 15

• Bi-directional Sanger sequence analysis of all 13 exons of the PAH gene.
• Flanking intronic sequence is included.
  Sequence from the branch point at the 5’ end of each exon to +20 3’ of the exon. The position of the branch point is predicted using human splicing finder.
• The PKU gene is amenable to sequence analysis as the gene is fairly small (190-500bp) and it is not very G:C rich.
• The technique is good for detecting point mutations, small insertions and deletions.
• Detection rate of approximately 96% as will not detect whole gene/large deletions.
• Will not detect deep intronic mutations.

Question 16

Outline the steps in a semi-automated bi-directional screening process for the PAH gene.

Answer 16

1) . Sequence requested by scientist.
2) . Automated PCR set up - 13 PCRs per patient.
3) . Enzymatic PCR clean up (Exo/SAp).
4) . Automated sequence reaction set up. Each PCR split into 1F and 1R reaction. 26 sequence reactions per patient.
5) . Clean up of sequence reaction.
6) . Run on alalyser.
7) . Data analysis and interpretation.

Question 17

Describe the interpretation of known mutations in PKU.

Answer 17

Once we have identified the 2 mutations we then need to report the genotype to the clinician. Follow a number of steps for this.

1). Check in house database for identified mutations. Look at previous reports for mutation information and patient origin.

2) . Check web-based database.
- PAH knowledgebase
- HGMD
- BIOPKU database - phenotype info and details of BH4 responsiveness.

3) . Check literature for further information.
- Key papers regarding predicted phenotype of mutations, residual enzyme activity - functional studies, affects on protein function, population data, BH4 responsiveness. Search for other literature if necessary.

• Always request parental samples to confirm mutations are on opposite chromosomes.

Question 18

In about 4% of cases of PKU we only detect 1 or 0 mutations on genetic analysis, why might this be?

Answer 18

1) . Biochemical diagnosis:
- HPA caused by BH4 deficiency - biochemical test for DHPR.
- Patient has had a blood transfusion - but most transfusions are white cell depleted so low risk of the DNA from the blood donor being amplified, but must be considered.
- Request repeat sample.

2) . Second allele carries a deletion:
- If a mutation or identified polymorphisms across the gene are seen in the homozygous state; or only a single mutation has been identified then the possibility of a large deletion must be considered.

3). Second mutation in deep intronic sequence.

Question 19

Outline analysis for large deletions in the PAH gene.

Answer 19

To date only large deletions have been described in the PAH gene and no duplications:

• Whole gene deletions
• Single exon deletions
• Exon 5 in three patients
• Exon 6 in six patients

In BGL 4% of PKU chromosomes uncharacterised, i.e. only one mutation detected.

Zygosity of common polymorphisms and family studies can provide evidence that a deletion is present.

Large deletions can’t be detected by sequence analysis. Quantitative analysis required for detection of large deletions (and duplications).

In Bristol a number of quantitative techniques have been tried:

• Semi-quantitative dosage multiplex PCR - old technique and not robust, superseded by MLPA.
• MLPA - problematic due to patient DNA samples being very old and so not robust. Analysis by comparison of all PAH exons against control probes.
• Deletions detected by both of these methods had to be confirmed by a second method.
• cDNA analysis using Southern blotting confirmed exon 6 deletions but not exon 5 deletions.
• Long-range PCR - confirmed exon 6 deletions but not exon 5 deletions.
• Exon 5 deletions were confirmed using semi-quantitative dosage multiplex PCR and MLPA.
• As PAH gene deletions are so rare we now send DNA to a German lab for MLPA testing.
• In house we can test for deletions of exon 6 by long range PCR.

Question 20

Outline the process for the interpretation of unclassified variants in the PAH gene.

Answer 20

• A number of mutations detected are novel - difficult to prove if they are pathogenic or benign variants.
• Missense mutations particularly problematic.
• Silent variants may still be pathogenic - may effect splicing.
• Ideally need functional studies (or family studies) to prove if a variant is pathogenic.
• Functional studies not feasible for diagnostic laboratories.
• Use various in silico tools for assessment of variants - collect evidence. Web based tool - Alamut.
• BGPs produced by the CMGS for interpretation of UVs.

Investigating UVs:

• Mutations databases
• PubMed
• dbSNP (NCBI)
• Sequence conservation
• Physiochemical differences of amino acids - grantham score
• Online prediction tools - SIFT - Polyphen - Align GVGD
• Splicing predictions
• Family studies

Question 21

What problems may arise when investigating unclassified variants?

Answer 21

Investigating UVs:

• Mutations databases
• PubMed
• dbSNP (NCBI)
• Sequence conservation
• Physiochemical differences of amino acids - grantham score
• Online prediction tools - SIFT - Polyphen - Align GVGD
• Splicing predictions
• Family studies
• Often the bioinformatic tools do not agree.
  This leads to uncertainty in reporting.
• Cannot be completely sure that variant is pathogenic.
• Cannot be used for prediction of phenotype or BH4 responsiveness.
• Need to take general consensus of the bioinformatic results to assess likely pathogenicity.
• Remember - biochemical diagnosis of PKU so if bioinformatics suggest pathogenicity then likely that it is pathogenic. Family studies important.

Question 22

Briefly summarise PKU.

Answer 22

• PKU is caused by mutations in the PAH gene leading to the deficiency of the PAH enzyme.
• PKU can be treated using dietary control and BH4 supplements.
• A wide spectrum of mutation types have been identified in the PAH gene.
• Unclassified variants are difficult to interpret, but most patients have a biochemical diagnosis.
• PAH genotype useful in predicting phenotype/genotype correlations and response to treatment.

Question 23

Describe the common exon 12 PAH mutation c.1222C>T p.(Arg408Trp).

Answer 23

The common exon 12 PAH mutation c.1222C>T p.(Arg408Trp):

• Reported in literature and to PAH database.
• Mutation associated with a classical PKU phenotype.
• It accounts for approximately 41% of PKU chromosomes in Ireland.
• The mutation is listed as BH4 non-responsive.

Question 24

Describe the exon 2 PAH mutation c.177C>G p.(Phe39Leu).

Answer 24

The exon 2 PAH mutation c.177C>G p.(Phe39Leu):

• Reported in literature and to PAH database.
• Associated with variable phenotype from mild to classical PKU, with the majority being moderate PKU.
• This mutation is associated with some residual activity and it listed as BH4 responsive.