PKU by jizhen1947


									      Approach to IEM
         AA - PKU

               Annette Feigenbaum
Division of Clinical and Metabolic Genetics, HSC

• 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.
       Functional proteins

• Catalytic enzymes
• Transport - Famililal hypercholesterolemia,
  Cystic Fibrosis
• Structural - DMDystrophy, Osteogenesis I
• Homeostasis - immune response, clotting
• Growth and Differentiation
• Communication - receptors, hormones,
• Usually autosomal
• Substrate
  accumulation and/or
  product deficiency
• Small or large
• Multiple enzymes can
  be affected in cofactor
• Deficiencies affecting dimerization
• Deficiencies of Modifying enzymes
• Organelle biosynthesis defects e.g
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
       Neonatal or later onset
• Amino acids
  – MSUD-acute, chronic ataxia, intermittent
  – 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
     PKU / Phenylketonuria 1934

• Commonest IEM AA metabolism Caucasians-
  British, N. Europe
• Phenylalanine hydroxylase deficiency liver
• Autosomal recessive
• Phenylalanine+BH4 ---X--->tyrosine + BH4

-->Norepinephrine -->Epinephrine
                        The Enzyme Defect
 BH4 - tetrahydrobiopterin (cofactor)
 DHPR - dihydropteridine reductase (recycles BH4)
          •Catabolism          PAH

    Phenylalanine                                      Tyrosine

                         BH4                    qBH2
• Elevated Phenylalanine levels often
  >1000/even 2000uM
• normal:
  – adult 58+-15 uM
  – teenage 60 +-13
  – child 63+-18
  – newborn <120 uM ( 2mg/dl)
    Classical PKU-small molecule
• Untreated               severe MR, IQ <40, sz

• High Phe                      Dev delay
• Low Tyrosine                  Executive Fx
• Neurotransmitter deficiency ? Seizures
• Clinical
     -clinically different phenotypes caused
  by mutations at the same locus
• Genetic
     - same or similar phenotypes caused
  by different genetic mechanisms
   • 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
      Clinical - Genetic heterogeneity
• Classical PKU
  – “severe”
  – <1% 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 <600uM
  – no diet needed

• Type IV/Malignant PKU 2%
  – BH4 cofactor defect
  – need neurotransmitter replacement therapy
  – outcome often not good
           PKU MRI - abnormal white matter
Even in treated PKU there are neurological consequences-
learning, executive function problems etc
            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
•   Chromosome 12q24.1
•   Gene cloned 1983 90kB, 13 exons
•   >350 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
• transcription- promotor
• RNA splicing/cleavage
• Point mutation: nonsense, frameshift,
  missense- null
• Large mutations: frameshift deletion,
  insertions, duplications- all null
• 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
• Abnormal regulatory domain - altered
  level of expression
            PAH mutations

• Most common mutations (North America)
  – R408W             Classical PKU   (18.7%)
  – IVS12G-A+1        Classical PKU   (7.8%)
  – Y414C             Mild hyperphe   (5.4%)
  – 13 other mutations                (1-5%)    31.9%

  – 55 other mutations                (<1%)
• 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
• 2 mutations with similar severity may confer a
  milder phenotype than either would do alone
Mutation Classification
          Prenatal Diagnosis
• Available - direct mutation, linkage
• Possible outcomes ie. Classical, atypical

• ?Desired
     • perceived risk
     • burden
     • acceptable outcome
            Maternal PKU
Untreated Risks
• 92% MR
• 73% microcephaly
• 40% growth retardation
• 12% congenital anomalies
• maternal levels 120-360 mol/L
  preconception throughout pregnancy; diet
  must be closely monitored to avoid fetal
  damage from malnutrition
    PKU Neonatal screening

                               – Guthrie 1961
                               – Ontario Started 1965
                               – Blood spot (filter paper)
                                 samples using the Guthrie
                                 bacterial inhibition assay
                               – Normal plasma Phe<0.24 mM.
                               – Cost effective: 2.5-6.6 cost
Horst Bickel and Robert Guthrie benefit ratio
                               – Prevention maternal PKU
PKU- plasma TLC screen
Urine amino acid qualitative screen
         Maple Syrup Urine Disease
         -quantitative amino acids
         -High Performance Liquid Chromatography

   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 it’s variants 1/12,00 births
     • Severe (Classical) form PKU 1/21,000 births.
• Present status Ontario:
  – PKU : Phenylalanine hydroxylase deficiency
  – Congenital Hypothyroidism 1:4000
        Newborn population screening
Principles:                                           PKU
•   common                                   1:±12 000 LB NA
•   medically significant
•   effective treatment                      diet
•   test sensitive                           few false negatives <1%,
                                             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
•   access and support to follow-up and treatment
•   centralized, coordinated, controlled, monitored, egalitarian
        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 10:1 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
           Expanded Neonatal
• 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
• AAP recommends integrated program that incorporates
  screening, diagnosis, management and support
           Tandem Mass Spectrometry
        Liquid    Ion Spray
                                 Q1             Q2                   Q3       Detector
      injection                m/z mass   N2 Collision Cell        m/z mass

                                Q1               Q2           Q3

Janice Fletcher
Tandem Mass Spectroscopy

   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
        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
• Petition MOH Ontario to support expanded
  newborn screening by tandem MS and
  added tests as already exists in NS,
• Establish the Coordinated Genetic
  Screening centre at HSC
  – Service, resource, support, education
            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
              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
    What to do if initial screen
• You will get a report from MOH with the
  level asking for repeat sample within 5
  days (usually > 0.24)
• Repeat the sample – 10:1 will be normal
• If repeat still positive esp. if >0.36…….

     Explain to family and…..
              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
             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
        What to do….

Refer to the designated PKU
    centre for follow-up

  HSC, CHEO, KGH, CHWO, McMaster
    PKU follow-up at designated
•   Quantitative plasma amino acids on HPLC
•   Rule out biopterin synthesis defect
•   Counseling
•   Dietary intervention if needed
•   Follow-up and monitoring
              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

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