Neonatal Biochemistry Investigation for Inherited Metabolic - - PowerPoint PPT Presentation

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Neonatal Biochemistry Investigation for Inherited Metabolic - - PowerPoint PPT Presentation

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Neonatal Biochemistry Investigation for Inherited Metabolic Disorders (IMDs) Anne Green Birmingham Childrens Hospital Leeds May 2005 Birmingham Childrens Hospital NHS Trust United Kingdom Overview of how IMDs present cases

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Birmingham Childrens Hospital NHS Trust United Kingdom

Neonatal Biochemistry

Investigation for Inherited Metabolic Disorders (IMDs)

Anne Green

Birmingham Children’s Hospital

Leeds May 2005

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Birmingham Childrens Hospital NHS Trust United Kingdom

  • Overview of how IMDs present

– cases

  • Range of Disorders & Incidence

– disorders presenting in the newborn

  • Approach to Investigation
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Birmingham Childrens Hospital NHS Trust United Kingdom

Presentation of IMD

  • Intrauterine

– HELLP – AFLP

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Birmingham Childrens Hospital NHS Trust United Kingdom

  • HELLP
  • Haemolysis
  • Elevated Liver Enzymes
  • Low Platelets
  • AFLP
  • Acute Fatty Liver of Pregnancy
  • severe liver dysfunction
  • thrombocytopenia
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Birmingham Childrens Hospital NHS Trust United Kingdom

LCHADD

  • Long chain 3-hydroxy acyl CoA

dehydrogenase deficiency

  • defect of mitochondrial fat oxidation (1990)
  • hypoketotic hypoglycaemia
  • metabolic collapse (esp. liver)
  • death
  • ther problems include:
  • myopathy and cardiomyopathy
  • retinitis pigmentosa
  • FTT with diarrhoea and vomiting
  • common mutation G1528C
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Birmingham Childrens Hospital NHS Trust United Kingdom

HELLP/AFLP and LCHADD

(Wilcken et al, 1993)

  • 11 pregnancies in 5 mothers (HELLP)
  • 6 LCHADD babies

In pregnancies where foetus has LCHADD, the frequency of pre- eclampsia related conditions is high

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Birmingham Childrens Hospital NHS Trust United Kingdom

HELLP/AFLP and LCHADD (Ibdah et al 1996)

  • 16 families with LCHADD
  • 12 different mutations
  • G1528C found in 50% of mutant alleles
  • 11 women had AFLP
  • All these women were carrying foetuses with

G1528C (5 homozygous, 6 heterozygous)

  • LCHADD G1528C mutation

associated with AFLP

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Birmingham Childrens Hospital NHS Trust United Kingdom

Fatty Acid Oxidation Defects in the Foetus which can cause

AFLP/HELLP

  • LCHADD
  • MCADD
  • Carnitine Palmitoyl transferase type 1
  • SCADD
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 1

1 month

  • presented to A&E with hypoglycaemia
  • collapse → ITU Died
  • post mortem fatty acid changes in

liver/kidney

  • LCHADD diagnosed P.M. (skin

fibroblasts)

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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 1

  • Mother 35/40 HELLP
  • Cord blood diagnosis LCHADD (acyl

carnitines)

  • Treatment - MCT
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Birmingham Childrens Hospital NHS Trust United Kingdom

Investigation of HELLP and AFLP

  • Mother
  • organic acids (urine)
  • carnitine, acyl carnitines (blood)
  • glucose, lactate, (free fatty acids, 3-

hydroxybutyrate)

  • +/- LCHADD DNA
  • Baby
  • organic acids (urine)
  • carnitine, acyl carnitines (blood)
  • LCHADD DNA
  • ? fibroblasts fat oxidation ( esp if baby

dies)

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Birmingham Childrens Hospital NHS Trust United Kingdom

Presentation of IMD

  • Intrauterine
  • HELLP
  • AFLP
  • Birth

– Hydrops – Dysmorphism

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Birmingham Childrens Hospital NHS Trust United Kingdom

Fetal and Neonatal Hydrops

  • Hydrops

–

IMDs are present in approx. 1-2% of non- immune hydrops fetalis

  • Investigate if unexplained, familial or

history of still-births/neonatal deaths/ spontaneous abortions

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Birmingham Childrens Hospital NHS Trust United Kingdom

Investigation for Neonatal/Foetal Hydrops

– skin (chromosomes & ? enzymes) – blood- haemoglobinopathies – placenta - histology – urine (IMD) (amniotic fluid) – liver/muscle- histology/biochemistry – www.metbio.net

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Birmingham Childrens Hospital NHS Trust United Kingdom

Presentation of IMD

  • Intrauterine
  • HELLP
  • AFLP
  • Birth

– Hydrops – Dysmorphism

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Birmingham Childrens Hospital NHS Trust United Kingdom

IMD & Dysmorphism in the neonate

  • Menkes
  • Zellwegers and Z like (Perox disorders)
  • Lysosomal

– GM2,ML2,MPS,Multiple sulphatase

  • Congenital hypothyroidism
  • Maternal PKU
  • CDGS
  • Cholesterol synthesis defects
  • GA II
  • Sulphite/xanthine oxidase
  • Congenital lactic acidoses
  • Mevalonic kinase
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Birmingham Childrens Hospital NHS Trust United Kingdom

Presentation & IMD

  • Intrauterine
  • Birth
  • SUDI
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Birmingham Childrens Hospital NHS Trust United Kingdom

SUDI cases 1999/2000 - age at death

2 4 6 8 10 12 14 16 18 <1 1 2 3 4 5 6 7 8 9 10 >14 age in months

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Birmingham Childrens Hospital NHS Trust United Kingdom

Sudden Unexpected Death in Infancy ( SUDI)

  • Explained SUDI

– infection (respiratory,CNS, GI) – cardiovascular – accident/trauma – metabolic

  • SIDS

– no cause of death is found after a thorough post mortem examination

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Birmingham Childrens Hospital NHS Trust United Kingdom

Investigation of SUDI

  • Non accidental injury

– radiology exam – forensic investigation option

  • Infection
  • Metabolic
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Birmingham Childrens Hospital NHS Trust United Kingdom

Sudden Unexpected Death in Infancy

  • Working Group RCPath & RCPCH

( Sept 2004) Baroness Helena Kennedy

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Birmingham Childrens Hospital NHS Trust United Kingdom

SUDI Immediate Specimen Collection

  • Blood

– blood culture – blood spots - IMD – blood - lithium heparin Chromosomes – Blood – serum Toxicology

  • Nasopharyngeal aspirate & swabs
  • Urine Tox & IMD
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Birmingham Childrens Hospital NHS Trust United Kingdom

Biochemical Investigations

  • Amino acids ( blood &urine)
  • Organic acids (urine)
  • Acyl carnitines(blood spot)
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Birmingham Childrens Hospital NHS Trust United Kingdom

Metabolic Investigations Quantitative plasma amino acids

  • All show similar abnormalities

– grossly increased glutamine, glycine, alanine and proline consistent with acute collapse – taurine, aspartate, serine, ornithine increased consistent with haemolysis/autolysis

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Birmingham Childrens Hospital NHS Trust United Kingdom

Quantitative plasma amino acids (µM)

Upper limit of normal indicated by shaded area

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 tau thr glu pro ala AAB cys ile tyr

  • rn

his

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Birmingham Childrens Hospital NHS Trust United Kingdom

Acyl carnitines in SUDI cases

  • blood spots +/- plasma
  • analysed by tandem mass spectrometry
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Birmingham Childrens Hospital NHS Trust United Kingdom

Acyl carnitine results in SUDI

  • most specimens show

– increased free carnitine – increased short chain acyl carnitines – decreased long chain acyl carnitines

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Birmingham Childrens Hospital NHS Trust United Kingdom

Metabolic Investigations

( if indicated)

  • Cultured fibroblasts

– enzymes – DNA

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Birmingham Childrens Hospital NHS Trust United Kingdom

IMD diagnoses in SUDI cases

  • Carnitine transporter
  • LCHADD - 2
  • Citrullinaemia
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Birmingham Childrens Hospital NHS Trust United Kingdom

Sick Neonate

  • Well at birth/no signs or symptoms
  • Family History

– Consanguinity – Sibling illness/death

  • Presentation

– Non specific ( eg poor feeding, hypotonia) – Symptoms relate to feeding

  • Clues

– Smell – Hair/skin – ‘Biochemical’ features

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Birmingham Childrens Hospital NHS Trust United Kingdom

Presentation in the neonate

  • Hypoglycaemia
  • Acid base disturbance

– Metabolic acidosis – Respiratory alkalosis

  • Liver dysfunction/organomegaly

– Jaundice – Hepatitis – Liver failure

  • Neurological dysfunction

– Seizures – Hypotonia – Conscious level – Encepaholopathy

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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 2

  • Consanguinous parents, 2 older siblings

both well

  • 1 Sib died aged 4 days – no Dx
  • Normal birth 38/40
  • Well until 26 hours – jittery , not feeding
  • 40h – seizures, floppy
  • 45h ‘ hiccups’, required ventilation
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 2 ( cont’d)

  • Acid base normal
  • Plasma ammonia normal
  • Liver enzymes normal
  • Calcium, Magnesium ,Glucose normal
  • Hb & FBC normal
  • Lactate 3.5mmol/l ( sl increase)
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 2 ( cont’d)

  • Amino acids(urine)
  • Organic acids
  • Increased urine glycine
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Birmingham Childrens Hospital NHS Trust United Kingdom

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Birmingham Childrens Hospital NHS Trust United Kingdom

Glycinuria

  • Bacterial ( Hippuric acid)
  • Valproate therapy
  • Organic acid disorder eg MMA, PA , IVA
  • Non ketotic hyperglycinaemia
  • Iminoglycinuria
  • Prolinaemia / Hydroxyprolinaemia
  • Atypical persisitent hyperglycinaemia
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Birmingham Childrens Hospital NHS Trust United Kingdom

Metabolic investigation

  • csf glycine

205 µmol/l (<20)

  • plasma glycine

1626 µmol/l (<700)

  • csf:plasma glycine ratio

0.12 (<0.03) Consistent with non-ketotic hyperglycinaemia

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Birmingham Childrens Hospital NHS Trust United Kingdom

csf/plasma glycine ratio in NKH

100 200 300 400 500 600 700 0.00 0.10 0.20 0.30 0.40 0.50 0.60 csf glycine csf/plasma

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Birmingham Childrens Hospital NHS Trust United Kingdom

Progress

  • ventilation withdrawn
  • died 24h later
  • liver biopsy taken

– glycine cleavage enzyme undetectable

  • ante-natal diagnosis possible
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Birmingham Childrens Hospital NHS Trust United Kingdom

CSF quantitative amino acids

  • 0.5ml clear csf in plain bottle (non traumatic)
  • Glycine

– present at low concentrations (<20 µM) – non ketotic hyperglycinaemia

  • looking for high concentrations (usually >50 µM)
  • Serine

– present at low concentrations (approx 40 µM) – serine deficiency disorders

  • looking for low concentrations (usually <20 µM)
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 3

  • 1st child unrelated caucasian parents
  • 38/40 – well for first 24h
  • 28h – jittery ( plasma glucose 4mmol/l)
  • 40h – grunting respiration NNU

peripheral shut down

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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 3 ( cont’d)

  • CSF glucose & protein – normal
  • Na,K Ca ,Mg – normal
  • Started antibiotics ( infection screen awaited )
  • 50h – convulsions Rx phenobarb
  • 62 h – unresponsive, hyperventilating
  • 72h- further convulsions & Bradycardias
  • 74h – DIED ? Sepsis
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 3 cont’d

  • PM

– Fatty liver – Urine

  • Glutamine, alanine & citrulline +++
  • ASA anhydrides
  • Orotic acid ++
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Birmingham Childrens Hospital NHS Trust United Kingdom

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Birmingham Childrens Hospital NHS Trust United Kingdom

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Birmingham Childrens Hospital NHS Trust United Kingdom

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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 4

  • Previous sib died at 2 days – no diagnosis
  • Age 4 days

– 24 hour history of poor feeding and excessive sleeping – totally breast-fed – on admission

  • convulsing
  • hypothermic
  • hypoglycaemic (lab glucose 0.7 mmol/l)
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 4 - metabolic Ix

  • urine organic acids

– dicarboxylic aciduria – glycine conjugates (C6,C8)

  • plasma carnitine

– total 13 mmol/l (23-60) – free 4 mmol/l (15-53)

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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 4 - metabolic Ix

  • DNA

–G985 homozygote

∆ - medium chain acyl CoA dehydrogenase (MCAD) deficiency

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Birmingham Childrens Hospital NHS Trust United Kingdom

MCAD deficiency

  • commonest fatty acid oxidation disorder

– approx 1 in 10,000 births in UK

  • peak age of presentation 12-18m

– 25% die during first attack

  • readily treatable
  • fasting tolerance improves with age
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Birmingham Childrens Hospital NHS Trust United Kingdom

Case 4 - family history

  • previous sib died at 2 days
  • Coroner’s PM

– neonatal infection due to prolonged rupture of membranes; no metabolic Ix

  • tissue obtained for DNA analysis(retrospective)

– G985 homozygote

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Birmingham Childrens Hospital NHS Trust United Kingdom

Lessons

  • importance of family history
  • relationship between symptoms and

feeding

  • MCAD can present in the neonatal

period ( especially if breast fed)

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Birmingham Childrens Hospital NHS Trust United Kingdom

Range of Disorders

  • Intermediary metabolism ‘intoxication’

– Amino acids – Organic acids – Urea cycle – Carbohydrates – Purines/pyrimidines

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Birmingham Childrens Hospital NHS Trust United Kingdom

Range of Disorders

  • Intermediary metabolism ‘intoxication’

– Amino acids – Organic acids – Urea cycle – Carbohydrates – Purines/pyrimidines

  • Energy production ( liver,heart,muscle,brain)

– Mit resp chain/electron transport disorders – Fat oxidation – Gluconeogenesis – Glycogen storage

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Birmingham Childrens Hospital NHS Trust United Kingdom

IMD Diagnoses at BCH*

2000 - 2004

*excludes sibling diagnoses & at risk cases

11 17 20 Total 1 SLO 2 GSD 2 Elect Tr chain 3 2 Zellweger 1 6 Galactosaemia 2 1 AA (Tyr& MSUD) 2 3 Org acid 1 1 3 Fat Ox 3 2 6 NKH 7 UCD

15-28 days

7-14days 0-7 days

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Birmingham Childrens Hospital NHS Trust United Kingdom

  • NKH
  • UCD
  • Galactosaemia
  • Fat oxidation
  • Organic acids
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Birmingham Childrens Hospital NHS Trust United Kingdom

Approach to Investigation

  • Newborn screening
  • Sib testing
  • Clinical Presentation
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Birmingham Childrens Hospital NHS Trust United Kingdom

Newborn Screening Newborn Screening

  • Current universal screening

– PKU* – Congenital hypothyroidism – Duchenne ( Wales) – Cystic Fibrosis ( some areas only) – Sickle Cell disorders – Plus MCADD in ~50% UK – * plus other amino acids - MSUD, tyrosinaemia in some areas

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Birmingham Childrens Hospital NHS Trust United Kingdom

Importance of Sib Testing

  • When to test

– need to start treatment for baby – when does biochem abnormality appear?

  • How to test

– Metabolite – Enzyme – DNA

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Birmingham Childrens Hospital NHS Trust United Kingdom

Approach to Investigations

  • Ist line test

– ‘CLUES to further tests

  • 2nd line tests

– Metabolites

  • 3rd line tests

– Enymes – DNA

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Birmingham Childrens Hospital NHS Trust United Kingdom

First stage Investigations

  • BLOOD

– Calcium – Glucose – Blood count – Blood gases – Sodium, potassium – Liver function – Urea, uric acid – Lactate – Ammonia – FFA

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Birmingham Childrens Hospital NHS Trust United Kingdom

First stage Investigations

  • URINE

– Appearance & Smell – Colour – pH – Reducing substances/glucose – Ketones – Ferric Chloride – DNPH – Cyanide Nitroprusside – TLC MMA

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Birmingham Childrens Hospital NHS Trust United Kingdom

Metabolites

  • Amino acids (urine & plasma)
  • Organic acids (urine)
  • Acyl carnitines (blood)
  • +/-vlcfa
  • Discuss with Lab
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Birmingham Childrens Hospital NHS Trust United Kingdom

  • Consider IMD

– Intrauterine – SUDI – Sick baby

  • Commonest disorders

– Intermediary metabolism – Energy metabolism

  • Staged approach to Ix

Summary