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Approach to IEM AA - PKU

Approach to IEM AA - PKU. Annette Feigenbaum Division of Clinical and Metabolic Genetics, HSC 2002. Enzymes. Protein catalysts that rapidly mediate the chemical reactions in the body

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Approach to IEM AA - PKU

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  1. Approach to IEMAA - PKU Annette Feigenbaum Division of Clinical and Metabolic Genetics, HSC 2002

  2. 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.

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

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

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

  6. 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

  7. 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

  8. PKU / Phenylketonuria 1934 • Commonest IEM AA metabolism Caucasians-British, N. Europe • Phenylalanine hydroxylase deficiency liver • Autosomal recessive • Phenylalanine+BH4 ---X--->tyrosine + BH4 -->Dopamine -->Norepinephrine -->Epinephrine

  9. The Enzyme Defect BH4 - tetrahydrobiopterin (cofactor) DHPR - dihydropteridine reductase (recycles BH4) Protein • Ingested • Catabolism PAH Phenylalanine Tyrosine Phenylpyruvic Acid DHPR BH4 qBH2

  10. PKU-2 • Elevated Phenylalanine levels often >1000/even 2000uM • normal: • adult 58+-15 uM • teenage 60 +-13 • child 63+-18 • newborn <120 uM ( 2mg/dl)

  11. Classical PKU-small molecule disease • Untreated severe MR, IQ <40, sz • High Phe Dev delay • Low Tyrosine Executive Fx • Neurotransmitter deficiency ? Seizures

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

  13. 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

  14. 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

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

  16. 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

  17. PKU-3 • 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

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

  19. 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

  20. 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%) • 55 other mutations (<1%) 31.9%

  21. 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

  22. Mutation Classification

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

  24. 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

  25. 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 benefit ratio • Prevention maternal PKU syndrome Horst Bickel and Robert Guthrie

  26. PKU- plasma TLC screen

  27. Urine amino acid qualitative screen

  28. Maple Syrup Urine Disease • quantitative amino acids • High Performance Liquid Chromatography Normal

  29. 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

  30. 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,Guthriebloodspot, 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

  31. 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 diseases

  32. 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

  33. Tandem Mass Spectrometry Ionization Ion Spray Q1 m/z mass Q2 N2 Collision Cell Q3 m/z mass Detector Liquid injection Q1Q2Q3 JaniceFletcher

  34. Tandem Mass Spectroscopy MS/MS • 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:

  35. 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

  36. 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

  37. 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

  38. 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

  39. 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 > 0.24) • Repeat the sample – 10:1 will be normal • If repeat still positive esp. if >0.36……. Explain to family and…..

  40. 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

  41. 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

  42. What to do…. Refer to the designated PKU centre for follow-up HSC, CHEO, KGH, CHWO, McMaster

  43. 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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