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1
B Fowler
University Childrenrsquos Hospital
Basel Switzerland
Metabolic fates of methionine B12
and folates and their implications in
pathology (inborn errors of
metabolism
Basel
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Homocysteine
MeCbl
Vitamin B12
AdoCbl
Outline of Methionine Metabolism
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Methionine Metabolism Vitamins
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
MeCbl
Vitamin B6 (PLP)
Adenosine
Vitamin B12
GNMT
SAHH
MAT
Case AdoCbl
Vitamin B6 (PLP)
BHMT
Outline of Methionine Metabolism
Homocysteine
Methylation (DNARNA Hormones Lipids Proteins Creatine-P etc)
Proteins
Purines
(DNA RNA)
Thymidine
(DNA)
Hypermethioninaemias diagnosis
Deficiency MAT GNMT SAHH CBS
Metabolites in plasma
Methionine
AdoMet --
AdoHcy -- --
Sarcosine -- --
tHcy (--) -- (--)
2
Examples of transmethylation reactions
Group Methyl group acceptor function
DNA DNA-cytosine gene expression
regulation inactivation imprinting
RNA mRNA-guanine capping of mRNA
tRNA-cytosine -guanine -adenine
alteration of tRNA flexibilty
Proteins carboxyl groups L-isoaspartate (D-aspartate) in protein
repair
Proteins amino acid residues lysine in histones
arginine eg in basic myelin protein
Lipids Phosphatidylethanolamine synthesis
of phosphatidylcholine
N-methyltransferases Guanidinoacetic acid creatine
synthesis
O-methyltransferases Catechols eg norepinephrine
epinephrine dopamine inactivation
(9) S-adenosylmethionine decarboxylase (10) spermidine and spermine
synthases (11) methylthioadenosine phosphorylase (12) conversion of
methylthioribose to methionine
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
MeCbl
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
AdoCbl
Disorders of Methionine metabolism
Vitamin B6 (PLP)
Methionine Synthase
Cystathionine
szlig-synthase
Cystathionase
MeTHFolate reductase
S-AdoHcy Hydrolase
Glycine methyltransferase
Cobalamin defects cblA-F
Methionine
Adenosyltransferase
Betaine Me Transferase
Methionine Adenosyltransferase Deficiency -
Benign
Pungent odour of breath - 4-methyl-3-oxobutyrate
- Dimethylsulphide
Hepatology 200847000-000
Fig 1 Increase in oval
cells in
old Mat1a mice HampE
staining in
(A)undamaged control
(B) 18 month-old Mat1a
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
MeCbl
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
AdoCbl
Disorders of Methionine metabolism
Vitamin B6 (PLP)
Methionine Synthase
Cystathionine
szlig-synthase
Cystathionase
MeTHFolate reductase
S-AdoHcy Hydrolase
Glycine methyltransferase
Cobalamin defects cblA-F
Methionine
Adenosyltransferase
Betaine Me Transferase
Adenosine
monophosphate
ADK
Liver Appearance in ADK- Mice at 7 days
Adk ++ Adk --
1 cm
Neonatal hepatic steatosis by disruption of the adenosine
kinase gene Boison D Et al Fowler B PNAS 2002 996985-6990
Adenosine Kinase deficiency in the mouse
3
Phenotype
- Normal development during embryogenesis
- Within 4 days development of micro-vesicular hepatic
steatosis
- death by 14 days with fatty liver
- Apnoea and hypothermia
Adenosine Kinase deficiency in the mouse Liver Histology Evidence of micro- amp macro-
vesicular diffuse panlobular steatosis
Haematoxylin Haematoxylin Eosin Eosin
Embryonic
175d
Postnatal
05d
Postnatal
2d
Postnatal
4d
Intracellar
lipid
deposition
Elevated AdoHcy and decreased Adenine Nucleotide
levels
Adenosine Kinase deficiency in the mouse
Met
SAM
SAH
Hcy
Adenosine
Failure to thrive
profound psychomotor delay and liver dysfunction
epileptic seizures dysmorphic features with
macrocephalus
frontal bossing hypertelorism global psychomotor
delay with sparse or absent language
slowly progressive muscular weakness and
wasting
2011
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
Homocystinuria
Eye Damage
Short
sightedness
CBS deficiency
4
Clinical Features of CBS Deficency
Homocystinuria
System Feature Cause
Ocular Lens dislocation Cystine
Iridodonesis High level in
Zonular Fibres
Glaucoma
Homocystinuria human vs mouse phenotype
Human Mouse
Dislocated lens
Hepatopathy
Control
Mutant CBS --
Eyes ndashnormal
skeleton -normal
Homocystinuria
Blond Hair
after dietary treatment
Diffusion Weighted MRI of the Brain During high
Methionine Levels under Betaine Treatment CBS-def
Met
1200
micromoll
F Trefz
Tuumlbingen
Diffusion Weighted MRI of the Brain after
Normalization of Methionine stopping Betaine
F Trefz
Tuumlbingen
Treatment induced Pathology
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
5
Related Pathways Folic acid metabolism
Folic acid
DiH-Folate
TetraH-Folate
5-methyl-THF N10
formyl-THF
N5
N10
methylene-THF N5
N10
-methenyl-THF
Glycine
10 formyl-THF
synthetase
510 methenyl-THF
cyclohydrolase
510 methylene-THF
dehydrogenase
serine hydroxy-
methyltransferase
Homocysteine
Methionine
synthase
510 methylene-
THF reductase
Methionine
Serine
DHF-reductase
DHF-reductase
MethyleneTHF FormylTHF MethenylTHF MethylTHF
Formate +
THF
MethyleneTHF
DHF
MethenylTHF FormylTHF
Serine +
THF
THF +
Formate THF +
Serine
THF +
Glyine
THF
dUMP
FAICAR
AICAR
FGAR
GAR
Methionine
dTMP Homocysteine
AICART
DHFR
SHMT1
GART
TYMS
MTR
MTHFD1 MTHFD1 MTHFD1
MTHFD2 MTHFD2 MTHFD2L MTHFD2L MTHFD1L
SHMT2
FormiminoTHF
Histidine
Formiminoglutamate
FTCD
FTCD
GCS
SHMT2
MTHFR
IEM pathways Compartmentalized Folate metabolism
Mitochondrium
Cytosol
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
New Disorders of Folate Transport and
Metabolism
Steinfeld et al Nat Commun 2013 Jul 542123
Choroid plexus transcytosis and exosome shuttling
deliver folate into brain parenchyma
Methylene-THF Dehydrogenase Deficiency-
Mutations in MTHFD1
Patient 1 (Watkins 2011
Keller 2013)
Patient 2
-Neutropenia seizures infections
eczema
- Atypical haemolytic uraemic
syndrome (HUS)
- Megaloblastic anaemia
- Severe combined
immunodeficiency (SCID)
- Asthenia vomiting icteric skin
convergent strabism
- Severe arterial hypertension
- Dyserythropoesis with
megaloblastosis
- Atypical hemolytic uremic syndrome
with microangiopathy
- Plasma methylmalonic acid
elevated (Watkins 2011)
- Plasma methylmalonic acid
Normal (Keller 2013)
- Plasma total homocysteine 29
mmoll (refrange 6ndash9)
- Serum cobalamin and folate
levels normal
- Methylmalonic acid Normal
- Plasma methionine 16 mmoll
(normal 15-29)
- Plasma total homocysteine 50
mmoll (normal lt10)
- Serum cobalamin and folate levels
normal
MTHF Dehydrogenase Deficiency
Patient 1 (Watkins 2011 Keller
2013)
Patient 2
Fibroblast studies
- Mildly decreased synthesis of
methylcobalamin (29 of cellular Cbl
normal 52-54)
- Normal MTHFR activity
- Mildly decreased synthesis of
methylcobalamin (28 of cellular Cbl
normal 48-79)
- Normal MTHFR activity and kinetics
Mutations
c727+1GgtA affects the splice
acceptor site of intron 8
+ c517CgtT (pR173C) affects the
NADP-binding site
c1674GgtA leads to skipping of exon
17
+ c806CgtT (pT269I) affects a highly
conserved aa residue
MTHF Dehydrogenase Deficiency
Nancy pS105F + pG332R
(JL Gueant)
6
Incorporation of C14 Formate in Methionine
nmol16hmg)
- + OHCbl + 5-formyl-THF
controls 111 - 390 131 - 488 126
Patient 1 003 003
Patient 2 005 005 024
00
02
04
06
08
10
Meth
ionin
e form
ation
n
mo
l1
6 h
mg
pro
tein
-PEG +PEG
Complementation
analysis
Methylation
B12
Methionine
Homocysteine
Folate - Cycle
Purines
DNA
Anaemia Neuropathy CH3 (Myelin)
Consequences of Folate Defects
X
X
X
Glutamate-Formiminotransferase
Deficiency
Hereditary Folate Malabsorption
Methylene-THF-reductase
deficiency
Methionine Synthase deficiency
1991
Eur J Pediatr 1998 Apr157 Suppl 2S118-21
Demyelination and inborn errors of the single
carbon transfer pathway Surtees R
bull Inborn errors of the single-carbon transfer pathway are
complicated by demyelination resembling subacute
combined degeneration of the cord and brain
bull The study of CSF metabolites in patients with single-carbon
transfer defects suggests that S-adenosylmethionine
deficiency is a cause of the demyelination
bull Correction of deficiency causes clinical improvement and
remyelination
Disorders of intracellular
Cobalamin metabolism
Update
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
2
Examples of transmethylation reactions
Group Methyl group acceptor function
DNA DNA-cytosine gene expression
regulation inactivation imprinting
RNA mRNA-guanine capping of mRNA
tRNA-cytosine -guanine -adenine
alteration of tRNA flexibilty
Proteins carboxyl groups L-isoaspartate (D-aspartate) in protein
repair
Proteins amino acid residues lysine in histones
arginine eg in basic myelin protein
Lipids Phosphatidylethanolamine synthesis
of phosphatidylcholine
N-methyltransferases Guanidinoacetic acid creatine
synthesis
O-methyltransferases Catechols eg norepinephrine
epinephrine dopamine inactivation
(9) S-adenosylmethionine decarboxylase (10) spermidine and spermine
synthases (11) methylthioadenosine phosphorylase (12) conversion of
methylthioribose to methionine
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
MeCbl
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
AdoCbl
Disorders of Methionine metabolism
Vitamin B6 (PLP)
Methionine Synthase
Cystathionine
szlig-synthase
Cystathionase
MeTHFolate reductase
S-AdoHcy Hydrolase
Glycine methyltransferase
Cobalamin defects cblA-F
Methionine
Adenosyltransferase
Betaine Me Transferase
Methionine Adenosyltransferase Deficiency -
Benign
Pungent odour of breath - 4-methyl-3-oxobutyrate
- Dimethylsulphide
Hepatology 200847000-000
Fig 1 Increase in oval
cells in
old Mat1a mice HampE
staining in
(A)undamaged control
(B) 18 month-old Mat1a
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
MeCbl
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
AdoCbl
Disorders of Methionine metabolism
Vitamin B6 (PLP)
Methionine Synthase
Cystathionine
szlig-synthase
Cystathionase
MeTHFolate reductase
S-AdoHcy Hydrolase
Glycine methyltransferase
Cobalamin defects cblA-F
Methionine
Adenosyltransferase
Betaine Me Transferase
Adenosine
monophosphate
ADK
Liver Appearance in ADK- Mice at 7 days
Adk ++ Adk --
1 cm
Neonatal hepatic steatosis by disruption of the adenosine
kinase gene Boison D Et al Fowler B PNAS 2002 996985-6990
Adenosine Kinase deficiency in the mouse
3
Phenotype
- Normal development during embryogenesis
- Within 4 days development of micro-vesicular hepatic
steatosis
- death by 14 days with fatty liver
- Apnoea and hypothermia
Adenosine Kinase deficiency in the mouse Liver Histology Evidence of micro- amp macro-
vesicular diffuse panlobular steatosis
Haematoxylin Haematoxylin Eosin Eosin
Embryonic
175d
Postnatal
05d
Postnatal
2d
Postnatal
4d
Intracellar
lipid
deposition
Elevated AdoHcy and decreased Adenine Nucleotide
levels
Adenosine Kinase deficiency in the mouse
Met
SAM
SAH
Hcy
Adenosine
Failure to thrive
profound psychomotor delay and liver dysfunction
epileptic seizures dysmorphic features with
macrocephalus
frontal bossing hypertelorism global psychomotor
delay with sparse or absent language
slowly progressive muscular weakness and
wasting
2011
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
Homocystinuria
Eye Damage
Short
sightedness
CBS deficiency
4
Clinical Features of CBS Deficency
Homocystinuria
System Feature Cause
Ocular Lens dislocation Cystine
Iridodonesis High level in
Zonular Fibres
Glaucoma
Homocystinuria human vs mouse phenotype
Human Mouse
Dislocated lens
Hepatopathy
Control
Mutant CBS --
Eyes ndashnormal
skeleton -normal
Homocystinuria
Blond Hair
after dietary treatment
Diffusion Weighted MRI of the Brain During high
Methionine Levels under Betaine Treatment CBS-def
Met
1200
micromoll
F Trefz
Tuumlbingen
Diffusion Weighted MRI of the Brain after
Normalization of Methionine stopping Betaine
F Trefz
Tuumlbingen
Treatment induced Pathology
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
5
Related Pathways Folic acid metabolism
Folic acid
DiH-Folate
TetraH-Folate
5-methyl-THF N10
formyl-THF
N5
N10
methylene-THF N5
N10
-methenyl-THF
Glycine
10 formyl-THF
synthetase
510 methenyl-THF
cyclohydrolase
510 methylene-THF
dehydrogenase
serine hydroxy-
methyltransferase
Homocysteine
Methionine
synthase
510 methylene-
THF reductase
Methionine
Serine
DHF-reductase
DHF-reductase
MethyleneTHF FormylTHF MethenylTHF MethylTHF
Formate +
THF
MethyleneTHF
DHF
MethenylTHF FormylTHF
Serine +
THF
THF +
Formate THF +
Serine
THF +
Glyine
THF
dUMP
FAICAR
AICAR
FGAR
GAR
Methionine
dTMP Homocysteine
AICART
DHFR
SHMT1
GART
TYMS
MTR
MTHFD1 MTHFD1 MTHFD1
MTHFD2 MTHFD2 MTHFD2L MTHFD2L MTHFD1L
SHMT2
FormiminoTHF
Histidine
Formiminoglutamate
FTCD
FTCD
GCS
SHMT2
MTHFR
IEM pathways Compartmentalized Folate metabolism
Mitochondrium
Cytosol
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
New Disorders of Folate Transport and
Metabolism
Steinfeld et al Nat Commun 2013 Jul 542123
Choroid plexus transcytosis and exosome shuttling
deliver folate into brain parenchyma
Methylene-THF Dehydrogenase Deficiency-
Mutations in MTHFD1
Patient 1 (Watkins 2011
Keller 2013)
Patient 2
-Neutropenia seizures infections
eczema
- Atypical haemolytic uraemic
syndrome (HUS)
- Megaloblastic anaemia
- Severe combined
immunodeficiency (SCID)
- Asthenia vomiting icteric skin
convergent strabism
- Severe arterial hypertension
- Dyserythropoesis with
megaloblastosis
- Atypical hemolytic uremic syndrome
with microangiopathy
- Plasma methylmalonic acid
elevated (Watkins 2011)
- Plasma methylmalonic acid
Normal (Keller 2013)
- Plasma total homocysteine 29
mmoll (refrange 6ndash9)
- Serum cobalamin and folate
levels normal
- Methylmalonic acid Normal
- Plasma methionine 16 mmoll
(normal 15-29)
- Plasma total homocysteine 50
mmoll (normal lt10)
- Serum cobalamin and folate levels
normal
MTHF Dehydrogenase Deficiency
Patient 1 (Watkins 2011 Keller
2013)
Patient 2
Fibroblast studies
- Mildly decreased synthesis of
methylcobalamin (29 of cellular Cbl
normal 52-54)
- Normal MTHFR activity
- Mildly decreased synthesis of
methylcobalamin (28 of cellular Cbl
normal 48-79)
- Normal MTHFR activity and kinetics
Mutations
c727+1GgtA affects the splice
acceptor site of intron 8
+ c517CgtT (pR173C) affects the
NADP-binding site
c1674GgtA leads to skipping of exon
17
+ c806CgtT (pT269I) affects a highly
conserved aa residue
MTHF Dehydrogenase Deficiency
Nancy pS105F + pG332R
(JL Gueant)
6
Incorporation of C14 Formate in Methionine
nmol16hmg)
- + OHCbl + 5-formyl-THF
controls 111 - 390 131 - 488 126
Patient 1 003 003
Patient 2 005 005 024
00
02
04
06
08
10
Meth
ionin
e form
ation
n
mo
l1
6 h
mg
pro
tein
-PEG +PEG
Complementation
analysis
Methylation
B12
Methionine
Homocysteine
Folate - Cycle
Purines
DNA
Anaemia Neuropathy CH3 (Myelin)
Consequences of Folate Defects
X
X
X
Glutamate-Formiminotransferase
Deficiency
Hereditary Folate Malabsorption
Methylene-THF-reductase
deficiency
Methionine Synthase deficiency
1991
Eur J Pediatr 1998 Apr157 Suppl 2S118-21
Demyelination and inborn errors of the single
carbon transfer pathway Surtees R
bull Inborn errors of the single-carbon transfer pathway are
complicated by demyelination resembling subacute
combined degeneration of the cord and brain
bull The study of CSF metabolites in patients with single-carbon
transfer defects suggests that S-adenosylmethionine
deficiency is a cause of the demyelination
bull Correction of deficiency causes clinical improvement and
remyelination
Disorders of intracellular
Cobalamin metabolism
Update
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
3
Phenotype
- Normal development during embryogenesis
- Within 4 days development of micro-vesicular hepatic
steatosis
- death by 14 days with fatty liver
- Apnoea and hypothermia
Adenosine Kinase deficiency in the mouse Liver Histology Evidence of micro- amp macro-
vesicular diffuse panlobular steatosis
Haematoxylin Haematoxylin Eosin Eosin
Embryonic
175d
Postnatal
05d
Postnatal
2d
Postnatal
4d
Intracellar
lipid
deposition
Elevated AdoHcy and decreased Adenine Nucleotide
levels
Adenosine Kinase deficiency in the mouse
Met
SAM
SAH
Hcy
Adenosine
Failure to thrive
profound psychomotor delay and liver dysfunction
epileptic seizures dysmorphic features with
macrocephalus
frontal bossing hypertelorism global psychomotor
delay with sparse or absent language
slowly progressive muscular weakness and
wasting
2011
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
Homocystinuria
Eye Damage
Short
sightedness
CBS deficiency
4
Clinical Features of CBS Deficency
Homocystinuria
System Feature Cause
Ocular Lens dislocation Cystine
Iridodonesis High level in
Zonular Fibres
Glaucoma
Homocystinuria human vs mouse phenotype
Human Mouse
Dislocated lens
Hepatopathy
Control
Mutant CBS --
Eyes ndashnormal
skeleton -normal
Homocystinuria
Blond Hair
after dietary treatment
Diffusion Weighted MRI of the Brain During high
Methionine Levels under Betaine Treatment CBS-def
Met
1200
micromoll
F Trefz
Tuumlbingen
Diffusion Weighted MRI of the Brain after
Normalization of Methionine stopping Betaine
F Trefz
Tuumlbingen
Treatment induced Pathology
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
5
Related Pathways Folic acid metabolism
Folic acid
DiH-Folate
TetraH-Folate
5-methyl-THF N10
formyl-THF
N5
N10
methylene-THF N5
N10
-methenyl-THF
Glycine
10 formyl-THF
synthetase
510 methenyl-THF
cyclohydrolase
510 methylene-THF
dehydrogenase
serine hydroxy-
methyltransferase
Homocysteine
Methionine
synthase
510 methylene-
THF reductase
Methionine
Serine
DHF-reductase
DHF-reductase
MethyleneTHF FormylTHF MethenylTHF MethylTHF
Formate +
THF
MethyleneTHF
DHF
MethenylTHF FormylTHF
Serine +
THF
THF +
Formate THF +
Serine
THF +
Glyine
THF
dUMP
FAICAR
AICAR
FGAR
GAR
Methionine
dTMP Homocysteine
AICART
DHFR
SHMT1
GART
TYMS
MTR
MTHFD1 MTHFD1 MTHFD1
MTHFD2 MTHFD2 MTHFD2L MTHFD2L MTHFD1L
SHMT2
FormiminoTHF
Histidine
Formiminoglutamate
FTCD
FTCD
GCS
SHMT2
MTHFR
IEM pathways Compartmentalized Folate metabolism
Mitochondrium
Cytosol
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
New Disorders of Folate Transport and
Metabolism
Steinfeld et al Nat Commun 2013 Jul 542123
Choroid plexus transcytosis and exosome shuttling
deliver folate into brain parenchyma
Methylene-THF Dehydrogenase Deficiency-
Mutations in MTHFD1
Patient 1 (Watkins 2011
Keller 2013)
Patient 2
-Neutropenia seizures infections
eczema
- Atypical haemolytic uraemic
syndrome (HUS)
- Megaloblastic anaemia
- Severe combined
immunodeficiency (SCID)
- Asthenia vomiting icteric skin
convergent strabism
- Severe arterial hypertension
- Dyserythropoesis with
megaloblastosis
- Atypical hemolytic uremic syndrome
with microangiopathy
- Plasma methylmalonic acid
elevated (Watkins 2011)
- Plasma methylmalonic acid
Normal (Keller 2013)
- Plasma total homocysteine 29
mmoll (refrange 6ndash9)
- Serum cobalamin and folate
levels normal
- Methylmalonic acid Normal
- Plasma methionine 16 mmoll
(normal 15-29)
- Plasma total homocysteine 50
mmoll (normal lt10)
- Serum cobalamin and folate levels
normal
MTHF Dehydrogenase Deficiency
Patient 1 (Watkins 2011 Keller
2013)
Patient 2
Fibroblast studies
- Mildly decreased synthesis of
methylcobalamin (29 of cellular Cbl
normal 52-54)
- Normal MTHFR activity
- Mildly decreased synthesis of
methylcobalamin (28 of cellular Cbl
normal 48-79)
- Normal MTHFR activity and kinetics
Mutations
c727+1GgtA affects the splice
acceptor site of intron 8
+ c517CgtT (pR173C) affects the
NADP-binding site
c1674GgtA leads to skipping of exon
17
+ c806CgtT (pT269I) affects a highly
conserved aa residue
MTHF Dehydrogenase Deficiency
Nancy pS105F + pG332R
(JL Gueant)
6
Incorporation of C14 Formate in Methionine
nmol16hmg)
- + OHCbl + 5-formyl-THF
controls 111 - 390 131 - 488 126
Patient 1 003 003
Patient 2 005 005 024
00
02
04
06
08
10
Meth
ionin
e form
ation
n
mo
l1
6 h
mg
pro
tein
-PEG +PEG
Complementation
analysis
Methylation
B12
Methionine
Homocysteine
Folate - Cycle
Purines
DNA
Anaemia Neuropathy CH3 (Myelin)
Consequences of Folate Defects
X
X
X
Glutamate-Formiminotransferase
Deficiency
Hereditary Folate Malabsorption
Methylene-THF-reductase
deficiency
Methionine Synthase deficiency
1991
Eur J Pediatr 1998 Apr157 Suppl 2S118-21
Demyelination and inborn errors of the single
carbon transfer pathway Surtees R
bull Inborn errors of the single-carbon transfer pathway are
complicated by demyelination resembling subacute
combined degeneration of the cord and brain
bull The study of CSF metabolites in patients with single-carbon
transfer defects suggests that S-adenosylmethionine
deficiency is a cause of the demyelination
bull Correction of deficiency causes clinical improvement and
remyelination
Disorders of intracellular
Cobalamin metabolism
Update
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
4
Clinical Features of CBS Deficency
Homocystinuria
System Feature Cause
Ocular Lens dislocation Cystine
Iridodonesis High level in
Zonular Fibres
Glaucoma
Homocystinuria human vs mouse phenotype
Human Mouse
Dislocated lens
Hepatopathy
Control
Mutant CBS --
Eyes ndashnormal
skeleton -normal
Homocystinuria
Blond Hair
after dietary treatment
Diffusion Weighted MRI of the Brain During high
Methionine Levels under Betaine Treatment CBS-def
Met
1200
micromoll
F Trefz
Tuumlbingen
Diffusion Weighted MRI of the Brain after
Normalization of Methionine stopping Betaine
F Trefz
Tuumlbingen
Treatment induced Pathology
Homocystinurias
Main Features amp Neurological abnormalities CBS deficiency
bull Ocular changes
Mental retardation
bull Seizures
Psychiatric
disorders
bull Osteoporosis
bull Scoliosis
bull Thromboembolism
of Arteries amp Veins
methionine
vascular pathology
metabolite toxicity
MTHFR deficiency
bull Presentation 23d
- 16y
bull severe neurolog
abnormalities
bull Psychomotor
retardation
bull gait abnormalities
bull seizurespsychiatric
disturbance
methionine
Me-Folate
vascular pathol
AdoMet deficiency
Met Synth deficiency
(cbl disorders)
bull hyper- hypotonia
bull abnormal movements
bull seizures
bull developmental
delay
bull sub-acute degen of
spinal cord brain
bull megaloblastic anaemia
methionine
Me-Folate
AdoMet deficiency
trapping of folates
5
Related Pathways Folic acid metabolism
Folic acid
DiH-Folate
TetraH-Folate
5-methyl-THF N10
formyl-THF
N5
N10
methylene-THF N5
N10
-methenyl-THF
Glycine
10 formyl-THF
synthetase
510 methenyl-THF
cyclohydrolase
510 methylene-THF
dehydrogenase
serine hydroxy-
methyltransferase
Homocysteine
Methionine
synthase
510 methylene-
THF reductase
Methionine
Serine
DHF-reductase
DHF-reductase
MethyleneTHF FormylTHF MethenylTHF MethylTHF
Formate +
THF
MethyleneTHF
DHF
MethenylTHF FormylTHF
Serine +
THF
THF +
Formate THF +
Serine
THF +
Glyine
THF
dUMP
FAICAR
AICAR
FGAR
GAR
Methionine
dTMP Homocysteine
AICART
DHFR
SHMT1
GART
TYMS
MTR
MTHFD1 MTHFD1 MTHFD1
MTHFD2 MTHFD2 MTHFD2L MTHFD2L MTHFD1L
SHMT2
FormiminoTHF
Histidine
Formiminoglutamate
FTCD
FTCD
GCS
SHMT2
MTHFR
IEM pathways Compartmentalized Folate metabolism
Mitochondrium
Cytosol
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
New Disorders of Folate Transport and
Metabolism
Steinfeld et al Nat Commun 2013 Jul 542123
Choroid plexus transcytosis and exosome shuttling
deliver folate into brain parenchyma
Methylene-THF Dehydrogenase Deficiency-
Mutations in MTHFD1
Patient 1 (Watkins 2011
Keller 2013)
Patient 2
-Neutropenia seizures infections
eczema
- Atypical haemolytic uraemic
syndrome (HUS)
- Megaloblastic anaemia
- Severe combined
immunodeficiency (SCID)
- Asthenia vomiting icteric skin
convergent strabism
- Severe arterial hypertension
- Dyserythropoesis with
megaloblastosis
- Atypical hemolytic uremic syndrome
with microangiopathy
- Plasma methylmalonic acid
elevated (Watkins 2011)
- Plasma methylmalonic acid
Normal (Keller 2013)
- Plasma total homocysteine 29
mmoll (refrange 6ndash9)
- Serum cobalamin and folate
levels normal
- Methylmalonic acid Normal
- Plasma methionine 16 mmoll
(normal 15-29)
- Plasma total homocysteine 50
mmoll (normal lt10)
- Serum cobalamin and folate levels
normal
MTHF Dehydrogenase Deficiency
Patient 1 (Watkins 2011 Keller
2013)
Patient 2
Fibroblast studies
- Mildly decreased synthesis of
methylcobalamin (29 of cellular Cbl
normal 52-54)
- Normal MTHFR activity
- Mildly decreased synthesis of
methylcobalamin (28 of cellular Cbl
normal 48-79)
- Normal MTHFR activity and kinetics
Mutations
c727+1GgtA affects the splice
acceptor site of intron 8
+ c517CgtT (pR173C) affects the
NADP-binding site
c1674GgtA leads to skipping of exon
17
+ c806CgtT (pT269I) affects a highly
conserved aa residue
MTHF Dehydrogenase Deficiency
Nancy pS105F + pG332R
(JL Gueant)
6
Incorporation of C14 Formate in Methionine
nmol16hmg)
- + OHCbl + 5-formyl-THF
controls 111 - 390 131 - 488 126
Patient 1 003 003
Patient 2 005 005 024
00
02
04
06
08
10
Meth
ionin
e form
ation
n
mo
l1
6 h
mg
pro
tein
-PEG +PEG
Complementation
analysis
Methylation
B12
Methionine
Homocysteine
Folate - Cycle
Purines
DNA
Anaemia Neuropathy CH3 (Myelin)
Consequences of Folate Defects
X
X
X
Glutamate-Formiminotransferase
Deficiency
Hereditary Folate Malabsorption
Methylene-THF-reductase
deficiency
Methionine Synthase deficiency
1991
Eur J Pediatr 1998 Apr157 Suppl 2S118-21
Demyelination and inborn errors of the single
carbon transfer pathway Surtees R
bull Inborn errors of the single-carbon transfer pathway are
complicated by demyelination resembling subacute
combined degeneration of the cord and brain
bull The study of CSF metabolites in patients with single-carbon
transfer defects suggests that S-adenosylmethionine
deficiency is a cause of the demyelination
bull Correction of deficiency causes clinical improvement and
remyelination
Disorders of intracellular
Cobalamin metabolism
Update
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
5
Related Pathways Folic acid metabolism
Folic acid
DiH-Folate
TetraH-Folate
5-methyl-THF N10
formyl-THF
N5
N10
methylene-THF N5
N10
-methenyl-THF
Glycine
10 formyl-THF
synthetase
510 methenyl-THF
cyclohydrolase
510 methylene-THF
dehydrogenase
serine hydroxy-
methyltransferase
Homocysteine
Methionine
synthase
510 methylene-
THF reductase
Methionine
Serine
DHF-reductase
DHF-reductase
MethyleneTHF FormylTHF MethenylTHF MethylTHF
Formate +
THF
MethyleneTHF
DHF
MethenylTHF FormylTHF
Serine +
THF
THF +
Formate THF +
Serine
THF +
Glyine
THF
dUMP
FAICAR
AICAR
FGAR
GAR
Methionine
dTMP Homocysteine
AICART
DHFR
SHMT1
GART
TYMS
MTR
MTHFD1 MTHFD1 MTHFD1
MTHFD2 MTHFD2 MTHFD2L MTHFD2L MTHFD1L
SHMT2
FormiminoTHF
Histidine
Formiminoglutamate
FTCD
FTCD
GCS
SHMT2
MTHFR
IEM pathways Compartmentalized Folate metabolism
Mitochondrium
Cytosol
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
New Disorders of Folate Transport and
Metabolism
Steinfeld et al Nat Commun 2013 Jul 542123
Choroid plexus transcytosis and exosome shuttling
deliver folate into brain parenchyma
Methylene-THF Dehydrogenase Deficiency-
Mutations in MTHFD1
Patient 1 (Watkins 2011
Keller 2013)
Patient 2
-Neutropenia seizures infections
eczema
- Atypical haemolytic uraemic
syndrome (HUS)
- Megaloblastic anaemia
- Severe combined
immunodeficiency (SCID)
- Asthenia vomiting icteric skin
convergent strabism
- Severe arterial hypertension
- Dyserythropoesis with
megaloblastosis
- Atypical hemolytic uremic syndrome
with microangiopathy
- Plasma methylmalonic acid
elevated (Watkins 2011)
- Plasma methylmalonic acid
Normal (Keller 2013)
- Plasma total homocysteine 29
mmoll (refrange 6ndash9)
- Serum cobalamin and folate
levels normal
- Methylmalonic acid Normal
- Plasma methionine 16 mmoll
(normal 15-29)
- Plasma total homocysteine 50
mmoll (normal lt10)
- Serum cobalamin and folate levels
normal
MTHF Dehydrogenase Deficiency
Patient 1 (Watkins 2011 Keller
2013)
Patient 2
Fibroblast studies
- Mildly decreased synthesis of
methylcobalamin (29 of cellular Cbl
normal 52-54)
- Normal MTHFR activity
- Mildly decreased synthesis of
methylcobalamin (28 of cellular Cbl
normal 48-79)
- Normal MTHFR activity and kinetics
Mutations
c727+1GgtA affects the splice
acceptor site of intron 8
+ c517CgtT (pR173C) affects the
NADP-binding site
c1674GgtA leads to skipping of exon
17
+ c806CgtT (pT269I) affects a highly
conserved aa residue
MTHF Dehydrogenase Deficiency
Nancy pS105F + pG332R
(JL Gueant)
6
Incorporation of C14 Formate in Methionine
nmol16hmg)
- + OHCbl + 5-formyl-THF
controls 111 - 390 131 - 488 126
Patient 1 003 003
Patient 2 005 005 024
00
02
04
06
08
10
Meth
ionin
e form
ation
n
mo
l1
6 h
mg
pro
tein
-PEG +PEG
Complementation
analysis
Methylation
B12
Methionine
Homocysteine
Folate - Cycle
Purines
DNA
Anaemia Neuropathy CH3 (Myelin)
Consequences of Folate Defects
X
X
X
Glutamate-Formiminotransferase
Deficiency
Hereditary Folate Malabsorption
Methylene-THF-reductase
deficiency
Methionine Synthase deficiency
1991
Eur J Pediatr 1998 Apr157 Suppl 2S118-21
Demyelination and inborn errors of the single
carbon transfer pathway Surtees R
bull Inborn errors of the single-carbon transfer pathway are
complicated by demyelination resembling subacute
combined degeneration of the cord and brain
bull The study of CSF metabolites in patients with single-carbon
transfer defects suggests that S-adenosylmethionine
deficiency is a cause of the demyelination
bull Correction of deficiency causes clinical improvement and
remyelination
Disorders of intracellular
Cobalamin metabolism
Update
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
6
Incorporation of C14 Formate in Methionine
nmol16hmg)
- + OHCbl + 5-formyl-THF
controls 111 - 390 131 - 488 126
Patient 1 003 003
Patient 2 005 005 024
00
02
04
06
08
10
Meth
ionin
e form
ation
n
mo
l1
6 h
mg
pro
tein
-PEG +PEG
Complementation
analysis
Methylation
B12
Methionine
Homocysteine
Folate - Cycle
Purines
DNA
Anaemia Neuropathy CH3 (Myelin)
Consequences of Folate Defects
X
X
X
Glutamate-Formiminotransferase
Deficiency
Hereditary Folate Malabsorption
Methylene-THF-reductase
deficiency
Methionine Synthase deficiency
1991
Eur J Pediatr 1998 Apr157 Suppl 2S118-21
Demyelination and inborn errors of the single
carbon transfer pathway Surtees R
bull Inborn errors of the single-carbon transfer pathway are
complicated by demyelination resembling subacute
combined degeneration of the cord and brain
bull The study of CSF metabolites in patients with single-carbon
transfer defects suggests that S-adenosylmethionine
deficiency is a cause of the demyelination
bull Correction of deficiency causes clinical improvement and
remyelination
Disorders of intracellular
Cobalamin metabolism
Update
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
7
Structure of Vitamin B12 (Cobalamin Cbl)
Essential cofactor for 2
Enzymes
Methyl- Methionine Synthase
Methyl-Cbl
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
Adenosyl Co Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
cblJ
The cblC gene
bull In 204 individuals 42 different mutations
bull One mutation 271dupA accounted for 40 of all disease
alleles
cblC genotype phenotype correlations
Early-onset disease
c271dupA
c331CgtT
Late-onset disease
c394CgtT (stop codon)
bull In further 118 individuals 11 additional mutations
42 271dupA (Lerner-EllishellipFowler Hum Mutation 2009)
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
8
Function of the cblC protein
conversion of propionyl-cbl MeCbl AdoCbl
Hannibal et al Molecular Genetics and Metabolism 2009
Function of the cblC protein
bull Similar motifs to those seen in bacterial genes with
cobalamin-related functions eg TonB
bull Recombinant MMACHC binds cobalamin and functions in
vitro as a CNCbl decyanase (Kim et al 2008)
bull X-ray structure of cblC Koutmos et al JBC 2011 28629780
Froese SD et al Biochemistry 2012 515083
MMACHC dimer formation enhanced by AdoCbl binding
and FMN Pocket found for Glutathione binding and
dealkylation activity (It is not like TonB)
bull Exact function of MMACHC
Dealkylation function
cobalamin trafficking chaperone
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy) bull Our study (2004 Suormala Coelho Fowler et al J B Chem279 42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
The CblD defect of cellular Cbl metabolism
one gene ndash three phenotypes
bull Original cblD
2 siblings with combined defect methylmalonic-
aciduria (MMA) and homocystinuria (Hcy)
bull Our study (2004 Suormala Coelho Fowler et al J B Chem279
42742)
- 2 patients with isolated Hcy - normal MMA
- 1 patient with isolated MMA ndashnormal Hcy
Complementation studies proved that these patients
belong to the cblD complementation group = gene
bull Now gt17 patients known
6 combined defect (2 described 1980 2 new ones)
6 isolated homocystinuria
5 isolated Methylmalonic acidaemia
Terttu
Suormala
CblD-MMAHC
5 patients
CblD-HC
6 patients
CblD-MMA
6 patients
(+4 Madrid)
Plasma
Hcy
Methionine
Hcy
Methionine
Hcy normal
Methionine normal
Clinical findings
Encephalopathy
Feeding difficulties
Development delay
Seizureshypotonia
Megaloblastic anemia
Development delay
Ataxia hypotonia
Nystagmus
Megaloblastic anemia
Severe ketoacidosis
Hyperammonemia
Leucopenia Tc-penia
Seizures
Urine
MMA
MMA normal
MMA
Classification of cblD patients
Splice site deletion Missense
FrameshiftStop codon
Inframe duplication
Nonsense
cblD-MMAHC
cblD-HC cblD-MMA
2 3 4 5 6 7 8
F2
04
_A
23
2d
el
R2
50
X
C1
9fs
X2
0
R5
4X
L1
03
_S
10
8d
up
Y1
40
X
L2
0fs
X2
2
T1
82
N
Y2
49
W
L2
59
P
S2
28
X
T1
52
fsX
16
2
D2
46
G
A4
5fs
X5
9
MMADHC mutations
N7
7E
fsX
81
N C
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
9
MMADHC mutations in cblD-MMA
cblD-MMA
2 3 4 5 6 7 8
C1
9fs
X2
0
R5
4X
L2
0fs
X2
1
Start codon
Start codon
Met 62
Start codon
Met 116
N7
7E
fsX
81
N C
Identification of AdoCbl and MeCbl functional
domains
Expression of site directed mutagenic
MMADHC and in cblD-MMAHC cells
HMG 2012 vol 21 1410-1418
2 3 4 5 6 7 8
Start codon
Met 62
Met 116 292
mitochondrial
targeting adenosyl-cbl synthesis
methyl-Cbl synthesis
Gln 118
Identification of AdoCbl and MeCbl functional
domains
2009 41234
Homozygosity mapping
cblF defect Mutations in the LMBRD1 gene (encoding LMBD1 a lysosomal
membrane protein) found in cblF patients
Helix number and orientation corresponds to the published description for
lipocalin receptor LIMR (Fluckinger et al 2007) and to the annotation for
the prediction of LMBD1 in the database (Q9NUN5 UniProtEMBL)
the exact start and termination of the helices is slightly different for the individual
prediction methods Prediction and numbering used here are from TMHMM
(TMHMM Server v 20 from the Center for Biological Sequence Analysis
Technical University Denmark)
The helix lengths differs not dramatically therefore they are shown with equal length
in the scheme The figure can certainly be presented in graywhite without colors
A corresponding legend could be as follows
FigX Schematic representation of the membrane spanning topology and putative glycosylation
sites for LMBD1 The loci of the four frameshift mutations described in this paper are
indicated (T137Ifsx14 T172RfsX9 E283GfsX2 L352LfsX18)
Membrane spanning helices were predicted using TMHMM (TMHMM Server v 20
from the Center for Biological Sequence Analysis Technical University Denmark)
1 2 3 4 5 6 7 8 9
cytosolic
Intra-
lysosomal
N-terminus
C-terminusT134fs
L352fs
G88
G78
G170
G347 G
448
G457
10 66 101 165 202 328 368 432 489
540
32 44 123 143 224 306 390 410 511
G Putative glycosylation site
Site of mutation (fs = frameshift)
T172fs
K281fs
T237X
No 2
No 5
No 1
No 3
No 4
The 1056delG (L352fs) allele (No 5) leading to a frameshift and
premature termination codon in exon 11 was present on 18 of the 24
disease chromosomes (common 134 Mb haplotype)
bull6q13
bull18 exons
bull614 kDa
protein
bull540 amino
acids
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
10
A new Cbl complementation class mimicking
cblF is caused by mutations of ABCD4 Coelho D et al Basel Zuumlrich colleagues from Montreal Muumlnster
Nat Genet 2012 Oct441152-5
Two patients with cblF phenotype but new complementation group
N American case 1 European case 2
ndash whole exome capture microcell mediated chromosome transfer
2 mutations of ABCD4 exome sequencing of chr 14
2 other mutations of ABCD4
ABCD4 presumed ABC transporter
Complementation analysis in Case 2 with
combined MeCbl and AdoCbl synthesis defect cblF and cblJ defects
Biochemical features
- Homocystinuria and methylmalonic aciduria
- High uptake of CNCbl
- Free Cbl (not protein bound)
Protein
- Lysosomal
- Unknown function
LMBD1 (cblF) Homology with a membrane receptor (LIMR)
ABCD4 (cblJ) Classified as ABC transporter
Mutations identified in ABCD4 cDNA
CblJ01 patient c1456 GgtT (pGly486Cys)
c 542+1 GgtT (exon skipping)
CblJ02 patient c955AgtG (pTyr319Cys)
c1747_1748insCT (pGlu583LeufsX9)
What is ABCD4
-protein of unknown function
member of the subfamily D of the ATP Binding
Cassette transporters
- ABCD4 localisation is controversial and has been described
as a peroxisomal or as an endoplasmic reticulum protein
Lysosomal location of ABCD4
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
11
Membrane
C ytos olic
Intra-lys os omal
C OOH
81
37 98
54 189 293 313
160
177
276 330206
606
421ATP binding(Walker A)
As p143_S er181del
ATP binding(Walker B )
H2N 536
Tyr319C ys
G lu583L eufs 9
As p143
S er181
S er485G ly443
G ly443_S er485del
Asp548Asn
ABCD4 Structure and Mutations
0
5
10
15
20
25
30
35
Vec
tor on
ly
ABCD4-
wt
ABCD4-
pLy
s427
Leu
ABCD4-
pAsp
548A
snABCD4-
pG
lu54
9Gln
o
f to
tal co
ba
lam
ins
AdoCbl MeCbl
ATPase activity
Level of
Rescue of Cbl
coenzyme
synthesis by
wild type and
mutated
ABCD4 alleles
Asp548Asn
Open questions about ABCD4
What is the exact function of ABCD4 and LMBD1
Which protein is the cbl transporter
rarr is ABCD4 a human ABC importer (almost all
importers are prokaryotic)
Does ABCD4 interact with LMBD1
ADP + Pi ATP
lysosome
cytosol
LMBD1 ABCD4 ABCD4
ADP + Pi ATP
LMBD1 LMBD1 ABCD4 ABCD4
Coelho D Suormala T Stucki M Lerner-Ellis JP Rosenblatt DS Newbold R Baumgartner M Fowler B N Engl J Med 35814 April 3 2008
TCR
Intracellular Vitamin B12 metabolism
Membrane protein
Chaperone
Enzyme deficiency
Trafficking cblJ
Named as cblX but behaves as cblC in complementation
analysis
More a HCFC1 disorder than cobalamin disorder
Vascular changes in remethylation defects
Section of the narrowed coronary artery of a 4
month old patient with the cblC defect
Disruption
of inner
elastic
membrane
Intimal
proliferation
63 x
Baumgartner et al 1979 Helv Pediat Acta 34465
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
12
Section of the small renal cortical artery of a 4
month patient with the cblC defect
Swollen endothelial
cells
Subendothelial
deposition of
fibrinous material
400 x
Baumgartner et al 1979 Helv Pediat Acta 34465
Vascular changes in remethylation defects Mild Hyperhomocysteinaemia Mouse Model
ldquoEndothelial dysfunction in a murine model of mild
hyperhomocyst(e)inemiardquo
(RT Eberhardt et al JCI 2000 106483-491)
mice heterozygous for cystathionine szlig-synthase deletion
- plasma homocysteine levels 9 micromolL (control 4 micromolL)
- endothelial function and oxidant burden disturbed
Vessel architecture
Endothelial damage smooth muscle cell
proliferation Collagen synthesis media fibrosis
Cell structure damage
mitochondrial damage Endoplasmic-Reticulum
stress
Endothelial Dysfunction
NO system disturbance
NO availability
asymmetrical dimethyl arginine
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Transcription factors
activation of regulatory proteins
Oxidative Stress
Lipid peroxidation
Leucocyte adhesion
Clotting Factors
Thrombin
Fibrinolysis
Platelet aggregation
Homocysteine risk factor for vascular disease
Postulated mechanisms of damage
Acknowledgements
Collaborators
Jordan Lerner-Ellis David Rosenblatt (Montreal)
Petra Zavadakova Viktor Kožich (Prague)
Frank Rutsch (Muumlnster)
D Boison (Zuumlrich)
Jan Kraus
A Kuster (Nantes)
Many colleagues who sent us fibroblasts
Swiss National Science Foundation grant No 3200-066878
Acknowledgements
Terttu Suormala David Coelho (Basel Zuumlrich)
Matthias Baumgartner Martin Stucki Michele Frapolli (Zuumlrich) Patricie Burda
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
13
Fluoresence microscopy of expressed tagged
wild type MMADHC and mitochondria
37 kDa
25 kDa
20 kDa
Wild type C19fs_X20
bull Cell extracts immunoprecipitated with a polyclonal rabbit anti-human MMADHC antibody bull BSA 1 in PBST 01
bull Monoclonal antibody mouse anti-human MMADHC 15000
bull Horse Radish Peroxidase conjugated antibody rabbit anti-mouse 1100 000
Electrophoresis of fibroblast extracts
detected with monoclonal antibody
144 Mb 152 Mb 151 Mb 148 Mb 149 Mb 146 Mb 147 Mb 145 Mb
Telomere Centromere
153 Mb
MMADHC
M132
M151
M2335
M2324
150 Mb
M2275
M2301
M2236
M2313
M2184
Identification of the cblD gene
Significant homology with bacterial genes related to ABC transporters
Encodes a polypeptide of 296 aminoacids (328 kDa)
Mutations were found in all cblD patients
rarr MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC)
Maps to chromosome 2q232
David
Coelho
Possible function of the cblD Protein
Protein sequence highly conserved in mammalian species MMADHC not member of previously identified gene family Shares similarity with putative ATPase component of bacterial ABC transporter Lacks critical domains of most ABC transporters Possible mitochondrial leader sequence
0
10
20
30
40
50
60
70
Vector
only
1 2 3 4 5 6 7 8 9 10
o
f to
tal cob
ala
min
s
AdoCbl
MeCbl
1 Wildtype
10 Gln118X
9 Ser89X
5 Met1-Thr61del
7 Met1-Val115del
8 ALDH2_MLS_at_Met116
3 Met62Gln_Met116Gln
4 ALDH2_MLS_Val12+Met62Gln_Met116Gln
6 ALDH2_MLS_at_Met62
2 ALDH2_MLS_Val12
Met62 Met116
Met62 Met116 Val12
Met62Gln Met116Gln
Met62Gln Met116Gln Val12
Met62 Met116
Met62 Met116
Met116
Met116
Met116 Met62 Ser89X
Gln118X Met62
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
14
Identification of AdoCbl and MeCbl functional
domains
0
5
10
15
20
25
30
35
1 2 3
Cb
l fo
rme
d
to
tal
Ado-Cbl
Me-Cbl
Wild type Met 62 Met 116
Met 62 Met 116 ALDH2_MLS_Val12
Val 12
Gln 62 Gln 116
Met62Gln_Met116Gln
Adenosyl-Cbl
Me-Cbl
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
Mitochondrion
cblF
Cbl OHCbl
TC
Cbl
TC
Lysosome
cblC
Cbl
cblD Cbl
Cbl
CblD-HC
CblD-MMA
cblE
cblB cblA Cbl
Mutase
OHCbl
Cytoplasm
Cbl
MeCbl
Homocysteine
Methionine
Cbl
Methylmalonyl-CoA Succinyl-CoA
AdoCbl
Mitochondrion
Cbl
Cbl
OHCbl
TC
OHCbl
TC
Lysosome
Related pathways
Intracellular Cobalamin Metabolism The CblC defect of cellular cobalamin
metabolism
NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100
bull Discovered by homozygosity mapping
bull Located on chromosome 1p
bull Codes for approx 30 kDa protein
Types of Disorders CblC severe presentation
35 w Feeding problems
temperature dysregulation
Pale irritable unconscious dystrophic
poor growth
neurological abnormalities tachycardia
anaemia
Prof Sengers Nijmegen
Plasma Hcy Methionine
Urine Methylmalonic acid
Treated OH-Cbl im 1mgd betaine carnitine
4 months re-admitted to hospital
died one day later with multi-organ failure hyperthermia
Clinical
12y- 21y
Unsteady gait urinary incontinence
Spinal cord involvement neuropathy
inability to walk
respiratory insufficiency (respirator)
Thought to have multiple sclerosis Steroid treatment
Gold et al 1995
Laboratory
Urine MMA
Plasma total homocysteine
Treated im OH-Cbl 500microg d - 10mg week
Types of Disorders CblC late presentation
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
15
M Heumer Bregenz - Austria
International network
bull define patients subgroups bull define natural history bull genotypephenotype correlations bull evaluate therapeutic measures bull identify possible prognostic indicators bull identify possible measures to improve the natural course
Dr C Dionisi-Vici Rome-Italy
B Fowler SFischer Basel-Switzerland
aims
Clinical aspects of cblC questionnaire survey of 88 patients Clinical aspects of cblC questionnaire survey of 88 patients
Clinical aspects of cblC questionnaire survey of 88 patients
bull define patients subgroups
neonatal - early onset - late onset
bull define natural history
yes addition of new important components
bull genotypephenotype correlations
confirmed previous studies
Clinical aspects of cblC Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions
bull evaluate therapeutic measures
the study shows clearly that although some features
respond to treatment others do not
indicating that conventional treatment has little effect
bull identify possible measures to improve the natural course
back to pathogenesis
Long-term outcome is still dominated by severe neurological and ocular impairment
Clinical aspects of cblC questionnaire survey of 88 patients
Conclusions JIMD Rep 201311149-63
Metabolic profiling of total homocysteine and related
compounds in hyperhomocysteinemia utility and limitations
in diagnosing the cause of puzzling thrombophilia in a family
Stabler SP Korson M Jethva R Allen RH Kraus JP Spector EB
Wagner C Mudd SH
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
16
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
The Homocysteine Metabolome
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Heat map of
metabolites in
untargetted
metabolomics
Metabolic
crosstalk
Moonlight
functions
Other Pathways related
to homocysteine
Diagnosis of the cobalamin defects
Defect No patients
MMA-CoA mutase apo-enzyme defect mut 199
cblA defect 45
cblB defect 42
cblAB defects (combined group) 19
cblCD defect 124
cblF defect 6
cblE defect (MS reductase def) 14
cblG defect (MS apo-enzyme def) 12
Unexplained HC 13
Unexplained MMA 12
MTHFR 47
Manchester 1978 ndash Basel from 1990
The Homocysteine Metabolome
How should it look
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
17
Cerebral Folate Deficiency (OMIM 613068)
Mutations in the FOLR1 gene coding for Folate Receptor FRα Steinfeld R et al 2009 described three patients - movement disorders - psychomotor deterioration - epilepsy - MRI ndash hypomyelinisation low Choline Inositol
Fibroblasts - low methotrexate dependent folate binding - rescue by transfection with wild type FRα Treatment with 5mgkgday folinic acid - clinical brain abnormalities and function improved other cases described (Cario et al 2009 Peacuterez-Duenas et al 2010
Dihydrofolate Reductase (DHFR) Deficiency
(OMIM 613839)
bull Neurologic disease childhood absence epilepsy
bull Macrocytosis megaloblastic anemia andor pancytopenia
bull Red cell folate Low (but plasma folate normal)
bull Cerebral folate tetrahydrobiopterin deficiency
bull Cerebral and cerebellar atrophy
bull No elevation of homocysteine or phenylalanine
bull Low DHPR activity in transformed lymphoblasts
bull Homozygous missense mutations in DHFR
bull Treatment with reduced folate (folinic acid) improved haematology CSF folate and neurologic symptoms
Banka S et al Am J Hum Genet 88216 2011
Cario H et al Am J Hum Genet 88226 2011
bull Hereditary Folate Malabsorption
bull Glutamate Formiminotransferase Deficiency
bull Methylenetetrahydrofolate Reductase Deficiency
bull Methionine Synthase Reductase Deficiency (cblE)
bull Methionine Synthase Deficiency (cblG)
bull Cerebral Folate Deficiency (2009)
bull Dihydrofolate Reductase Deficiency (2011)
bull MTHFD1 (Trifunctional Enzyme) Deficiency (2011)
Disorders of Folate Transport and
Metabolism
Disorders of Cobalamin metabolism
bull none-genetic Nutritional deficiency
(strict vegetarian diet Vegans)
reduced intestinal absorption (elderly persons)
bull genetic Disorders of Absorption and Transport
Hereditary Intrinsic Factor Deficiency
Defective Transport of Cbl by Enterocytes
(Imerslund-Graumlsbeck Syndrome)
Haptocorrin (R Binder) Deficiency
Transcobalamin (TC) Deficiency
Disorders of Intracellular Utilization
of Cbl Combined Deficiencies
of AdoCbl and MeCbl
AdoCbl Deficiency
MeCbl Deficiency
Richard E Monteoliva L Juarez S Perez B Desviat LR Ugarte M Albar
JP Quantitative analysis
of mitochondrial protein expression in methylmalonic acidemia by two-
dimensional difference gel
electrophoresis J Proteome Res 2006 51602ndash1610 [PubMed
16823967]
cblD Ref 33 from Hannibal proteomics Methods for Diagnosis of
intracellular cobalamin
defects
5 m
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
18
Disease Findings Clinical Presentation
Childhood JuvenileAdult
Methionine-S-
Adenosyltransferase
deficiency
Met
N
SAM
N
HCy
Sarc
Unpleasant odor due to excretion
of methionine metabolites
Majority of subjects without other
clinical features rarely
neurological involvement
Glycine N-
methyltransferase
deficiency
Met
SAM
N
HCy
N
SAH
N Sarc
Only 3 patients
described with mild
hepatomegaly and
elevated
transaminases at
12 and 5 years of
age
S-Adenosyl-
homocysteine
hydrolase deficiency
Met
SAM
SAH
HCy
Sarc
One patient
presenting in first
year with severely
delayed
psychomotor
develop-ment
muscular hypotonia
lack of tendon
reflexes elevated
creatinine kinase
transaminases and
delayed myelination
g-Cystathionase
deficiency
Ca
UCa
N
UHC
N
MMA
Most likely a benign disorder
Transiently seen in newborns
secondary finding in liver disease
neural tumors and severe vitamin
B6 deficiency
Methods for Diagnosis of HC MMA disorders
Metabolite measurements
Urine
- Homocystine methionine
Thin layer chromatography electrophoresis
- Methylmalonic acid
Gas chromatography-mass spectrometry
Plasma
- Free homocystine methionine cystine Cys-Hcys
disulphide
Ion exchange chromatography
- Total homocysteine (mild hyperhomocysteinaemia)
- MMA by stable isotope dilution method
Routine laboratory parameters
Haematological folate cobalamin
HPLC Analysis of Homocysteine in plasma
Homocysteine Cysteine
Cys-Gly
Internal Standard
Time (minutes)
9 8 7 6 5 4 3 2 1
derivatised with Fluoro-Benzo-oxa-Diazole-Sulphonic acid
(SBDF)
GC-MS Chromatogram Methylmalonic aciduria
Tota
l Io
n C
urr
ent R
esponse
Elution time in minutes
MeCitrate
MethylMalonic acid
Methods for Diagnosis of the Homocystinurias
Specific Enzyme Assays
Cystathionine szlig synthase plusmn Pyrdoxal 5 phosphate
(Fibroblasts) plusmn S adenosylmethionine
Methylene THF reductase plusmn Flavin-adenine dinucleotide
(Fibros Leukocytes) plusmn Heating at 46deg (Thermolabile
Mutation)
Methionine synthase plusmn varying reducing agent for cblE
(Fibros Leukocytes)
Good discrimination between control and homozygous affected
individuals in most cases
Exceptions known variant mild forms
Methods for Diagnosis of the Homocystinurias
Indirect Pathway Assays in Intact Cultured
Fibroblasts
Formation of methionine and serine from [14C] formate
Remethylation defects (MR MS cblCD)
Complementation analysis
Cobalamin uptake and coenzyme formation
cblCD cblF cblE cblG (MTHFR def)
[14C] propionate incorporation into cell proteins
plusmn Hydroxo-Cbl
cblCD Methylmalonic acidurias
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
19
Methylmalonic acidaemias assays in fibroblasts
bull incorporation of [14C] propionate into cell proteins
bull specific activity of MMCoA mutase
bull specific activity of cobalamin adenosyltransferase
bull cellular uptake of CN-[57Co] cobalamin and its conversion to
cobalamin coenzymes
bull Somatic cell complementation analysis
Vitamin
B12
Adenosyl-Cbl
mitochondrion
Me-Cbl
cytosol
Intracellular Conversion of Vitamin
B12 to Active Coenzymes
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
Outline of Homocysteine Metabolism Enzymes
Homocysteine
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
20
MTHFR
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Homocysteine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
CBS
MS
Vitamin B6 (PLP)
Folic acid
Adenosine
Vitamin B12
GNMT
SAHH
MAT
BHMT
Riboflavin
Case Vitamin B6 (PLP)
MeCbl
AdoCbl
Outline of Homocysteine Metabolism Vitamins
MeTHFReduct
Methionine
S-adenosyl
methionine
S-adenosyl
homocysteine
Glycine
Sarcosine
Cystathionine
Cysteine
Sulphate
Serine
Tetrahydrofolate
5-Methyl-THF
510-MethyleneTHF
Cysta szlig-Synthase
Met Synthase
Adenosine
Gly N Me Transf
S-AdoHcy Hydrol
Met Adenosyl transf
Cystathionase
Betaine
HMT
IEM Pathways Homocysteine Metabolism
Homocysteine
Deficiencies of
each enzyme
known
The Homocysteine Metabolome
Main Branch lines metabolites
Homocysteine
Betaine Choline
etc(10) methylated
cmpds
(50 -85 )
B12 Vitamers (5)
MMA energy cmpds
cmpds (30-40)
Transulphuration
AdoMet AdoHcy
Sarcosine (10)
Cysteine
Glutathione
Taurine
cmpds (15)
DNA
precursors (5)
Folates
cmpds (gt 70)
Polyamines
precursors
(10)
Metabolite estimate
205 -265
Succinate
Fumarate
Malate
Oxaloacetate Citrate
Isocitrate
a-Ketoglutarate
Succinyl-CoA
Acetyl-CoA
Pyruvate Lactate
L-Methylmalony-CoA D-Methylmalonyl-CoA
free Methylmalonic Acid
Mutase
Propionyl-CoA
Valine Isoleucine
Methionine
Threonine
Odd-chain fatty acids
Cholesterol
AdoCbl
Epimerase
Succinate-CoA
Ligase ATP
GDP GTP
Cobalamin
ADP
Methylmalonic acid metabolism
7
Structure of Vitamin B12 (Cobalamin Cbl)
In man
essential cofactor for 2
enzymes
Methionine Synthase
Methyl-Cbl (MW 1344)
Methylmalonyl CoA-Mutase
Adenosyl-Cbl
(MW 1579)
Co Methyl Adenosyl
Microcell Mediated Chromosome Transfer (MMCT) (Hunt JD Anal Biochem1996)
Mouse donor cell line (23)
cytochalasin B
centrifugation
cell fusion (PEG)
filtration
PHA-P
selection
by antibiotic
TEST for phenotype
change
Hybrid cell line
Patient cell line
colcemid
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges
21
102 Mb chromosomal interval
containing 28 genes
8 uncharacterized genes
cM Markers Size HET ()
1544 150 218-242 83
1544 2286 218-264 70
1544 2334 259-273 77
1546 349 129-135 47
1548 129 162-180 77
1559 132 189-213 76
1564 381 298-312 60
1564 2270 209-221 56
1564 151 211-229 80
1569 2335 153-173 76
157 2301 108-135 73
1586 2277 245-259 58
1586 2324 130-156 71
Mapping analysis of chromosome 2 Content
A bit of history
Scope and overview
Methionine to Cysteine
Folate metabolism
Cobalamin
Newer IEMs
ADK mouse model precedes human disease
Betaine
MTHFD
CblD Cbl J (cblX )
Treatment challenges