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

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

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

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

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

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

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

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

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

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

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

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

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

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