Approach to IEM AA - PKU

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Approach to IEMAA - PKU

• Annette Feigenbaum
• Division of Clinical and Metabolic Genetics, HSC
• 2002

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Enzymes

• Protein catalysts that rapidly mediate the
  chemical reactions in the body
• The clinical phenotype of an IEM is caused by
  metabolic disturbances resulting from the
  deficiency of a catalytic or transport protein.

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Functional proteins

• Catalytic enzymes
• Transport - Famililal hypercholesterolemia,
  Cystic Fibrosis
• Structural - DMDystrophy, Osteogenesis I
• Homeostasis - immune response, clotting
• Growth and Differentiation
• Communication - receptors, hormones, transducers

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Enzymopathies

• Usually autosomal recessive
• Substrate accumulation and/or product deficiency
• Small or large molecules
• Multiple enzymes can be affected in cofactor
  deficiencies

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Also

• Deficiencies affecting dimerization
• Deficiencies of Modifying enzymes
• Organelle biosynthesis defects e.g peroxisomal

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Amino and organic acidopathies

• small diffusible molecules disease
• acute encephalopathy
• catastrophic newborn disease or late onset subtle
  dev delay
• Importance
• treatable
• prenatal available
• difficult to diagnose early to avoid brain damage
• mimic common medical problems e.g. sepsis

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ACUTE ENCEPHALOPATHY Neonatal or later onset

• Amino acids
• MSUD-acute, chronic ataxia, intermittent variants
• NKHG-seizures, spastic, dev delay
• Homocystinemia MMA (cobalamin)
• Organic acids
• MMA, PA, IVA - acute/chronic/mild/severe
• Hypoglycemia Fatty Acid Oxidation Defects e.g.
  MCAD
• Mitochondrial-acute/chronic
• Hyperammonemia - UCED, FAOD, OA, PC

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PKU / Phenylketonuria 1934

• Commonest IEM AA metabolism Caucasians-British,
  N. Europe
• Phenylalanine hydroxylase deficiency liver
• Autosomal recessive
• PhenylalanineBH4 ---X---gttyrosine BH4
• --gtDopamine
• --gtNorepinephrine --gtEpinephrine

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The Enzyme Defect

• BH4 - tetrahydrobiopterin (cofactor)
• DHPR - dihydropteridine reductase (recycles BH4)

Protein

• Ingested
• Catabolism

PAH
Phenylalanine
Tyrosine
Phenylpyruvic Acid
DHPR
BH4
qBH2
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PKU-2

• Elevated Phenylalanine levels often gt1000/even
  2000uM
• normal
• adult 58-15 uM
• teenage 60 -13
• child 63-18
• newborn lt120 uM ( 2mg/dl)

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Classical PKU-small molecule disease

• Untreated severe MR, IQ lt40, sz
• High Phe Dev delay
• Low Tyrosine Executive Fx
• Neurotransmitter deficiency ? Seizures

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Heterogeneity

• Clinical -clinically different phenotypes
  caused by mutations at the same locus
• Genetic - same or similar phenotypes
  caused by different genetic mechanisms

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Heterogeneity

• Genetic - same or similar phenotypes
  caused by different genetic mechanisms
• Allelic - different mutant alleles at the
  same locus, each capable of producing the
  abnormal phenotype e.g PKU, Hurler Scheie
• Locus - mutations at more than one locus
  /gene can produce similar phenotypes e.g Tay
  Sachs vs Sandhoff disease San Filippo

Note need specific diagnosis to allow accurate
carrier and prenatal testing
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Clinical - Genetic heterogeneity

• Classical PKU
• severe
• lt1 residual enzyme activity
• very high levels PHE-strict diet for life
• Type II/Atypical PKU
• milder
• tolerate more liberal protein diet
• Type III/Mild/Benign persistent Hyperphe
• 5 residual activity
• levels lt600uM
• no diet needed
• Type IV/Malignant PKU 2
• BH4 cofactor defect
• need neurotransmitter replacement therapy
• outcome often not good

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PKU MRI - abnormal white matter
Even in treated PKU there are neurological
consequences- learning, executive function
problems etc
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Treatment PKU

• Protein restriction 1954 ( Bickel) Phe free
  formulas/lo pro foods FOR LIFE
• Maternal PKU syndrome 1957
• in -utero teratogenic effect of hyperPHE
• micocephaly, MR, birth defects incl cardiac,
  Cpalate, dysmorphic
• Gene therapy
• Drug therapy - PAL
• Liver transplant

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PKU-3

• Chromosome 12q24.1
• Gene cloned 1983 90kB, 13 exons
• gt350 mutations described-some common
• Little genotype- phenotype correlation -
• combined/compund heterozygosity
• mutations in modifying genes
• variability of therapies and outcome measures
  used- IQ, MRI, neuropsych testing
• environmental factors

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Mutations

• transcription- promotor
• RNA splicing/cleavage
• Point mutation nonsense, frameshift, missense-
  null
• Large mutations frameshift deletion,
  insertions, duplications- all null

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Mutations-2

• Abnormal amount or function of RNA
• Abnormal/absent protein
• loss/reduction of function
• enzyme deficiency
• defect active site
• abnormal multimeric assembly
• impaired cofactor bindng
• abnormal targetting/interaction
• Rarely gain of function e.g Huntington disease
• Abnormal regulatory domain - altered level of
  expression

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PAH mutations

• Most common mutations (North America)
• R408W Classical PKU (18.7)
• IVS12G-A1 Classical PKU (7.8)
• Y414C Mild hyperphe (5.4)
• 13 other mutations (1-5)
• 55 other mutations (lt1)

31.9
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Genotype/Phenotype correlation

• Classical PKU no good genotype phenotype
  relationship in most patients
• Complete or near complete enzyme deficiency leads
  to classical PKU
• Atypical/benign forms disease severity in most
  determined by the least severe of 2 PAH mutations
• 2 mutations with similar severity may confer a
  milder phenotype than either would do alone

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Mutation Classification
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Prenatal Diagnosis

• Available - direct mutation, linkage
• Possible outcomes ie. Classical, atypical PKU
• ?Desired
• perceived risk
• burden
• acceptable outcome

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Maternal PKU

• Untreated Risks
• 92 MR
• 73 microcephaly
• 40 growth retardation
• 12 congenital anomalies
• Recommendations
• maternal levels 120-360 ?mol/L preconception
  throughout pregnancy diet must be closely
  monitored to avoid fetal damage from malnutrition

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PKU Neonatal screening

• Guthrie 1961
• Ontario Started 1965
• Blood spot (filter paper) samples using the
  Guthrie bacterial inhibition assay
• Normal plasma Phelt0.24 mM.
• Cost effective 2.5-6.6 cost benefit ratio
• Prevention maternal PKU syndrome

Horst Bickel and Robert Guthrie
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PKU- plasma TLC screen
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Urine amino acid qualitative screen
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• Maple Syrup Urine Disease
• quantitative amino acids
• High Performance Liquid Chromatography

Normal
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Neonatal screening Ontario

• The newborn screening program is a voluntary
  program not mandated by legislation
• Ontario Public Health Laboratories Branch and
  Public Health Branch
• The incidence in Ontario
• PKU and its variants 1/12,00 births
• Severe (Classical) form PKU 1/21,000 births.
• Present status Ontario
• PKU Phenylalanine hydroxylase deficiency
• Congenital Hypothyroidism 14000

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Newborn population screening

• Principles PKU
• common 112 000 LB NA
• medically significant
• effective treatment diet
• test sensitive few false negatives
  lt1, may miss Type III
• test specific false positives
• easy to do, rapid Bacterial inhibition,Guthrie
  bloodspot, semiquantitative fluorometric
  better
• confirmatory test available blood amino acid
  analysis
• cost effective vs. cost of MR avoidance
  of maternal PKU prenatal
• access and support to follow-up and treatment
• centralized, coordinated, controlled, monitored,
  egalitarian

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Costs of newborn screening

• Organisation, Administration
• Sample collection and transportation to central
  lab
• Laboratory- equipment, reagents, salaries
• Program - data collection, record keeping,
  epidemiology, quality assurance, check system
• Confirmatory tests 101 false positive for PKU
• Interprogram considerations
• Communication - documentation, public education,
  parent education, training health care personnel,
  staff training
• Research and development- new techniques, new
  tests, new diseases

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Expanded Neonatal Screening

• By tandem MS
• Organic / amino acidopathies-MSUD,
  homocystinuria, tyrosinemia
• Fatty acid oxidation defects e.g. MCAD
• Other
• Biotinidase deficiency
• Galactosemia
• Other CF, DMD, sickle cell, other
  Hemoglobinopathies
• AAP recommends integrated program that
  incorporates screening, diagnosis, management and
  support

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Janice Fletcher
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Tandem Mass Spectroscopy MS/MS
Advantages

• profile approach
• screening for a wider group of disorders-39
• shorter analytical time and high throughput
• increased analytical sensitivity and specificity
• earlier and more accurate screening in the post
  natal period

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Advantages of Tandem MS

• profile approach
• screening for a 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

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Plans

• Petition MOH Ontario to support expanded newborn
  screening by tandem MS and added tests as already
  exists in NS, Saskatchewan
• Establish the Coordinated Genetic Screening
  centre at HSC
• Service, resource, support, education

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Screening Populations at risk

• Ethnic based carrier screening-ADULTS
• Black sickle cell anemia
• Oriental, Mediterranean Thalassemia
• Caucasian Cystic fibrosis
• Ashkenazi Jewish Tay Sachs, Canavan disease,
  Familial Dysautonomia, others
• Population screening for affected - CHILDREN
• Sickle cell
• CF
• Medical, Ethical, Legal, Social, Government,
  Insurance implications
• Tri-council mandate

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Questions - 1

• What to do with PKU - a positive newborn
  screen?
• A) stop breast feeds
• B) change formula
• C) refer to a genetic centre
• D) repeat the screen

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What to do if initial screen positive?

• You will get a report from MOH with the level
  asking for repeat sample within 5 days (usually gt
  0.24)
• Repeat the sample 101 will be normal
• If repeat still positive esp. if
  gt0.36. Explain to family and..

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Questions - 2

• What to tell the family with a second positive
  PKU screen?
• A) call and refer to local PKU centre
• B) restrict protein immediately
• C) the child will be mentally retarded or die
• D) do not have more children

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What not to do.

• Do not stop or restrict feeds
• Do not stop breastfeeds
• Do not change to soya milk
• Do not tell family the child will be retarded or
  die

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Refer to the designated PKU centre for follow-up
What to do.

• HSC, CHEO, KGH, CHWO, McMaster

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PKU follow-up at designated centre

• Quantitative plasma amino acids on HPLC
• Rule out biopterin synthesis defect
• Counseling
• Dietary intervention if needed
• Follow-up and monitoring

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Questions - 3

• What is screened for in newborn screening? True
  or false
• A) PKU
• B) Hypothyroidism
• C) Galactosemia
• D) Organic acidopathies
• E) Urea cycle defects
• F) Fatty acid oxidation defects