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
• 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 CaucasiansBritish, N. Europe
• Phenylalanine hydroxylase deficiency liver
• Autosomal recessive
• Phenylalanine+BH4 ---X--->tyrosine + BH4
-->Dopamine
-->Norepinephrine -->Epinephrine
The Enzyme Defect
BH4 - tetrahydrobiopterin (cofactor)
DHPR - dihydropteridine reductase (recycles BH4)
Protein
•Ingested
•Catabolism
PAH
Phenylalanine
Tyrosine
Phenylpyruvic
Acid
BH4
DHPR
qBH2
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 consequenceslearning, 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
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•
•
•
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
– IVS12G-A+1
Classical PKU
– Y414C
Mild hyperphe
– 13 other mutations
– 55 other mutations
(18.7%)
(7.8%)
(5.4%)
(1-5%)
(<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 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
Horst Bickel and Robert Guthrie benefit ratio
– Prevention maternal PKU
syndrome
PKU- plasma TLC screen
Urine 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 screening
Principles:
•
•
•
•
common
medically significant
effective treatment
test sensitive
• test specific
• easy to do, rapid
•
•
•
•
PKU
1:±12 000 LB NA
diet
few false negatives <1%,
may miss Type III
false positives
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
Costs of newborn screening
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•
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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
Tandem Mass Spectrometry
Liquid
injection
Ionization
Ion Spray
Q1
Q2
Q3
m/z mass
N2 Collision Cell
m/z mass
Q1
Janice Fletcher
Q2
Q3
Detector
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
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
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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….
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•
•
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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
centre
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•
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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