Transcript PUDAC Facilities

IDENTIFICATION OF DISEASE CAUSING MUTATIONS IN
HYPERGLYCINEMIC CAPTIVE BRED VERVET MONKEYS
(CHLOROCEBUS AETHIOPS)
Presenter: Zandisiwe Magwebu ([email protected])
Primate Unit and Delft Animal Centre (PUDAC)
13 September 2016
Berlin, Germany
PUDAC FACILITIES
PUDAC
VERVET
MONKEYS
INDOOR
FACILITY
OUTDOOR,
SEMIINDOOR
FACILITY
PRIMATE
UNIT
(Tygerberg)
DELFT
ANIMAL
CENTRE
(Delft)
RODENTS
RHESUS
MONKEYS
HORSES
PUDAC
Vervet monkey (Chlorocebus aethiops)
Rhesus macaques (Macaca mulatta)
Rodents for obese and diabetic research projects
Horse breeding at Delft Animal Centre
RANGE OF SERVICES AND RESEARCH SUPPORT
HYPERGLYCINEMIA IN VERVET MONKEYS
•
Cataract individuals are hyperglycinemic:
– Plasma: 740µmol/L, normal ≤450 µmol/L
– CSF: >12µmol/L, normal <8µmol/L
•
In humans, CSF/plasma ratio >0.02 ~ Nonketotic hyperglycinemia (NKH).
•
Association between cataract and NKH?
CATARACT MONKEYS
Fig. 1: Total cataract phenotype in captive-bred vervet monkeys.
NONKETOTIC HYPERGLYCINEMIA (NKH)
•
NKH is caused by defective glycine cleavage system (GCS).
–
GCS regulates extracellular glycine concentration
– Enzyme responsible for glycine breakdown.
•
CO2 and NH4+
– Has 3 components responsible for NKH.
•
GLDC (80%), AMT (15%) and GCSH (<1%)
– Three type of NKH in humans
•
•
•
Neonatal
Infantile
Late onset
NKH CLASSIFICATION
Table 1: Different forms of NKH based on glycine levels in CSF and plasma.
Laboratory data
Normal levels
Classic NKH
Late onset NKH
Transient NKH
Glycine in plasma
<450
420-4090
354-961
240-2285
3-10*
40-1440
7.4-68
16-463
0.012-0.04
0.09-0.25
0.02-0.07
0.04-0.88
(µmol/L)
Glycine in CSF
(µmol/L)
CSF-plasma ratio
Adapted from (Hennermann, 2006).
NONKETOTIC HYPERGLYCINEMIA (NKH)
•
•
NKH is also caused by defective glycine transporters GlyT1 (SLC6A9)
–
Removes glycine from extracellular space to avoid saturation at NMDA receptors
–
Failure to remove glycine results in hyperglycinemia
NKH can be mechanically induced by valproate
–
Anticonvulsant
NKH TREATMENT
•
There is no cure for NKH.
•
Alternative treatment: sodium benzoate and dextromethorphan.
•
The treatment reduces glycine levels and minimizes seizures.
GLYCINE CONJUGATION
Fig. 2: Metabolism of benzoate in the mitochondria (Lennerz et al., 2015)
AIMS/OBJECTIVES
•
Aim
– To determine the underlying cause of hyperglycinemia in cataract vervet
monkeys.
•
Objectives
– Screen for sequence variants in SLC6A9.
– Gene expression: GLDC, AMT and SLC6A9.
MATERIALS AND METHODS
Animal selection
Group 1: Control
Group 2: Spontaneous
DNA
RNA
SLC6A9
PCR
RT-PCR
Mutation
analysis
Expression
analysis
RESULTS: SLC6A9 ANALYSIS
Spontaneous
Control
C1419 >T
*
A473A
Fig. 1: SLC6A9 genotyping and mRNA gene expression in the control vs spontaneous group.
Sequence chromatogram displaying heterozygous transition silent mutation (C1419>T) in exon 11 of
SLC6A9. The gene expression data was expressed as mean ± SD and mRNA expression in a.u. (arbitrary
units). * represent significant level (p<0.05)
RESULTS: GLDC & AMT ANALYSIS
GLDC
AMT
*
*
Fig. 2: GLDC and AMT mRNA gene expression in the control vs spontaneous group. The data was
expressed as mean ± SD and mRNA expression in a.u. (arbitrary units). * represent significant level
(p<0.05)
DISCUSSION
•
The presence of hyperglycinemia and cataract formation in NHP is unique
•
Genotyping analysis of GLDC, AMT and SLC6A9 revealed eight novel singlebase substitutions.
•
Expression of GLDC and AMT was upregulated while SLC6A9 was
downregulated
•
Mutations in any of the GCS complex genes affect overall activity of the
complex
•
It has been suggested that mutations in (SLC6A9) are responsible for a
different form of NKH
CONCLUSION
•
Genotyping and gene expression findings confirmed that there is an
association between the two disorders
•
The findings also confirmed that both defective GCS and GlyT1 contributed to
hyperglycinemia
•
Since GlyT1 is reported for the first time, findings can be extrapolated to
humans
•
Future studies are aimed at using multiplex ligation-dependent probe
amplification (MLPA)
ACKNOWLEDGEMENTS
Supervisors: Dr Chesa Chauke (PUDAC) and Dr Abdul-Rasool (UWC)
Financial support: SAMRC-PUDAC
CSF collection: Prof Ali Dhansay
Glycine Analysis: NHLS/UCT and PathCare
PUDAC Staff: Dr. Charon de Villiers, Joritha van Heerden, Timothy Collop and Abe Davids
PUDAC CONTACT DETAILS
• ADDRESS:
SAMRC-PUDAC
NIVS/RIND Building
P.O Box 19070
Francie van zijl drive
Tygerberg
7505
• Email:
[email protected] or
[email protected]
• Tel:
+27 21 938 0369 or ext. 0671
AIMS/OBJECTIVES
• To determine the induced effect of valproate
(50mg/kg) in vervet monkeys and to assess
the treatment effect of sodium benzoate and
dextromethorphan in valproate induced as
compared to spontaneous monkeys.
STUDY DESIGN
Table 2: Different forms of NKH based on glycine levels in CSF and plasma.
PHASE: 1
Groups
No. of
Subjects
Treatment
Daily Dose
(mg/kg bwt)
Group 1
Control
4
Maintenance diet
None
Duration
3 weeks
4
Valproate
50 mg/kg/day
Group 2:
Induced
Blood (2 ml), 0.5 ml CSF and urine was collected at baseline. To monitor glycine level, blood
and urine sampling was performed every week except for CSF which was collected at the end
of week three valproate induction.
PHASE: 2
Group 1
Control
4
Controls
Group 2
Induced
4
Sodium benzoate
and
Dextromethorphan
Group 3
Spontaneous
4
Sodium benzoate
and
Dextromethorphan
Maintenance diet
250mg/kg/day
5mg/kg/day
4 weeks
250mg/kg/day
5mg/kg/day
Blood and urine was collected every week of the treatment to monitor the efficacy of the
compounds, whereas CSF was only collected at the end of the treatment and washout period.
GLYCINE ANALYSIS
Plasma
CSF
10
Control
Induced
Spontaneous
5
Control
Induced
Spontaneous
600
400
200
0
0
Groups
Groups
200
ou
s
Control
Induced
Spontaneous
Sp
on
ta
ne
5
*
400
ed
Baseline
on
tr
ol
10
C
Control
Induced
Spontaneous
600
Plasma (mol/L)
CSF (mol/L)
15
In
du
c
CSF (mol/L)
*
Plasma (mol/L)
800
15
0
0
Groups
Groups
ou
s
Sp
on
ta
ne
ed
In
du
c
on
tr
ol
C
ou
s
Sp
on
ta
ne
ed
In
du
c
C
on
tr
ol
Fig.3: The effect of NKH treatment in vervet monkeys. A) CSF (µmol/L). B) Plasma (µmol/L). * represent significant
level (p<0.05)
GLYCINE ANALYSIS
Induced
Spontaneous
10
6
CSF (mol/L)
8
CSF (mol/L)
15
Baseline
Induction
Treatment
Washout
4
Baseline
Treatment
Washout
*
10
5
2
0
0
Periods
Periods
W
0
as
ho
ut
W
*
en
t
400
Tr
ea
tm
lin
e
600
as
e
as
ho
ut
en
t
Tr
ea
tm
tio
n
In
du
c
200
as
e
lin
e
400
Baseline
Treatment
Washout
B
*
800
Plasma (mol/L)
Baseline
Induction
Treatment
Washout
600
B
Plasma (mol/L)
800
200
0
Periods
Periods
as
ho
ut
W
Tr
ea
tm
en
t
as
e
B
as
ho
ut
W
en
t
Tr
ea
tm
tio
n
In
du
c
B
as
e
lin
e
lin
e
Fig.4: The effect of NKH treatment in spontaneous vervet monkeys. A) CSF (µmol/L). B) Plasma (µmol/L).
* represent significant level (p<0.05)
Relative gene expression (a.u)
GENE EXPRESSION
0.25
Control
Induced
0.20
*
0.15
0.10
0.05
0.00
Groups
Fig. 5: GLDC mRNA gene expression in the induced vs spontaneous group. The expression of control compared to
the induced group. The control group received a maintenance diet throughout the study while the induced group received
50mg/kg/day of valproate and treated with sodium benzoate (250mg/kg/day) and dextromethorphan (5mg/kg/day)
together with the spontaneous group. The data was expressed as mean ± SD and mRNA expression in a.u. (arbitrary
units). * represent significant level (p<0.05)
PUDAC CONTACT DETAILS
• ADDRESS:
SAMRC-PUDAC
NIVS/RIND Building
P.O Box 19070
Francie van zijl drive
Tygerberg
7505
• Email:
[email protected] or
[email protected]
• Tel:
+27 21 938 0369 or ext. 0671