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Inborn Errors of Metabolism Dr B.Vahabi 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 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. Metabolism Food Enzyme A Amino acids Carbohydrates Lipids Nucleic acids Enzyme B Protein Carriers Energy Biomolecules 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 Transporters Metabolite D Enzyme 1 Enzyme 2 Substrate Metabolite A Accumulation of substances present in small amount Deficiency of critical intermediary products Metabolite B Deficiency of specific final products • The concept of inborn errors of metabolism (IEM) was first introduced by Archibald Garrod in 1908. 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 Inheritance •Majority IEMs are autosomal recessive •Some IEMs are X-linked (Mothers are carriers) •Mitochondrial diseases have also been detected Categories of IEMs •Amino acid metabolism disorders •Carbohydrate metabolism disorders •Lysosomal storage disorders •Fatty acid oxidation disorders •Urea cycle defects •Peroxisomal disorders •Mitochondrial disorders 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 Amino acid metabolism disorders Examples: •Phenylketonuria- phenylalanine • Homocysteinuria- methionine •Maple syrup urine disease- Leucine, isoleuscine and valine •Tyrosinaemia- Tyrosine 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 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 Molecular genetics and classification contd… • PKU is classified by the severity of hyperphenylalaninaemia: – Blood Phenylalanine concentrations of: • 50-110 µmol/Lnormal • 600-1200µ Mol/L Mild • >1200 µMol/L classic PKU • Classification difficult in newborn babies • Classification can also be made on the basis of tolerance for dietary phenylalanin Pathophysiology of PKU • Phenylalanine’s entry into the brain is mediated by the large neutral aminoacid carrier Laminoacid transporter (LAT1) • Raised phenylalanine concentration can induce damage in the brain by: – Reducing formation of myeline in brain’s 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-3methylglutaryl coenzyme reductase. 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. 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 Carbohydrate metabolism disorders Examples: • • • • Glycogen storage diseases Galactosemia Fructose intolerance Fructose 1,6-diphosphate deficiency 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.) Pathways of liver glucose production Von Gierke disease (GSD type I) • Caused by defective liver glucose 6phosphatase activity • Mutations can either be in: – Gene coding for the liver glucose-6phosphatase – Gene coding for endoplasmic reticulum substrate – Product transport proteins of the glucose-6-phosphatase system 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 Presentation of Von Gierke 1b disease • In addition to clinical symptoms seen in GSD1a: – – – – – – – Recurrent infections Neutropenia Neutrophil dysfunction Inflammatory bowl disease Fever Diarrhea Perioral and anal ulcers 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 Lysosomal storage disorders Examples • Tay-Sachs • Niemann-Pick disease • Gaucher’s disease Tay-Sachs disease • An autosomal recessive disorder with an overall prevalence of 1:300 • More prevalent in Ashkenazi Jews and Ferench Canadians • Lack of lysosomal β-hexosaminidase A (Hex-A) enzyme activity • Mutations in the α-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 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 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. Diagnosis and Management There are 3 important steps in the diagnosis and management of IEM: 1. Suspicion 2. Evaluation 3. Treatment 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 Usual clinical presentation of IEMs Neonates Young Children • • • • • • •Recurring vomiting •Dysmorphic features (characteristic facial expression, slant of eyes) •Developmental delay (milestones) •Seizures •Mental retardation Poor feeding Vomiting Apnoea (breathing disorder) Irritability Abnormal tone Seizures Developmental delay 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 Evaluation-2 • There are two different types of testing for metabolic conditions: screening tests and diseasespecific 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 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. 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 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 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 Odours attributed to IEMs Phenylketonuria (PKU) Musty, mousy Tyrosinemia Musty, Cabbage like Maple syrup urine disease Sweet, Maple syrup Isovaleric acidemia Multiple carboxylase deficiency Sweaty feet Cat urine 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 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 Brain in Tay-Sachs disease • 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: 1) Prevent Catabolism 2) Limit the intake of the offending substance 3) Increase excretion of toxic metabolites 4) Enzyme-replacement therapy 5) Increase the residual enzyme activity 6) Reduce substrate synthesis 7) Replacement of the end products 8) Transplantation and gene therapy Treatments/ Management of IEMs 1) 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 Treatments/ Management of IEMs 4) Enzyme replacement therapy Replacement of the deficient enzyme E.g.Human alpha glucosidase enzyme is used for treatment of pompe’s 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. 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 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 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 Summary • Individually rare but collectively important • Present a wide variety of metabolic disorders • Can be present at different stages of development • Can be fatal!! Key references • Blau, N. et al (2010) Phenyketonuria. The Lancet. 376:1417-1427 • Martins, A.M. (1999) Inborn errors of metabolism: a clinical overview. Sao Paulo Med J/Rev Paul Med. 117:251-65 • Myerowitz, R (1997) Tay-Sachs Disease-Causing Mutations and Neutral Polymorphisms in the Hex A Gene. Human Mutation. 9:195-208 • Bayraktar, Y. (2007) Glycogen storage diseases:New perspective. World J Gastreonterol. 13:2541-2553 • 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. 2:274-281 • Saudubray, J.M. et al (2002) Clinical approach to inherited metabolic disorders in neonates: an overview. 7:3-15 • 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. 21202122