Inborn Errors of Metabolism

Dr Mick Henderson

Biochemical Genetics Leeds Teaching Hospitals

What this seminar will cover

• Overview of principles in IEM • Neonatal screening • Approach to diagnosis • Investigating suspected cases – Examples from disorders of intermediary metabolism It won’t and cant be a comprehensive review of all IEM

Inborn Errors of Metabolism

• Individually rare, collectively a significant health problem • Approx 1:1-2,000 live births • 25-50 in Yorkshire p.a.

• Most present in childhood

Enzyme Deficiencies z a

b

cofactor

c c’ase active c’ase inactive IEM are not just enzyme defects, can also be transport proteins, receptors or structural components

d

Mechanisms of Disease

• accumulation of a toxin • energy deficiency • deficient production of essential metabolite / structural component

Investigation of IEM

’

s

•

Population Screening

-Neonatal screening programme •

Individual Case Investigation

- based in clinical presentation

Neonatal screening in the UK

• Phenylketonuria • Hypothyroidism • Cystic fibrosis • MCAD deficiency • Sickle disease Leeds now involved in pilot for expanded screening, GA1, IVA, LCHADD, Hcys, MSUD

www.expandedscreening.org

Neonatal screening card

Guthrie sample: Good sample

Front Back

Front

Guthrie sample: repeat needed

Back

Leeds Neonatal Screening Pathway

Approach to Diagnosis

Investigations likely to be directed by presentation

Acute vs Chronic

Acute

: may be medical emergency, hypoglycaemia, hyperammonaemia, metabolic acidosis

Chronic

: more difficult, need to decide how many investigations to persue • •

Specific

clinical features that immediately suggest a disorder or group e.g.

Dislocated optic lenses Acute abdominal pain, red urine, hyponatraemia

Metabolic Investigations

• Most lab’s have a urine “ metabolic screen ” • Common initial profile: Urine; Organic acids Amino acids Sugar Chromatography Oligosaccharides Mucopolysaccharides Blood; Amino acids Acylcarnitines

Further tests

Mutation analysis

CF mutations

Enzyme assays

White cells

Biopsies

Fibroblast studies

N.B. Genetic testing is changing fast, increasing use of NGS for arrays and whole exome sequencing

Examples from different IEM groups

Amino acid metabolism:

phenylketonuria (PKU) maple Syrup Urine Disease (MSUD) homocystinuria

Urea cycle disorders

arginino succinnic aciduria OTC deficiency

Organic Acidaemias

propionic acidaemia methyl malonic aciduria isovaleric acidaemia

Fat Oxidation Defects:

MCAD deficiency

Carbohydrate Metabolism

: glycogen storage disorders galactosaemia Lysosomal storage disorders: gaucher and Fabry diseases mucopolysaccharidoses

Transport protein defects:

cystic Fibrosis cystinuria cystinosis

Mitochondrial disorders:

Pearson syndrome cytochrome oxidase def

Phenylketonuria

• Affects 1: 10,000 Caucasian births • Severe mental retardation untreated • Excellent prognosis if treated from birth • Screening test: bloodspot phenylalanine • Confirm diagnosis with plasma phe measurements –

no need to meas enzyme or DNA

Phenylketones

 

Classical PKU

Dihydrobiopterin reductase

Phenylalanine

 

Pre-block metabolite increases

Tetrahydrobiopterin (reduced)

Phenylalanine

X Dihydrobiopterin (Oxidised)

Tyrosine



Post-block metabolite decreases

Treatment

• Low phenylalanine diet – requires careful monitoring – risk of tyrosine insufficiency – risk vitamin and trace element deficiencies • ? biopterin supplementation (sapropterin) • Large Neutral Amino Acids (val, leu, ileu) supplements • Diet for life • Management of PKU pregnancies

Low tyrosine High phe

Metabolism of homocysteine

tetrahydrofolate methionine cobalamin

MTHF reductase

5-me tetrahydrofolate homocystine betaine Methyl donor reactions

homocysteine

pyridoxine

cystathionine synthase

cystathionine cysteine

Molecular Forms

COOH CHCH2 CH2SH NH 2 Homocysteine COOH COOH CHCH 2 CH 2 SSCH 2 CH 2 CH NH 2 NH 2 Homocystine

• • • •

Natural History of Clasical Homocystinuria

Mudd et al Am J Hum Genet 1985; 31: 1-31

Lens dislocation:

– 82% dislocated by age 10 years

Osteoporosis (x-ray):

– 64% with osteoporosis by age 15 yrs

Vascular events:

– 27% had an event by age 15 years

Death:

– 23% will not survive to age 30 years • Mental Retardation – approx 50%

A tragic Case

Female, 21 years old Pregnant Normal delivery Died 3 days post partum Saggital vein thrombosis

Tests Performed

Urine Positive cyanide-nitroprusside test Chromatography: homocystine Plasma total homocysteine: 152  mol/L (ref <18)

No comment on haematology report as to significance

Follow Up

Aunt

had similar event some years before Siblings : F, 20y: F, 18y: M,16y: M, 7y:

Total homocysteine (



mol/L)

3 268 263 4

Urea cycle & Hyperammonaemia

Hyperammonaemia variable depends on: nature of IEM - nutrition, protein intake - renal clearance of metabolites - Lyonisation (for x-linked OTC)

Plasma Ammonia

• Lithium heparin • Phone lab to request urgently • Transport immediately to lab Delays cause falsely high ammonia • Avoid contamination: smoking

The Urea Cycle

Converts highly toxic ammonia to less toxic urea

Ornithine NH 3 + Bicarbonate

Carbamyl phosphate synthetase

Carbamyl Phosphate

Ornithine Transcarbamylase (OTC)

Citrulline Urea

Argininosuccinate Synthase Arginase

Arginine Argininosuccinic acid

Argininosuccinate Lyase

Disorders of urea cycle

Side pathway utilised

NH 3

 Marked hyperammonaemia

Carbamyl phosphate synthetase

Orotic acid Carbamyl Phosphate

Ornithine Transcarbamylase (OTC)

Citrulline OTC deficiency Urea

 Impaired urea synthesis

Ornithine

Arginase

Argininosuccinic acid Arginine

Argininosuccinate Lyase

Arginino succinic aciduria

OTC deficiency

Side pathway utilised

Orotic acid

Marked hyperammonaemia

NH 3



Carbamyl phosphate synthetase

Urea

 Impaired urea synthesis

Ornithine Carbamyl Phosphate

Ornithine Transcarbamylase (OTC)

X

Citrulline

Argininosuccinate Synthase Arginase

Argininosuccinic acid Arginine

Argininosuccinate Lyase

OTC deficiency

• OTC deficiency is x-linked • Males and female homozygotes are severely affected • Female heterozygotes are variably affected due to random x inactivation (Lyonisation)

OTC presentation - infancy

– 12- 72 hours of age – Lethargic and poor feeding – Abnormal respirations – vomiting – Seizure – Decreasing conscious level – If untreated die

Differential diagnosis is sepsis / meningitis – Initial investigations • Gas –

respiratory alkalosis

• FBC • U+E, Ca2+, glucose –

increase anion gap

(anion gap Na – (HCO3+Cl) normally 8 to 11) • Lactate, ammonia • LFT (often abnormal) • Urine and blood cultures and ?LP (also test urine for ketones) – Initial management • commence IV antibiotics... • review management with results of investigations –

Interpreting Ammonia

• Abnormal • >200 µmol/l premature neonates • >100 µmol/l term neonates • >40 µmol/l in older infants Prognosis depends on duration and degree of hyperammonaemia • <500 = 94% surivival • >1000 = 34% survival

Hyperammonaemia Treatment • Protein restriction • Antibiotics • Benzoate • Phenylbutyrate /Phenylacetate • Arginine • Carbaglu • Dialysis

Alternative pathway treatment

• Benzoate • phenylbutyrate

Benzoate Benzoate Therapy + Glycine Hippurate

Phenylbutyrate/acetate Therapy Phenylbutyrate

Phenylbutyryl CoA

Phenylacetate Glutamine Phenylacetylglutamine Urine

N-Carbamoyl-L-glutamic acid • Carbaglu • Marketed by Orphan • N-acetylglutamate analogue • Stimulates Carbamyl phosphate synthetase • Particularly useful in NAGS def

Arginine supplementation

Arginine deficiency common in many urea cycle defects Neonates and young infants have high requirement for arginine

Argininosuccinic aciduria

Why can ammonia be normal ?

NH 3 normal,



or



Carbamyl phosphate synthetase

Urea



Ornithine Carbamyl Phosphate

Ornithine Transcarbamylase (OTC)

Citrulline



Argininosuccinate Synthase Arginase



Arginine Argininosuccinic acid

 X

Argininosuccinate Lyase

Arginino succinic aciduria

Ornithine NH 3

Carbamyl phosphate synthetase

Carbamyl Phosphate

Ornithine Transcarbamylase (OTC)

Citrulline Urea

Argininosuccinate Synthase Arginase

Arginine Urea cycle effectively becomes a

“

linear pathway

”

provided arginine intake is adequate Argininosuccinic acid (ASA)



Renal clearance of ASA is much higher than for citrulline. NH 3 is excreted as ASA

Small MW organic acids are intermediates in most metabolic pathways amino acids neurotransmitters carbohydrates drugs, diet Organic acids micro organisms cholesterol purines pyrimidines fatty acids

Clinical indications Acute, intoxication

• Unexplained metabolic acidosis • Hyperammonaemia • Hypoglycaemia • Lactic acidaemia • ketonuria

Chronic

• Developmental delay • Fits or seizures • Liver disease

Branched chain amino acid catabolism Leucine 2-Oxoisocaproic Valine 2-Oxoisvaleric Isoleucine 2-Oxo-3-methylvaleric Isovaleryl-CoA 3-Methylcrotonyl-CoA 3-Methylglutaconyl-CoA 3-OH-3-Methylglutaryl CoA 2-Methylmalonic acid semialdehyde Propionyl CoA 2-Methylmalonyl-CoA Succinyl CoA 2-Methylbutyryl Triglyl-CoA 2-Methyl-3OHbutyryl-CoA Acetyl-CoA

Case history

• Male baby • Hx of miscarriages Parents First cousins 3 other children • Admitted Local District Hospital @ 5days • Feeding difficulty • Lethargy • ?sepsis • ?IEM

Day 7: Na K Urea Creatinine Bicarb Glu

Case

cont

156 3.5

24.5

123 12 22 Ammonia 1850  mol/L (ref<40) Transferred to Leeds Urine organic acids: Gross inc. in isovalerylglycine and 3OHisovaleric acid Diagnosis Isovaleric acidaemia Day 9 Died in hospital

Hyperammonaemia in organic acidaemias glutamate + acetyl CoA propionyl CoA methylmalonyl CoA -ve N-acetyl glutamate ATP + CO 2 + NH 3 +ve

CPS synthetase NAG synthetase

Carbamoyl phosphate

UREA CYCLE

Methyl Malonic Acidaemia

• Episodes of metabolic acidosis triggered by intercurrent illness • Hyperammonaemia, ketoacidosis • Vomiting • Poor weight gain • Progressive loss of renal function • Hypotonia and later learning difficulties • Seizures

Methyl Malonic Acidaemia: B12 Treatment

• Vitamin B12 is a co-factor

Test for B12 responsiveness

• Pre-B12, MMA in urine… • Post-B12, MMA in urine… 4359 3332 5181 279 982 472 (units= umol/mmol creatinine)

Methyl Malonic Acidaemia: other treatments

• Protein restriction • Carnitine supplements • Antibiotics • Management of CRF

MCAD deficiency

• A fat oxidation defect • Unable to mobilise full energy from fat during fasting • Prolonged fasting → hypoglycaemia • Impaired ketone production – Hypoketotic response to hypoglycaemia • Incidence  1 in 10,000

MCAD; the biochemical defect Acyl carnitines fatty acids carnitine ketones

mitochondria

acetyl CoA fatty acid oxidation TCA cycle dicarboxylic acids URINE

Ketone production pathways

Triglycerides Glucagon, Cortisol Growth hormone, Adrenaline Insulin Free fatty acids + Glycerol Mitochondrial  -oxidation Acetyl CoA TCA cycle Ketones (3-hydroxybutyrate, acetoacetate)

MCAD deficiency

• Before screening  25% of diagnoses were post mortem • Crisis often follows D&V, chest infections, etc i.e. prolonged fasting when present with lethargy and decrease conscious level or seizures • Hypoglycaemia severe (→ zero) and no ketones in urine • Neonatal screening now mandated UK-wide • Untreated ongoing problems liver and brain damage, coma, and sudden death.

MCAD deficiency

• Treatment in crisis: ABC in A&E • Check BM for all unconsious children if low then bloods hypoglycaemia screen • IV dextrose • Recovery time relatively high • Slow recovery is partly due to accumulation of toxic metabolites • Dieticians: Emergency advice/packs

Classical Galactosaemia: Initially

• 1 week old, F, term delivery • Milk feeds established, poor feeder and failing to thrive • Vomiting, diarrhoea, jaundice, hepatomegaly • LFT ’ s: Bilirubin – 371 Conj Bili – 136 (ie around 30%) ALT – 199 Alk Phos – 2293 • Cataracts • Deranged clotting

Metabolic Investigations

Prolonged jaundice screen - Obstructive jaundice – remember Gal-1-PUTas well as biliary atresia Metabolic investigations • Urine Organic acids: NAD • Urine amino acids:

generalised amino aciduria

• Urine sugar chromatography: NAD Galactosaemia screen: Absent activity

Galactosaemia Diagnosis: Pitfalls

• Galactosuria: False Neg & Pos results • False Neg: If no galactose intake • Galactosaemia blood test is red cell enzyme • Invalid if child has been blood transfused

Galactosaemia management

• Primary source of galactose is lactose therefore: – Stop breast feeding – Involve a dietician – Lactose free formula (or soy formula) – Lactose free diet • Good prognosis but even if well treated there are long term complications – short stature – Female infertility

Nephropathic Cystinosis



Presents with:

Fanconi syndrome: generalised aminoaciduria glycosuria phosphaturia   Polyuria, Polydipsia Failure to thrive   Renal failure Rickets  Fair complexion  Photophobia – Cysteine crystals on slit lamp examination  Incidence: ~ 1 in 200,000 live births  Defective lysosomal membrane transport protein for cystine,

cystinosin

Cystinosis: Diagnosis

Clinical - Opthalmology

 Corneal cystine crystals on slit-lamp examination of eye

Biochemical

 White cell cystine (definitive)  CVS / amniotic fluid prenatal available

Lysosomal metabolism

Amino acids Specific TP

’

s Amino acids

Hydrolysis

Proteins

Treatment of cystinosis with cystagon

Cystine Cysteamine Cystine Cysteamine Cysteine Cysteamine -cysteine

Lysine TP

Cysteamine -cysteine