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STRUCTURE OF TETRAHYDROFOLATE. STRUCTURE OF FOLIC ACID AND REDUCED FOLATES INVOLVED IN ONE-CARBON METABOLISM. FOLATE PATHWAY. Inborn Errors of Folate Transport and Metabolism. Hereditary Folate Malabsorption Glutamate Formiminotransferase Deficiency

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Folate pathway l.jpg
FOLATE PATHWAY ONE-CARBON METABOLISM


Inborn errors of folate transport and metabolism l.jpg
Inborn Errors of Folate Transport and Metabolism ONE-CARBON METABOLISM

  • Hereditary Folate Malabsorption

  • Glutamate Formiminotransferase Deficiency

  • Methylenetetrahydrofolate Reductase Deficiency

  • Methionine Synthase Reductase Deficiency (cblE)

  • Methionine Syntase Deficiency (cblG)


Slide8 l.jpg

Histidine ONE-CARBON METABOLISM

Formiminoglutamate

2

Glutamateformiminotransferase

Formate + THF

5-Formimino-THF

2

Cyclodeaminase

5-Formyl-THF

NAD+ NADH

10-Formyl-THF

5, 10-Methenyl-THF

NADP+

NADPH

NADP+ NADPH

Purine nucleotides

5, 10-Methylene-THF

Methylene-THF reductase

dUMP

3

Glycine

dTMP

5-Methyl-THF

DHF

Serine

Pyrimidine nucleotides

1

Transport across intestine + CP

NADPH

THF

4

5

SAM

MeCbl

Methionine

synthase

Homocysteine

Methionine + THF

Figure 1: Summary of major reactions of folate pathway. DHF= dihydrofolate, THF= tetrahydrofolate, dUM= deoxy-uridine phosphate, dTMP= deoxy-thymidine phosphate, CP= choroid plexus, SAM= S-adenosylmethionine, MeCbl= methylcobalamin. Disorders are indicated by circled numbers. 1= Hereditary folate malabsorption, 2= Glutamate formiminotransferase-cyclodeaminase deficiency, 3= Severe Methylenetetrahydrofolate reductase deficiency, 4= Methionine synthase deficiency (cblG) (see Intracellular Cobalamin Metabolism section), 5= Methionine synthase reductase deficiency (cblE) (see Intracellular Cobalamin Metabolism section).


Herditary folate malabsorption l.jpg
HERDITARY FOLATE MALABSORPTION ONE-CARBON METABOLISM


Slide10 l.jpg

Histidine ONE-CARBON METABOLISM

Formiminoglutamate

2

Glutamateformiminotransferase

Formate + THF

5-Formimino-THF

2

Cyclodeaminase

5-Formyl-THF

NAD+ NADH

10-Formyl-THF

5, 10-Methenyl-THF

NADP+

NADPH

NADP+ NADPH

Purine nucleotides

5, 10-Methylene-THF

Methylene-THF reductase

dUMP

3

Glycine

dTMP

5-Methyl-THF

DHF

Serine

Pyrimidine nucleotides

1

Transport across intestine + CP

NADPH

THF

4

5

SAM

MeCbl

Methionine

synthase

Homocysteine

Methionine + THF

Figure 1: Summary of major reactions of folate pathway. DHF= dihydrofolate, THF= tetrahydrofolate, dUM= deoxy-uridine phosphate, dTMP= deoxy-thymidine phosphate, CP= choroid plexus, SAM= S-adenosylmethionine, MeCbl= methylcobalamin. Disorders are indicated by circled numbers. 1= Hereditary folate malabsorption, 2= Glutamate formiminotransferase-cyclodeaminase deficiency, 3= Severe Methylenetetrahydrofolate reductase deficiency, 4= Methionine synthase deficiency (cblG) (see Intracellular Cobalamin Metabolism section), 5= Methionine synthase reductase deficiency (cblE) (see Intracellular Cobalamin Metabolism section).


Hereditary folate malabsorption l.jpg
Hereditary Folate Malabsorption ONE-CARBON METABOLISM

  • Hereditary folate malabsorption (HFM) (OMIM 229050) is a rare autosomal recessive disorder caused by impaired intestinal folate absorption with folate deficiency characterized by anemia, hypoimmunoglobulinemia with recurrent infections, such as Pneumocystis carinii pneumonitis, and recurrent or chronic diarrhea. In many patients, neurological abnormalities such as seizures or mental retardation emerge at some point in early childhood, attributed to impaired transport of folates into the central nervous system 1. When this disorder is diagnosed early, signs and symptoms of HFM can be obviated by parental administration of folates or with higher doses of folates by the oral route 1, 2. If untreated, the disease is fatal and, if treatment is delayed, the neurological deficits can become permanent


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Hereditary Folate Malabsorption ONE-CARBON METABOLISM

  • Qui A et al. Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption. Cell 127, 917-928, December 1, 2006

  • Proton coupled, high affinity folate transporter operating at low pH.

  • Loss of function mutations in HFM

  • PCFT/HCP1


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Histidine ONE-CARBON METABOLISM

Formiminoglutamate

2

Glutamateformiminotransferase

Formate + THF

5-Formimino-THF

2

Cyclodeaminase

5-Formyl-THF

NAD+ NADH

10-Formyl-THF

5, 10-Methenyl-THF

NADP+

NADPH

NADP+ NADPH

Purine nucleotides

5, 10-Methylene-THF

Methylene-THF reductase

dUMP

3

Glycine

dTMP

5-Methyl-THF

DHF

Serine

Pyrimidine nucleotides

1

Transport across intestine + CP

NADPH

THF

4

5

SAM

MeCbl

Methionine

synthase

Homocysteine

Methionine + THF

Figure 1: Summary of major reactions of folate pathway. DHF= dihydrofolate, THF= tetrahydrofolate, dUM= deoxy-uridine phosphate, dTMP= deoxy-thymidine phosphate, CP= choroid plexus, SAM= S-adenosylmethionine, MeCbl= methylcobalamin. Disorders are indicated by circled numbers. 1= Hereditary folate malabsorption, 2= Glutamate formiminotransferase-cyclodeaminase deficiency, 3= Severe Methylenetetrahydrofolate reductase deficiency, 4= Methionine synthase deficiency (cblG) (see Intracellular Cobalamin Metabolism section), 5= Methionine synthase reductase deficiency (cblE) (see Intracellular Cobalamin Metabolism section).



Methylenetetrahydrofolate reductase deficiency severe l.jpg
Methylenetetrahydrofolate Reductase Deficiency (Severe) DEFICIENCY

  • Hyperhomocysteinemia and homocystinuria

  • Low or normal plasma methionine

  • No megaloblastic anemia !!

  • Variable clinical manifestations including: 1) death in the first year of life; 2) developmental delay; 3) neurologic and psychiatric disease; 4) thrombotic events; 5) asymptomatic

  • Gene/location: MTHFR/ Chr. 1p36.3

  • Common polymorphisms: 677CT; 1298AC



Mthfr 677c t l.jpg
MTHFR 677C DEFICIENCYT

  • Originally discovered because specific activity of MTHFR in cell extracts was thermolabile

  • 50-60% decrease in specific activity of MTHFR

  • First postulated association (Kang et al) was between thermolability of MTHFR and heart disease


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MTHFR 677C DEFICIENCYT

  • After cloning of the gene, the cause of thermolability of MTHFR was shown to be this common polymorphism in the catalytic domain that results in the change of an alanine to a valine.

  • Gene frequency of the T allele varies with ethnic groups (30% in Europeans and Japanese, 11% in African Americans).


Mthfr 677c t20 l.jpg
MTHFR 677C DEFICIENCYT

  • T allele is associated with elevated levels of total homocysteine (tHcy).

  • Effect is much more prominent in TT individuals

  • Dietary folate (multivitamins, fortification of cereal grains) can mask the effect of the T allele.


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MTHFR 677C DEFICIENCYTDisease Associations (Incomplete)

  • Cardiovascular Disease

  • Alzheimer Disease

  • Colon Cancer

  • Diabetes Mellitus

  • Down Syndrome

  • Leukemia

  • Neural Tube Defects (NTD)

  • Pregnancy Complications


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MTHFR 1298A DEFICIENCYC

  • Associated with 35% decrease in MTHFR specific activity

  • Not associated with enzyme thermolability

  • Frequency of C allele: 30% Western Europe and 18% in Asians

  • 1298C and 677T rarely found together in cis

  • Fewer studies have looked at this polymorphism



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Histidine DEFICIENCY

Formiminoglutamate

2

Glutamateformiminotransferase

Formate + THF

5-Formimino-THF

2

Cyclodeaminase

5-Formyl-THF

NAD+ NADH

10-Formyl-THF

5, 10-Methenyl-THF

NADP+

NADPH

NADP+ NADPH

Purine nucleotides

5, 10-Methylene-THF

Methylene-THF reductase

dUMP

3

Glycine

dTMP

5-Methyl-THF

DHF

Serine

Pyrimidine nucleotides

1

Transport across intestine + CP

NADPH

THF

4

5

SAM

MeCbl

Methionine

synthase

Homocysteine

Methionine + THF

Figure 1: Summary of major reactions of folate pathway. DHF= dihydrofolate, THF= tetrahydrofolate, dUM= deoxy-uridine phosphate, dTMP= deoxy-thymidine phosphate, CP= choroid plexus, SAM= S-adenosylmethionine, MeCbl= methylcobalamin. Disorders are indicated by circled numbers. 1= Hereditary folate malabsorption, 2= Glutamate formiminotransferase-cyclodeaminase deficiency, 3= Severe Methylenetetrahydrofolate reductase deficiency, 4= Methionine synthase deficiency (cblG) (see Intracellular Cobalamin Metabolism section), 5= Methionine synthase reductase deficiency (cblE) (see Intracellular Cobalamin Metabolism section).


Glutamate formimotransferase deficiency l.jpg
Glutamate Formimotransferase Deficiency DEFICIENCY

  • Autosomal Recessive (<20 patients)

  • Formiminoglutamate (FIGLU) excretion

  • Clinical heterogeneity: 1) developmental delay, elevated serum folate, FIGLU excretion 2) mild speech delay, high levels of FIGLU excretion.

  • Note that GFTD activity cannot be measured in cultured cells-present only in liver.


Human ftcd l.jpg
Human FTCD DEFICIENCY

  • Discovered by examination of EST’s on chromosome 21 as part of a study assessing the molecular basis of Down Syndrome

  • EST compared to porcine FTCD

  • Human 21q22.3

  • 15 exons

  • 541 amino acid residues with 84% homology to the pig.

  • Five different transcripts


Gft patients l.jpg
GFT Patients DEFICIENCY

  • Siblings: 1) Age 2 1/2 years - speech delay, some growth delay, hypotonia, increased FIGLU excretion 2) Age 8 years-hypotonia, abnormal EEG, increased FIGLU excretion

  • Two missense mutations: c457 c->T (R135C) and c940 C->G (R299P). Not found in 200 control alleles.


Third gft patient l.jpg
Third GFT Patient DEFICIENCY

  • Apnea in the first year of life

  • Recurrent infections

  • At age 2, mild developmental delay, hypotonia, breathing difficulties

  • Hypersegmented neutrophils

  • Increased FIGLU excretion

  • One mutation: c1033 insG (not found in 200 control alleles)


Southern blot l.jpg
Southern Blot DEFICIENCY

HindIII

BamHI

Kpn I

MCH24

WG1795

MCH39

WG1191

MCH24

WG1795

MCH39

WG1191

MCH24

WG1795

MCH39

WG1191

10 ug of genomic DNA (5 ug for MCH 39) was digested with the indicated enzymes, run on a 0.8% agarose gel at 25V and transferred to Hybond N+. The blot was probed with random-primed P32 labelled hFTCD (B-form) probe.


Western blot l.jpg
Western Blot DEFICIENCY

c1033insG

FTCDH6

CD333H6

S407L

FTH6

R135C

R299P

A438E

175 kDa

83.0 kDa

62.0 kDa

47.5 kDa

32.5 kDa

25.0 kDa

16.5 kDa

25 ? Ug of protein (crude extract) was run on 12%SDS-PAGE and transferred to nitrocelluose. The blot was probed with polyclonal rabbit anti-pFTCD followed by HRP-conjugated goat anti-rabbit IgG.


Ftcd assay l.jpg
FTCD Assay DEFICIENCY


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FTCD Assay DEFICIENCY


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Conclusions DEFICIENCY

  • First mutations in Human FTCD in three patients with glutamate formiminotransferase deficiency.


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FUNCTIONAL METHIONINE SYNTASE DEFICIENCY DEFICIENCY

Overlap in Folate and Cobalamin Metabolism:

One phenotype

Two Genotypes: cblE (Methionine synthase reductase deficiency)

cblG (Methionine synthase deficiency)



Methionine synthase reductase deficiency cble46 l.jpg
Methionine Synthase Reductase Deficiency- DEFICIENCYcblE

  • Megaloblastic anemia, hyperhomocysteinemia and homocystinuria

  • Low plasma methionine

  • Cerebral atrophy, nystagmus, blindness, altered tone

  • Reduced methionine synthase activity in the absence of an exogenous reducing system

  • Gene/ location: MTRR/ 5p15.2-15.3

  • Polymorphism: 66AG


Methylcobalamin dependent methionine synthase in e coli l.jpg
Methylcobalamin-Dependent Methionine Synthase in DEFICIENCYE. Coli

  • 2 component flavoprotein system

  • flavodoxin

  • NADPH-ferredoxin (flavodoxin) oxidoreductase, a member of electron transferases termed the “FNR family”


Methionine synthase reductase l.jpg
Methionine Synthase Reductase DEFICIENCY

  • Findings suggest evolution of the two genes specifying flavodoxin/flavodoxin reductase to a single gene encoding a fused version of the two proteins in man.

  • This new gene has been called MTRR since the gene for methionine synthase is MTR.


Methionine synthase reductase49 l.jpg
Methionine Synthase Reductase DEFICIENCY

  • Localized to chromosome 5p15.2-p15.3

  • 2094 bp - 698 amino acids

  • Predicted molecular mass 77,000 Da

  • Prominent RNA species of 3.6 kb with an additional smaller 3.1 kb species in brain

  • 38% identity (49% similarity) with human cytochrome P-450 reductase


Slide50 l.jpg

Lysosome DEFICIENCY

Mitochondrion

Methylmalonyl-CoA

TCII-Cob(III)alamin

mut

cblB

TCII

Methylmalonyl-CoAMutase

Cob(I)alamin

AdoCbl

Cob(III)alamin

cblF

cblA

Succinyl-CoA

cblH

Cob(III)alamin

Cob(II)alamin

cblC

cblD

Cob(I)alamin

Methionine

5-MethylTHF

MTHFR

Methionine Synthase

Cob(II)alamin

cblG

cblG

5,10-methyleneTHF

Methionine SynthaseReductase

AdoMet

cblE

Extracellular Space

Homocysteine

THF

Cytoplasm

Methylcobalamin



Methionine synthase deficiency cblg53 l.jpg
Methionine Synthase Deficiency- DEFICIENCYcblG

  • Hyperhomocysteinemia and homocystinuria

  • Low plasma methionine; Megaloblastic anemia

  • Cerebral atrophy, nystagmus, blindness, altered tone. Some patients present in adult life!!

  • Reduced methionine synthase activity

  • Gene/Location: MTR/ Chr. 1q43

  • Polymorphism: 2756AG



Slide61 l.jpg

I-F-Cobalamin Receptor Deficiency DEFICIENCY

(Imerslund –Gräsbeck Syndrome) (MGA1)

Example of One Phenotype, 2 Genes


I f cobalamin receptor deficiency imerslund gr sbeck mga1 l.jpg
I-F-Cobalamin Receptor Deficiency (Imerslund -Gräsbeck) (MGA1)

  • Early onset megaloblastic anemia, low serum cobalamin levels, and proteinuria

  • Homocystinuria and methylmalonic aciduria may be found but are not prominent

  • Decreased absorption of cobalamin in the presence of normal synthesis of intrinsic factor

  • Common in Finland, Norway and the Middle East

  • Defects in CUBN (cubilin) & AMN (amnionless)

  • Genes/ Locations: Chrs. 10p12.1 & 14q32


I f cobalamin receptor deficiency imerslund gr sbeck mga163 l.jpg
I-F-Cobalamin Receptor Deficiency (Imerslund -Gräsbeck) (MGA1)

Fyfe et al. Blood Online October 2003:

Interaction of cubilin and amnionless to form a complex (cubam) that functions as the cobalamin-IF receptor.

Without amnionless, cubilin does not reach the cell membrane.


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Intracellular Cobalamin Metabolism: (MGA1)

Endocytosis

Reduction

Mitochondrial Transport & Adenosylation-AdoCbl

Methylation-MeCbl


Vitamin b 12 pathway overview l.jpg
Vitamin B (MGA1)12 Pathway Overview

TCblR



Transcobalamin receptor67 l.jpg
Transcobalamin Receptor (MGA1)

Jacobsen and Glushchenko, 2009


Transcobalamin receptor68 l.jpg
Transcobalamin Receptor (MGA1)

  • TC Receptor-First Inborn Error

    • Infant with methylmalonic aciduria detected on newborn screening.

    • Response to treatment with cobalamin but low level MMA persisted.


Transcobalamin receptor69 l.jpg
Transcobalamin Receptor (MGA1)

  • Total Cobalamin Uptake

Control

WG3733


Transcobalamin receptor70 l.jpg
Transcobalamin Receptor (MGA1)

Uptake and accumulation of B12 in fibroblasts during six days in culture


Transcobalamin receptor71 l.jpg
Transcobalamin Receptor (MGA1)

Binding kinetics of TC-Cbl to normal and mutant fibroblasts


Transcobalamin receptor72 l.jpg
Transcobalamin Receptor (MGA1)

Amino acid sequence of TCblR

(Deletion is shown in red and the polymorphisms in green)


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Transcobalamin Receptor (MGA1)

Ribbon Diagram depicting the secondary structure of the wild type and mutant TCblR


Slide74 l.jpg
cblF (MGA1)

  • Combined homocystinuria and methylmalonic

    aciduria

  • Accumulation of free cobalamin in lysosomes

  • Postulated defect in efflux of cobalamin from lysosomes


Slide75 l.jpg

cblF (MGA1)

  • Microcell-mediated chromosome transfer

    • Transferred chromosome 2-7, 10, 12 and 16 into cblF patient fibroblasts.


Microcell mediated chromosome transfer colonies tested l.jpg

cblF (MGA1)

Microcell-mediated chromosome transfer:Colonies tested


Slide77 l.jpg
cblF (MGA1)

Chromosome 6 Propionate Incorporation

Normal


Slide78 l.jpg
cblF (MGA1)

  • Mapping of a locus for cblF on chromosome 6q13

Rutsch et al, 2009


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cblF (MGA1)

  • Mapping of a locus for cblF on chromosome 6q13

Rutsch et al, 2009


Slide80 l.jpg
cblF (MGA1)

  • Localization of LMBD1 to lysosome


Slide81 l.jpg
MMA (MGA1)

  • Is the MMA isolated? Is tHcy elevated?

  • Low serum cobalamin levels should lead one to expect a disorder of intake or transport: Breast –fed infant of vegan mother or mother with subclinical PA

  • Imersund-Grasbeck (MGA1)-mutations in cublin or amnionless (Stephan Tanner-Ohio)

  • Combined MMA and Homocystinuria (cblC, cblD, cblF)


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MUT (MGA1)


Mut mma l.jpg
mut MMA (MGA1)

  • At least 178 different mutations

  • Difficult to make genotype/phenotype correlations. Many patients are compound heterozygotes and different patients homozygous for the same mutation may have different phenotypes

  • There are a number of mutations that are more common in specific ethnic groups and a number of common mutations.


Slide84 l.jpg
MUT (MGA1)

 seen in more than one patient

 seen in only one family

Missense Mutations

1 2 3 4 5 6 7 8 9 10 11 12 13

        

            

   

     

 

      

   

       

Nonsense Mutations



   

 



  

Deletions and insertions

   

  

    

  

 

 

Splice Mutations


Cobalamin responsive mma l.jpg
Cobalamin-responsive (MGA1) MMA

  • Two genes cloned on the basis of homology:

  • MMAA: cblA complementation group

  • MMAB: cblB complementation group


Slide87 l.jpg

MMAA (MGA1)


Slide88 l.jpg

c.64C>T (R22X) (MGA1)

c.161G>A (W54X)

c.260-267dupATAAACTT

c.266T>C (L89P )

c.283C>T (Q95X)

c.387C>A (Y129X)

c.742C>T (Q248X)

c.433C>T (R145X)

c.959G>A (W320X)

c.434G>A (R145Q)

c.439+1_4delGTCA Splice

c.970-2A>T Splice

1

Exon

4

2

3

5

6

7

c.733+1G>A Splice

c.620A>G (Y207C)

c.653G>A (G218E)

c.592_595delACTG

c.988C>T (R330X)

c.503delC

c.1076G>A (R359Q)

c.450_451insG

c.440G>A (E147G)

c.1089_1090delGA


Slide89 l.jpg
MMAA (MGA1)

  • At least 29 mutations known

  • C.433C>T accounts for 43% alleles in one North American Study

  • c503delC more frequent in Japan (8 of 14 mutant alleles)


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MMAB (MGA1)


Slide91 l.jpg

MMAB (MGA1)

c.572_576 del GGGCC

c.56-57 GC>AA (R19Q)

c.575 G>A (E193K)

c.716 T>A (M239K)

c.IVS2-1 G>T

c.571 C>T (R191W)

c.700 C>T (Q234X)

c.569 G>A (R190H)

c.656 A>G (Y219C)

c.IVS7-2 A>C

c.556 C>T (R186W)

c.654_657 del CTAT

c.403 G>A (A135T)

c.IVS3-1 G>A


Mmab mutations l.jpg
MMAB (MGA1)Mutations

  • 22 mutations Identified

  • Most predicted to affect the active site of the enzyme, identified from the crystal structure of is bacterial ortholog

  • C.556C>T (p.R186W) represents 33% of affected alleles.


Mmadhc l.jpg

MMADHC (MGA1)


Mmadhc cbld variant cblh l.jpg
MMADHC- (MGA1)cblDvariant=cblH

  • Associated with isolated MMA

  • Decreased propionate incorporation

  • Decreased AdoCbl synthesis

  • Novel gene MMADHC isolated by Brian Fowler in Switzerland

  • Identical to cblH

  • Mutations in N-terminal regions associated with isolated MMA


Slide95 l.jpg

cblD-HC (MGA1)

cblD-MMA

L20fsX21

T152fsX162

2

3

4

5

6

7

8

9

154

372

478

609

696

891

S228M

cblD-HC+MMA


Genes associated with isolated mma l.jpg
Genes Associated with Isolated MMA (MGA1)

  • MUT

  • MMAA

  • MMAB

  • MMADHC (NEJM in press)

  • MCEE-may not be related to clinical

  • SUCLA2-developmental delay

  • SUCLG1-fatal infantile lactic acidosis (Ostergaard E et al. Am J Hum Genet 81:383, 2007)


Slide97 l.jpg

5-Methyl-THF (MGA1)

THF

cblG

Methionine synthase

Homocysteine

Methionine

MeCbl

MTRR

cblE

Co(I)bl

cblD variant 1

cblC, cblD

cblF

Cbl

TC

TC/Cbl

Co(III)bl

Co(II)bl

cblD variant 2

Lysosome

cblA, cblH

Co(II)bl

Co(I)bl

cblB

AdoCbl

Cell membrane

Methylmalonyl-CoA Succinyl-CoA

Methylmalonyl-CoA

mutase

mut

Mitochondrion


Slide98 l.jpg
cblC (MGA1)

  • Most common inborn error of Vitamin B12 metabolism

  • Early-onset:

    • Feeding difficulties, hypotonia/hypertonia, lethargy

    • Abnormal movement, seizures

    • Multisystemic involvement

    • Pancytopenia or megaloblastic anemia

    • Salt-and-pepper retinopathy

    • Moderate to severe cognitive disability


Slide99 l.jpg
cblC (MGA1)

  • Late-onset (renal phenotype):

    • Chronic thrombotic microangiopathic syndrome

    • Absence of neurological involvement

  • Late-onset (neurological phenotype):

    • Sudden cognitive decline (confusion, dementia)

    • Extrapyramidal signs, ataxia, peripheral neuropathy

    • Milder hematological abnormalites


Diagnosis of cblc l.jpg
Diagnosis of cblC (MGA1)

  • Clinical history, physical exam

  • Laboratory investigations:

    • CBC with smear, ± bone marrow biospy

    • Plasma amino acids (elevated Hcy, low methionine)

    • Urine organic acids (elevated MMA)

    • Total plasma homocysteine

    • Others as clinically indicated (Normal serum cobalamin and folate levels).


Diagnosis of cblc101 l.jpg
Diagnosis of cblC (MGA1)

  • Special investigations: cultured fibroblasts

    • Incorporation of label from [14C]propionate and 5-[14C]methyltetrahydrofolate into cellular macromolecules

    • Cbl distribution studies

    • Complementation studies


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c.331C>T (MGA1)

c.440G>A

c.271dupA

c.3G>A

c.394C>T

c.608G>A

c.547_548delGT

c.609G>A


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  • 204 patients (MGA1)

  • 42 mutations

    • c.271dupA: 40%

    • c.331C>T: 9%

    • c.394C>T: 8%


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Phenotype-Genotype Correlations: (MGA1)Seeking Answers in Case-Reports

  • 37 previously published patients:

    • 25 early-onset cases

    • 12 late-onset cases:

      • 9: neurological phenotype

      • 3: renal phenotype


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Early-Onset Cases (MGA1)

  • 25 out of 37 patients

  • 9/25: homozygous for c.271dupA

  • 3/25: homozygous for c.331C>T

  • 5/25: c.271dupA / c.331C>T

  • 1/25: c.271dupA / c.394C>T

  • Remaining 8 patients either:

    • Compound heterozygous for different nonsense mutations

    • Homozygous for another nonsense mutation


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Late-Onset Cases (MGA1)

  • 12 of 37 patients

  • 9/12: neurological phenotype

  • 3/12: renal phenotype

  • Neurological phenotype:

    • 4/9: homozygous for c.394C>T

    • 2/9: c.271dupA and c.394C>T

    • 3/9: c.271dupA and a missense mutation

  • Renal phenotype:

    • 3/3: c.271dupA and c.82-9_-12delTTTC


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Observations on Ethnic Background (MGA1)

  • Homozygosity for c.271dupA: 9 patients

    • 5 White

    • 1 Hispanic

    • 1 Iranian

    • 1 Middle Eastern

    • 1 ? Ethnicity

    • In database: 44 other patients of various ethnic backgrounds

Therefore, not specific to one ethnic group


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Observations on Ethnic Background (MGA1)

  • Homozygosity for c.331C>T: 3 patients

    • “Cajun”

    • 3 unpublished French Canadian patients from Québec and New Brunswick

  • Compound heterozygosity c.331C>T/c.271dupA:

    • 5 patients:

      • 1: White (USA, “French” background on pedigree in lab)

      • 1: French Canadian from Québec

      • 3: Louisiana, USA (New Orleans)

      • In database: 5 additional patients of French-Canadian or Cajun background

Suggest possible founder effect/genetic drift


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Observations on Ethnic Background (MGA1)

  • Homozygosity for c.394C>T: 4 patients

    • 3: Asiatic-Indian (incl. 2 sibs)

    • 1: Middle Eastern

    • In database: 9 other patients, all Asiatic-Indian, Pakistani or Middle Eastern

  • Heterozygosity c.394C>T / c.271dupA: 3 patients

    • 1: Greek

    • 1: Portuguese

    • 1: ? Ethnicity

Mutational hot-spot: arose at least twice independently


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Observations on Ethnic Background (MGA1)

  • Homozygosity for c.440G>A: 2 patients

    • Native American (Southwestern)

    • In database: 1 unpublished Native American patient of the same tribe


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Compound heterozygosity: c.394C>T / c.271dupA (MGA1)

  • 2 late-onset published cases:

    • Ages 4.5 and 10 years

  • 1 early-onset published case:

    • Age 6 months

  • Intrafamilial phenotypic heterogeneity:

    • Augoustides-Savvopoulou P, Mylonas I, Sewell AC, Rosenblatt DS. Reversible dementia in an adolescent with cblC disease: clinical heterogeneity within the same family. J InheritMetab Dis 1999; 22(6):756-758

    • Late-onset AND early-onset in the same family!!!

Interpretation of anticipated phenotype based on this genotype may be unreliable


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Response to Cbl Supplementation (MGA1)

  • Homozygosity c.271dupA:

    • Tend to have progression of disease despite Tx

  • Homozygosity c.394C>T:

    • Almost complete reversal of psychiatric and neurological symptoms

  • Compound heterozygosity c.394C>T / c.271dupA

    • Almost complete reversal of psychiatric and neurological symptoms


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c.271dupA / missense mutations (MGA1)

  • Late-onset neurological phenotype:

    • c.271dupA / c.440G>C, 45 years

      Powers JM, Rosenblatt DS, Schmidt RE et al. Neurological and neuropathologic heterogeneity in two brothers with cobalamin C deficiency. Ann Neurol 2001; 49(3):396-400

    • c.271dupA / c.482G>A, 20 years

      Bodamer OA, Rosenblatt DS, Appel SH, Beaudet AL. Adult-onset combined methylmalonic aciduria and homocystinuria (cblC). Neurology 2001; 56(8):1113

    • c.271dupA / c.347T>C, 24 years

      Roze E, Gervais D, Demeret S et al. Neuropsychiatric disturbances in presumed late-onset cobalamin C disease. Arch Neurol 2003; 60(10):1457-1462


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  • The (MGA1)MMACHC protein is not a member of any previously identified gene family.

  • It is well conserved among mammals. However, the C-terminal end does not appear to be conserved in eukaryotes outside Mammalia, and no homologous protein was identified in prokaryotes

  • Motifs were identified in MMACHC that are homologous to motifs in bacterial genes with vitamin B12-related functions.


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  • It is possible that the (MGA1)MMACHC gene product plays a role, directly or indirectly, in removal of the upper axial ligand and/or reduction of Cbl, and this is a challenge for future studies.

  • MMACHC may be involved in the binding and intracellular trafficking of Cbl.

  • Further studies on co-localization and a search for novel binding partners may help us to better understand the early steps of cellular vitamin B12 metabolism.


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Cobalamin metabolism (MGA1)

Moras et al., 2006