Inborn Errors of Metabolism

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Inborn Errors of Metabolism

• Dr B.Vahabi

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Lecture outcomes

• Understand the general pathophysiology underlying
  the inborn errors of metabolism (IEMs)
• Review some important IEMs
• Understand the genetic inheritance of IEMs
• Review the general diagnostic methods used for
  detection of IEMs
• Discuss the current treatment options for people
  suffering from IEMs

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Metabolism

• Metabolism is the sum of all the chemical
  reactions in the body
• Some chemical reactions are involved in breaking
  down molecules, others are involved in building
  up (synthesis)
• A metabolic pathway consists of several stages
  involved in the conversion of one metabolite to
  another.

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Metabolism
Food
Enzyme A
Amino acids Carbohydrates Lipids Nucleic acids
Enzyme B
Protein Carriers
Energy Biomolecules
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Errors in Metabolism

• If an error occurs in the gene that codes for the
  enzyme a FAULT occurs.
• Subsequently the enzyme is not produced and the
  pathway breaks down.
• These are called INBORN ERRORS OF METABOLISM
  (IEMs).
• IEMs are uncommon but complicated medical
  conditions involving abnormalities in complex
  biochemical and metabolic pathways

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Transporters
Metabolite D
Enzyme 1
Enzyme 2
Substrate
Metabolite B
Metabolite A
Accumulation of substances present in small amount
Deficiency of specific final products
Deficiency of critical intermediary products
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• The concept of inborn errors of metabolism (IEM)
  was first introduced by Archibald Garrod in 1908.

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Incidence

• More than 1000 human diseases are known today
  that are caused by IEM
• Overall prevalence of 1 in 5000
• However the prevalence of each disease has many
  variables
• Certain IEMs have a race related prevalence
• e.g Tay-Sachs in Ashkenazi Jews

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Inheritance

• Majority IEMs are autosomal recessive

• Some IEMs are X-linked (Mothers are carriers)
• Mitochondrial diseases have also been detected

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Categories of IEMs

• Amino acid metabolism disorders
• Carbohydrate metabolism disorders
• Lysosomal storage disorders
• Fatty acid oxidation disorders
• Urea cycle defects
• Peroxisomal disorders
• Mitochondrial disorders

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Amino acid metabolism disorders

• A heterogeneous group of disorders
• Block at early step of metabolic pathway
  resulting in accumulation of amino acids
• Block at later stages of metabolic pathway
  resulting in accumulation of metabolites
• Defect in transport mechanism of amino acids
  resulting in decreased intestinal transport and
  increased urinary excretion

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Amino acid metabolism disorders

• Examples
• Phenylketonuria- phenylalanine
• Homocysteinuria- methionine
• Maple syrup urine disease- Leucine, isoleuscine
  and valine
• Tyrosinaemia- Tyrosine

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Phenylketonuria (PKU)

• Most prevalent disorder caused by inborn errors
  of amino acid metabolism
• Caused by mutations in phenyalanine hydroxylase
  (PAH) gene
• PAH converts phenyalanine into tyrosine and
  requires the cofactor tetrahydrobiopterin (BH4),
  molecular oxygen and iron
• Loss of PAH activity ? increased concentrations
  of phenyalanine in blood an d toxic
  concentrations in the brain

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Molecular genetics and classification

• The PAH gene consists of 13 exons
• PKU arises when both alleles are mutated (548
  separate mutations)
• Some mutations only partly inhibit the enzyme
  activity? mild PKU
• About 1-2 of cases of PKU are due to mutations
  in genes coding for enzymes involved in BH4
  biosynthesis

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Molecular genetics and classification contd

• PKU is classified by the severity of
  hyperphenylalaninaemia
• Blood Phenylalanine concentrations of
• 50-110 µmol/L?normal
• 600-1200µ Mol/L? Mild
• gt1200 µMol/L ?classic PKU
• Classification difficult in newborn babies
• Classification can also be made on the basis of
  tolerance for dietary phenylalanin

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Pathophysiology of PKU

• Phenylalanines entry into the brain is mediated
  by the large neutral aminoacid carrier
  L-aminoacid transporter (LAT1)
• Raised phenylalanine concentration can induce
  damage in the brain by
• Reducing formation of myeline in brains white
  matter
• Inhibition of LAT1 carriers and neutral amino
  acids from entering the brain
• Reduced activity of pyruvate kinase
• Disturbed glutamatergic neurotransmission
• Reduced activity of the enzyme 3-hydroxy-3-methylg
  lutaryl coenzyme reductase.

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Presentation of PKU

• Developmental Delay
• Behavioural abnormalities and motor dysfunction
• Reduced IQ levels
• Autism
• Hypopigmentation (decreased melanin)
• Musty odour
• Detected by newborn screening (heel prick test)
• Can be dietary controlled.

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Carbohydrate metabolism disorders

• A heterogeneous group of disorders
• Caused by inability to metabolize specific
  sugars, aberrant glycogen synthesis or disorders
  of gluconeogenesis
• Manifest with hypoglycemia, hepatosplenomegaly,
  lactic acidosis or ketosis

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Carbohydrate metabolism disorders

• Examples
• Glycogen storage diseases
• Galactosemia
• Fructose intolerance
• Fructose 1,6-diphosphate deficiency

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Glycogen storage diseases (GSDs)

• Characterized by abnormal inherited glycogen
  metabolism in the liver, muscle and brain.
• Lead to build up of glycogen in tissues
• Categorised numerically (0-X)
• (e.g. Type II, Type III etc.)

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Pathways of liver glucose production
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Von Gierke disease (GSD type I)

• Caused by defective liver glucose 6-phosphatase
  activity
• Mutations can either be in
• Gene coding for the liver glucose-6-phosphatase
• Gene coding for endoplasmic reticulum substrate
• Product transport proteins of the
  glucose-6-phosphatase system

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Presentation of Von Gierke 1a disease

• Initial symptoms are due to hypoglycaemia and
  include
• Tremor, irritability, hyperventilation, apnea,
  convulsions, paleness, sweating, cerebral edema,
  coma and death
• Older infants may present with
• Doll-like facial appearance, frequent lethargy,
  difficult arousal from sleep, overwhelming
  hunger, protuberant abdomen, relatively thin
  extremities.
• With ageing the patient presents
• Poor growth, short stature, and rachitic changes
• Most striking laboratory findings
• Hypoglycaemia, lactic acidosis, hyperlipidemia,
  hyperuricaemia, mosaic pattern of the liver, pale
  staining of the tissue and swollen hepatocytes

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Presentation of Von Gierke 1b disease

• In addition to clinical symptoms seen in GSD-1a
• Recurrent infections
• Neutropenia
• Neutrophil dysfunction
• Inflammatory bowl disease
• Fever
• Diarrhea
• Perioral and anal ulcers

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Lysosomal storage disorders

• Genetic disorder inherited in an autosomal
  recessive fashion
• Result from defective lysosomal acid hydrolysis
  of endogenous macromolecules? accumulation of
  glycoproteins, glycolipids or glycosaminoglycans
  within lysosomes in various tissues
• Usually present later in infancy with
  organomegaly, facial coarseness and
  neurodegeneration
• Show progressively degenerative course

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Lysosomal storage disorders

• Examples
• Tay-Sachs
• Niemann-Pick disease
• Gauchers disease

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Tay-Sachs disease

• An autosomal recessive disorder with an overall
  prevalence of 1300
• More prevalent in Ashkenazi Jews and Ferench
  Canadians
• Lack of lysosomal ß-hexosaminidase A (Hex-A)
  enzyme activity
• Mutations in the a-subunit of Hex-A are
  responsible for Hex-A deficiency
• Hex-A breaks down a fatty acid substance called
  GM2 ganglioside in nerve cells
• Accumulation of GM2-ganglioside has a toxic
  effect on cells? neuronal deterioration ? mental
  and motor retardation

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Tay-Sachs disease contd..

• The severity of the disease is inversely
  proportional to the amount of residual Hex-A
  activity
• No Hex-A activity? classic/infantile form? early
  age onset of disease? death early childhood
• Some residual Hex-A activity ? childhood/
  Juvenile/ adult forms? late onset? Less severe
• More than 100 mutations of the alpha-subunit
  have been described

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Presentation of Tay-Sachs disease

• Infant appears normal at birth but within few
  weeks may become less visually attentive,
  hypotonic and easily startled by sound, light or
  touch
• By 6-8 months developmental delay becomes obvious
• Fundiscopic examination of retina reveals a
  whitish surrounding? lipid deposition
• By 1 year ? marked reduction in purposeful
  movement, child becomes spastic and lethargic
• Vision deteriorates
• Frequent seizures
• By age 2 years the child is in a vegetative state
  and requires constant care
• Feeding difficulty
• A light cherry red spot in the middle of the eye
• The brain increases in weight and size but
  shows generalized atrophy and reduction
  in nerves
  and white matter
• Deafness
• Usually death before age of 5.

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Diagnosis and Management

• There are 3 important steps in the diagnosis and
  management of IEM
• Suspicion
• Evaluation
• Treatment

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Suspicion

• An important key to diagnosing IEM is thinking
  about the possibility in the first place
• The symptoms are very common and non-specific
• Screening allows for the differential diagnosis

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Usual clinical presentation of IEMs

• Young Children
• Recurring vomiting
• Dysmorphic features
• (characteristic facial expression, slant of eyes)
• Developmental delay (milestones)
• Seizures
• Mental retardation

• Neonates
• Poor feeding
• Vomiting
• Apnoea (breathing disorder)
• Irritability
• Abnormal tone
• Seizures

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Developmental delay
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Evaluation-1

• Once the possibility of an IEM is suspected,
  how should it be evaluated?
• History
• An important clue is a history of deterioration
  after an initial period of good health
• Developmental delay
• Change in diet and unusual dietary preferences
• Family history
• Most IEMs are autosomal recessive any other
  siblings with the same condition?
• Consanguineous marriages increases the
  incidence of recessive disease

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Evaluation-2

• There are two different types of testing for
  metabolic conditions screening tests and
  disease-specific diagnostic testing
• Initial screening tests
• Prenatal tests
• Ability to detect IEMs prenatally has increased
• Biochemical methods
• Detection of metabolites in amniotic fluid
• Enzyme assays
• DNA analysis
• Detection of genetic mutations

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• Prenatal tests
• Choice of sample can be dictated by which
  disorder is to be tested for.
• Amniocentesis
• Best carried out at 15-16 weeks
• Used for analysis of specific metabolites by gas
  chromatography with mass spectroscopy, tandem
  mass spectroscopy, etc
• Used for detection genetic defects using DNA
  technology
• Intended for diagnosis of some amino acid
  disorders, lysosomal storage disorders etc.

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• Prenatal tests
• Cultured amniotic fluid cells
• Used for measurement of specific enzyme activity
  using various enzyme assays
• Used for the study of various metabolic
  pathways
• Major disadvantage is the delay in waiting for
  sufficient number of cells to grow
• Chorionic villus sampling (CVS)
• Offers a greater advantage over amniocentesis
• Samples are taken at around 11-week gestation
• Used for determination of enzyme activity using
  various enzyme assays

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• Prenatal tests
• Foetal blood and Foetal tissue
• Foetal blood is rarely used
• Sample taken late in pregnancy
• Used when there has been a failure in amniotic
  fluid analysis
• Liver biopsies are used when enzyme deficiencies
  are not expressed in CVS
• Very risky
• Used for diagnosis of conditions where enzyme
  deficiency is expressed in the liver
• Testing of Pre-implantation embryos

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• Postnatal Tests
• The investigation of IEM should begin with simple
  urine and blood analysis.
• Screening tests allow you to detect the presence
  of a particular class of conditions and includes
• Serum electrolytes (looking for evidence of
  acidosis), glucose ammonia levels
• Blood and urine amino acids for disorders of
  amino acid metabolism
• Urine organic acids for disorders of organic acid
  metabolism, Acylcarnitine profile for disorders
  of fatty acid
• Blood lactate and pyruvate for disorders of
  carbohydrate metabolism and mitochondrial
  disorders

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Odours attributed to IEMs

• Phenylketonuria (PKU) Musty, mousy
• Tyrosinemia Musty, Cabbage like
• Maple syrup urine disease Sweet, Maple syrup
• Isovaleric acidemia Sweaty feet
• Multiple carboxylase deficiency Cat urine

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Examples of screening tests

• Tandem mass spectroscopy

• Used for measurement of amino acids and
  acylcarnitines in blood
• Used for detection of disorders of amino-acid,
  organic acid and fatty-acid metabolism.
• Potential of simultaneous multi-disease screening
• Blood taken from newborn babies are absorbed by
  filter paper (can also be used in the Guthrie
  test).
• A punched sample from the dried blood spot is
  extracted with solvent containing appropriate
  isotopes
• The extracted metabolites are identified and
  quantified with electrospray ionisation

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Disease specific diagnostic tests

• Key to exclusion or inclusion of an IEM and
  include
• MRI (Magnetic resonance imaging) can be used for
  detection of demyelination/neuron loss in the
  brain
• MRS (Magnetic resonance spectroscopy) can be used
  for detection of lactate levels in individuals
  with mitochondrial disorder
• Study of cells and tissues obtained via biopsies
  to establish the nature of accumulated material,
  organelle alterations and specific markers

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Treatments/ Management of IEMs

• Treatment depends on the clinical manifestation
  and type of metabolites accumulated
• The basic principal for treatment is reduction of
  the substrate that accumulates due to deficient
  enzyme activity
• This can be mediated by an increasing number of
  therapeutic approaches
• Prevent Catabolism
• Limit the intake of the offending substance
• Increase excretion of toxic metabolites
• Enzyme-replacement therapy
• Increase the residual enzyme activity
• Reduce substrate synthesis
• Replacement of the end products
• Transplantation and gene therapy

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Treatments/ Management of IEMs

• Prevent Catabolism
• Controlling the administration of calories used
  to prevent endogenous protein breakdown and
  induction of anabolism
• 2) Limit the intake of offending substance
• Restriction of certain dietary components.
• E.g. restriction of intake of galactose and
  fructose to prevent galactosaemia and fructose
  intolerance
• E.g. Neonates with PKU should be given protein
  substitute that is phenyalanine-free.
• 3) Increase the excretion of toxic metabolites
• Rapid removal of toxic metabolites can be
  achieved by exchange transfusion, peritoneal
  dialysis, haemodialysis, forced diuresis etc.
• E.g. Haemodialysis is considered mandatory for
  hyperammonaemia

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Treatments/ Management of IEMs

• 4) Enzyme replacement therapy
• Replacement of the deficient enzyme
• E.g.Human alpha glucosidase enzyme is used for
  treatment of pompes disease
• 5) Increase the residual enzyme activity (if
  possible)
• Usually accomplished by administration of
  pharmacological doses of vitamin cofactor for the
  defective enzyme
• 6) Reduce substrate synthesis
• Inhibiting the synthesis of a substrate that can
  not be converted to the end products
• E.g used for treating lysosomal storage disorders
  in order to reduce the rate of glycosphingolipid
  breakdown.

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Treatments/ Management of IEMs

• 7) Replacement of end products
• Replacement of a product due to an enzyme defect
• E.g. in patients with glycogen storage disease,
  hypoglycaemia is prevented with frequent feeds
  during the day and nasogastric feeding during
  night in infants and young children.
• 8) Transplantation and gene therapy
• Bone marrow transplantation (BMT) has been used
  as effective therapy for selected IEMs
• Mainly Lysosomal storage diseases and peroxisomal
  disorders are treated by BMT.
• The main rationale is based on provision of
  correcting enzymes by donor cells within and
  outside the blood compartment.
• In most gene therapy procedures a "normal" gene
  is inserted into the genome to replace an
  "abnormal," disease-causing gene

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Diagnosis and treatment of PKU

• Prenatal diagnosis
• Prenatal diagnosis is less commonly performed for
  PKU due to good prognosis on treatment
• Few have been undertaken by DNA analysis
• Postnatal diagnosis
• Gutherie test using the ability of phenylalanine
  to facilitate bacterial growth in a culture
  medium with an inhibitor.
• The Guthrie assay is sensitive enough to detect
  serum phenylalanine levels of 180-240 µmol/L (3-4
  mg/dL). In healthy normal people, phenylalanine
  levels are usually under 120 µmol/L.
• Tandem mass spectroscopy
• Have a sensitivity of 3umol/l for phenyalanine
• Discrimination is further enhanced by
  simultaneous measurement of tyrosine.
• Defects in BH4 synthesis should also be checked

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Treatment of PKU

• Treatment from birth with a low phenylalanine
  diet largely prevents the deviant cognitive
  phenotype
• Present treatment relies on a diet low in
  phenylalanine
• Tyrosine supplementation in the diet
• Enzyme replacement therapy is being investigated
• Pharmacological doses of exogenous BH4
• Drug based therapeutics using sapropterin
  dihydrochloride which is a synthetic cofactor for
  PAH.
• Gene therapy is being used in preclinical trials
  to deliver the PAH gene into liver

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Summary

• Individually rare but collectively important
• Present a wide variety of metabolic disorders
• Can be present at different stages of development
• Can be fatal!!

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Key references

• Blau, N. et al (2010) Phenyketonuria. The Lancet.
  3761417-1427
• Martins, A.M. (1999) Inborn errors of metabolism
  a clinical overview. Sao Paulo Med J/Rev Paul
  Med. 117251-65
• Myerowitz, R (1997) Tay-Sachs Disease-Causing
  Mutations and Neutral Polymorphisms in the Hex A
  Gene. Human Mutation. 9195-208
• Bayraktar, Y. (2007) Glycogen storage
  diseasesNew perspective. World J Gastreonterol.
  132541-2553
• Shin, Y.S. (2006) Glycogen Storage Disease
  Clinical, Biochemical, and Molecular
  Heterogeneity. Semin Pediatr Neurol. 13 115-120
• Low, L.C.K. (1996) Inborn errors of metabolism
  clinical approach and management. HKMJ. 2274-281
• Saudubray, J.M. et al (2002) Clinical approach to
  inherited metabolic disorders in neonates an
  overview. 73-15
• Besley, G.T.N in Walker, J.M Rapley, R. (Eds
  2001) Medical biomedthods handbook. Humana press
  Inc. Totowa, N.J.
• Burchell, A (2003) Von Gierke disease.
  Encyclopedia of Genetics. 2120-2122