Approach to IEMAA - PKU

Annette FeigenbaumDivision of Clinical and Metabolic Genetics, HSC

2002

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.

Functional proteins

• Catalytic enzymes

• Transport - Famililal hypercholesterolemia, Cystic Fibrosis

• Structural - DMDystrophy, Osteogenesis I

• Homeostasis - immune response, clotting

• Growth and Differentiation

• Communication - receptors, hormones, transducers

Enzymopathies

• Usually autosomal recessive

• Substrate accumulation and/or product deficiency

• Small or large molecules

• Multiple enzymes can be affected in cofactor deficiencies

Also

• Deficiencies affecting dimerization

• Deficiencies of Modifying enzymes

• Organelle biosynthesis defects e.g peroxisomal

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

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

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

The Enzyme Defect

Phenylalanine Tyrosine

Protein

PAH

DHPR

PhenylpyruvicAcid

BH4

BH4 - tetrahydrobiopterin (cofactor)

DHPR - dihydropteridine reductase (recycles BH4)

qBH2

•Ingested•Catabolism

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)

Classical PKU-small molecule disease

• Untreated severe MR, IQ <40, sz

• High Phe Dev delay

• Low Tyrosine Executive Fx

• Neurotransmitter deficiency ? Seizures

Heterogeneity

• Clinical-clinically different phenotypes

caused by mutations at the same locus

• Genetic- same or similar phenotypes caused

by different genetic mechanisms

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

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

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

Mutations

• transcription- promotor

• RNA splicing/cleavage

• Point mutation: nonsense, frameshift, missense- null

• Large mutations: frameshift deletion, insertions, duplications- all null

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

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%

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

Mutation ClassificationMutation Classification

Prenatal Diagnosis

• Available - direct mutation, linkage

• Possible outcomes ie. Classical, atypical PKU

• ?Desired• perceived risk• burden• acceptable outcome

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

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

PKU- plasma TLC screen

Urine amino acid qualitative screenUrine amino acid qualitative screen

Maple Syrup Urine Disease-quantitative amino acids -High Performance Liquid Chromatography

Normal

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 screeningPrinciples: 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 MRavoidance of maternal PKUprenatal

• 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

diseases

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

Liquid injection

Q1m/z mass

Q3m/z mass

Q2N2 Collision Cell

IonizationIon Spray Detector

Tandem Mass Spectrometry

Q1 Q2 Q3

Janice Fletcher

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:

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

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

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

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 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…..

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

Refer to the designated PKU centre for follow-up

HSC, CHEO, KGH, CHWO, McMaster

What to do….

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

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