“neurotransmitter” disorders a laboratory · pdf...
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“Neurotransmitter” Disorders
A Laboratory Approach
Panda SA Workshop,
Roodevallei, Pretoria,
May 2016
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
BH2
Tyrosine
Tryptophan
Phenylalanine
L-Dopa
5-HTP
Tyrosine
Dopamine
Serotonin
qBH2 BH4
HVA
5-HIAA PLP
O2
GTP
Dihydroneopterin Triphosphate
6-Pyruvoyltetrahydropterin
Tetrahydrobiopterin
Dihydroneopterin
GTP cyclohydrolase
Pyruvoyl tetrahydroptein synthase
Aldose reductase /Sepiapterin reductase
P3
-VE
BH4 Salvage
Tyr L-Dopa
BH4 qBH2 DHPR
NADH NAD+
PCD +
PCD = pterin carbinolamine dehydratase
DHPR = dihydropteridine reductase
BH2
• 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
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
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
GTP
Dihydroneopterin Triphosphate
6-Pyruvoyltetrahydropterin
Tetrahydrobiopterin
Sepiapterin Reductase Deficiency
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)
GTP
Dihydroneopterin Triphosphate
6-Pyruvoyltetrahydropterin
Tetrahydrobiopterin BH2 DHFR -Liver
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%.
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….
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.
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)
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)
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
Tyrosine
Tryptophan
L-Dopa
5-HTP
Dopamine
Serotonin
BH4
HVA
5-HIAA PLP
AADC
3-Methyldopa
Vanillactic acid
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)
Tyrosine
Tryptophan
L-Dopa
5-HTP
Dopamine
Serotonin
BH4
HVA
5-HIAA PLP
AADC
3-Methyldopa
Vanillactic acid
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
PNPO Deficiency
• Neonatal epileptic encephalopathy
• Fetal distress, prenatal seizures, low Apgar
• Pseudo AADC deficiency – Not consistent
• Glycine & Threonine – Not consistent
• Vanillactate excretion – Consistent ?
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)
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
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
•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.
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
5MTHF Endocytosis
Folate Polyglutamate
Pool
5MTHF FR1
RFC
sFR1
-ve
PLASMA CSF
1 2
O2
._
5MTHF ???
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
Secondary Causes
• Hypoxia
• Neurodegeneration
• Epileptic encephalopathy
• Lysosomal
• Mitochondrial
• Molybdenum cofactor deficiency
• Drugs – L-dopa
• Sample Processing
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