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“Neurotransmitter” Disorders A Laboratory Approach [email protected] Panda SA Workshop, Roodevallei, Pretoria, May 2016

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Page 1: “Neurotransmitter” Disorders A Laboratory · PDF file“Neurotransmitter” Disorders A Laboratory Approach ... convergent squint but no abnormal eye movements detected. ... Pseudo

“Neurotransmitter” Disorders

A Laboratory Approach

[email protected]

Panda SA Workshop,

Roodevallei, Pretoria,

May 2016

Page 2: “Neurotransmitter” Disorders A Laboratory · PDF file“Neurotransmitter” Disorders A Laboratory Approach ... convergent squint but no abnormal eye movements detected. ... Pseudo

Chemical Neurotransmission

• Neurotransmitters –

Substances that upon

release from nerve

terminals, act on

receptor sites at post-

synaptic membranes

to produce either

excitation or inhibition

of the target cell

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BH2

Tyrosine

Tryptophan

Phenylalanine

L-Dopa

5-HTP

Tyrosine

Dopamine

Serotonin

qBH2 BH4

HVA

5-HIAA PLP

O2

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GTP

Dihydroneopterin Triphosphate

6-Pyruvoyltetrahydropterin

Tetrahydrobiopterin

Dihydroneopterin

GTP cyclohydrolase

Pyruvoyl tetrahydroptein synthase

Aldose reductase /Sepiapterin reductase

P3

-VE

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BH4 Salvage

Tyr L-Dopa

BH4 qBH2 DHPR

NADH NAD+

PCD +

PCD = pterin carbinolamine dehydratase

DHPR = dihydropteridine reductase

BH2

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• Tube 1 0.5ml HVA & 5-HIAA

• Tube 2 0.5ml 5-MTHF & PLP

• Tube 3 1.0ml Pterins

CSF – Sample Requirements

(DTE/DETAPAC)

Collect at bedside and freeze immediately (not the form !)

Age related reference ranges

Clinical Details and Drugs

Strong Rostro-caudal Gradient

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With Hyperphenylalaninemia

GTP cyclohydrolase I (GTPCH) deficiency;

Phe = 90-1200 umol/L

6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency;

Phe = 240-2500 umol/L

Dihydropteridine reductase (DHPR) deficiency;

Phe = 180-2500 umol/L

Pterin-4a-carbinolamine dehydratase (PCD) deficiency;

Phe = 180-1200 umol/l

Without hyperphenylalaninemia

Sepiapterin reductase deficiency (SR).

Dopa-responsive dystonia (DRD) due to GTPCH deficiency;

Disorders of BH4 metabolism

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0

100

200

300

400

500

600

700

1.8 1.9

HVA 5-HIAA

DHPR Deficiency – Response to Treatment

Age (Years)

nm

ol/L

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GTP

Dihydroneopterin Triphosphate

6-Pyruvoyltetrahydropterin

Tetrahydrobiopterin

Sepiapterin Reductase Deficiency

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Sepiapterin Reductase Deficiency

Sex; Male. Dob; 31/12/1987. Sample; 09/05/2003. Dystonia

responsive to L-DOPA. No hyperphenylalaninaemia. DHPR normal.

HVA: 23 (71- 565 nmol/L)

5-HIAA: 2 (58- 220 nmol/L)

BH4: 11 (9- 39 nmol/L)

BH2: 64 (0.4- 13.9 nmol/L)

Total Neopterin: 19 (7- 65 nmol/L)

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GTP

Dihydroneopterin Triphosphate

6-Pyruvoyltetrahydropterin

Tetrahydrobiopterin BH2 DHFR -Liver

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L-Dopa Responsive Dystonia

•Hereditary progressive dystonia (Segawa et al., 1971).

•Autosomal Dominant – Female predominance (4:1).

•GTP cyclohydrolase – a causitive gene (Ichinose et al., 1994)

Mutations in gene cause at least 2 disorders:-

AR – present within 6 months, hyperphenylalaninaemia

& marked impaitment of dopamine and serotonin

turnover.

AD - DRD. Residual activity 2-20%.

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0

200

400

600

800

1000

1200

0 1 2 3 4 5 6

Time (hrs)

Phe

(um

ol/L

)

LLN Phe

ULN Phe

Phe

0

50

100

150

200

250

0 1 2 3 4 5 6

Time (hrs)

Tyr

(um

ol/L

)

LLN Tyr

ULN Tyr

Tyr

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Time (hrs)

Ph

e/T

yr

rati

o

LLN P/T ratio

ULN P/T ratio

P/T ratio

Phenylalanine load – DRD. DOB; 20/09/1966. Sample; 13/04/2004

Reported comment:„Phe response slightly

outside 95%CI and

conversion to Tyr

rather sluggish. These

results do not exclude

a pterin related defect.‟

Low CSF neopterin,

BH4, HVA and 5-HIAA

Outcome:

GTP cyclohydrolase

deficiency….

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Tyrosine Hydroxylase Deficiency

Tyr Dopa Dopamine HVA

• Parkinsonian, ptosis, drooling, myoclonic jerks, severe

head lag and trunkal hypotonia.

• L-Dopa marked and sustained improvement in

hypokinesia and parkinsonian symptoms.

• Identified from CSF analysis; Normal pterin & 5-HIAA concentration. Very low HVA. Mutation analysis also available.

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Tyrosine Hydoxylase Deficiency

Sex; Male. Dob; 17/05/2007.

Sample; 27/02/2008

HVA: <10 (154-867 nmol/L)

5-HIAA: 137 (68 -451 nmol/L)

BH4: 36 (19-56 nmol/L)

BH2: 8 (0.4-13.9 nmol/L)

Total Neopterin: 9 (7-65 nmol/L)

Serum Prolactin 706 (86 – 324 mU/ml)

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Increased Dopamine Turnover

First female child of consanguineous parents. 36 week gestation.

Feeding difficulties from birth. 6 months reduced movements and failure to

achieve milestones. 9 months able to smile but general paucity of

movements. Rigidity of all limbs suggestive of dopamine deficiency. Left

convergent squint but no abnormal eye movements detected.

HVA: 1705 (154–867 nmol/L)

5-HIAA: 250 (89-367 nmol/L)

Pterin profile and 5-MTHF status unremarkable

Elevated urinary HVA

Serum Prolactin; 915 (<500 mU/ml)

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Aromatic Amino Acid Decarboxylase Deficiency

Tyr L-Dopa Dopamine HVA

Trp 5-HTP Serotonin 5-HIAA

Clinical features resemble those of recessive BH4 deficiency;

hypotonia, occulogyric crises, ptosis and paucity of spontaneous

movement. Can be fatal

Urine: Vanillactic acid

CSF: Low HVA + 5-HIAA, but normal pterin profile and accumulation

of 3-O-methyldopa. Enzymatic analysis possible on plasma.

Treatment; B6, MAOI & dopamine agonists.

PLP

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Tyrosine

Tryptophan

L-Dopa

5-HTP

Dopamine

Serotonin

BH4

HVA

5-HIAA PLP

AADC

3-Methyldopa

Vanillactic acid

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Aromatic Amino Acid Decarboxylase

Deficiency

Male. Dob; 28/08/2007.

Sample; 10/01/2008

Floppy, episodes of dystonia, developmental delay

HVA 47 (362-955 nmol/L)

5-HIAA 14 (63- 503 nmol/L)

3-Methyldopa 1170 (<300 nmol/L)

PLP 32 (23-87 nmol/L)

Plasma AADC Activity 0.7 (36 -129 pmol/min/ml)

Serum Prolactin 900 (85 – 250 mU/ml)

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Tyrosine

Tryptophan

L-Dopa

5-HTP

Dopamine

Serotonin

BH4

HVA

5-HIAA PLP

AADC

3-Methyldopa

Vanillactic acid

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Vitamin B6 Metabolism

Pyridoxine-5’- phosphate Pyridoxamine-5’- phosphate

Pyridoxal-5’- phosphate

N

C H 2 O H

H O

H 3 C

CH2OPO3H2

CH2OPO3H2

CH2OPO3H2

N

C H 2 N H 2

H O

H 3 C

N

C H O

H O

H 3 C

PNPO PNPO

PNPO = Pyridox(am)ine-5‟-oxidase

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PNPO Deficiency

• Neonatal epileptic encephalopathy

• Fetal distress, prenatal seizures, low Apgar

• Pseudo AADC deficiency – Not consistent

• Glycine & Threonine – Not consistent

• Vanillactate excretion – Consistent ?

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CSF (PLP)

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16Age (Years)

PLP (

nm

ol/L)

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Methionine

Homocysteine

Adomet

Cystathionine

Cysteine

B12 Methyl-cbl MS

Methyl-THF

THF

Serine Diet

B6 CBS

MAT

Glycine

Betaine

DMG

X

Methyl-X

Cbl(II)

Methyl-cbl

Methyl transferases

SH

MT

Ado.hcy

Gly

Sar

GNMT

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CSF 5-MTHF Deficiency

• MTHFR deficiency • AADC deficiency • 3-Phosphoglycerate dehydrogenase def • Rett syndrome • Aicardi Goutieres • DHPR deficiency • Mitochondrial disorders • L-dopa treatment • Folic Acid treatment • Folate transport deficiency • Blocking antibodies • Methotrexate • Anticonvulsants • Steroids • Co-trimoxazole

Cerebral Folate Deficiency - Neurological syndrome associated with low CSF

5-MTHF and normal peripheral folate.

N

N

N

NH

NH2

NH

O

NH

O OH

OH

O

OH CH3

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•Leads to brain specific folate deficiency

•Loss of function mutations in the FOLR1.

•Gene coding for the FRa

•AR disorder manifests in late infancy with Severe developmental

regression, movement disturbances,epilepsy and leukodystrophy

•Beneficial effect of folinic acid.

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Cerebral Folate Deficiency & Mitochondrial

Disease

•Allen et al., 1983. Dougados et al., 1983. Association between cerebral

folate deficiency & Kearns-Sayre Syndrome.

•Pineda et al., 2006. Cerebral folate deficiency & leukoencephalopathy

caused by mitochondrial DNA deletion. Folinic acid treatment led, after

1 year, to remarkable clinical improvement and almost normal white

matter image.

•Responsive to folinic acid reported. 25% of ETC defects associated

with CSF 5-MTHF deficiency No apparent correlation with magnitude of

defect

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CSF 5-MTHF Deficiency & Mitochondrial

Disorders

Sex Age (years) 5-MTHF Ref Range (nmol/L)

F 15 29 46 - 160

M 9 5 72 - 172

M 8 44 72 - 172

F 2 17 52 - 178

F 6 7 72 - 172

No correlation between CSF 5-MTHF status and severity of respiratory

chain defect in muscle

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Secondary Causes

• Hypoxia

• Neurodegeneration

• Epileptic encephalopathy

• Lysosomal

• Mitochondrial

• Molybdenum cofactor deficiency

• Drugs – L-dopa

• Sample Processing

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Thank You & Thank You for Inviting Me

Simon Pope

Viruna Neergheen

Manju Kurian

Shamima Rahman

Sophie-Beth Aylett

Peter Clayton

Emma Footitt

Philippa Mills

Iain Hargreaves