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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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inborn errors of folate transport and metabolism
Inborn Errors of Folate Transport and Metabolism
  • Hereditary Folate Malabsorption
  • Glutamate Formiminotransferase Deficiency
  • Methylenetetrahydrofolate Reductase Deficiency
  • Methionine Synthase Reductase Deficiency (cblE)
  • Methionine Syntase Deficiency (cblG)
slide8

Histidine

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).

slide10

Histidine

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
Hereditary Folate Malabsorption
  • 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
hereditary folate malabsorption12
Hereditary Folate Malabsorption
  • 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
slide13

Histidine

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
Methylenetetrahydrofolate Reductase Deficiency (Severe)
  • 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
MTHFR 677C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
mthfr 677c t19
MTHFR 677C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
MTHFR 677C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.
mthfr 677c t disease associations incomplete
MTHFR 677CTDisease Associations (Incomplete)
  • Cardiovascular Disease
  • Alzheimer Disease
  • Colon Cancer
  • Diabetes Mellitus
  • Down Syndrome
  • Leukemia
  • Neural Tube Defects (NTD)
  • Pregnancy Complications
mthfr 1298a c
MTHFR 1298A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
slide24

Histidine

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
Glutamate Formimotransferase 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
Human FTCD
  • 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
GFT Patients
  • 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
Third GFT Patient
  • 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
Southern Blot

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
Western Blot

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.

conclusions
Conclusions
  • First mutations in Human FTCD in three patients with glutamate formiminotransferase deficiency.
functional methionine syntase deficiency
FUNCTIONAL METHIONINE SYNTASE 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
Methionine Synthase Reductase Deficiency-cblE
  • 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
Methylcobalamin-Dependent Methionine Synthase in E. Coli
  • 2 component flavoprotein system
  • flavodoxin
  • NADPH-ferredoxin (flavodoxin) oxidoreductase, a member of electron transferases termed the “FNR family”
methionine synthase reductase
Methionine Synthase Reductase
  • 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
Methionine Synthase Reductase
  • 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

Lysosome

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
Methionine Synthase Deficiency-cblG
  • 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
I-F-Cobalamin Receptor Deficiency

(Imerslund –Gräsbeck Syndrome) (MGA1)

Example of One Phenotype, 2 Genes

i f cobalamin receptor deficiency imerslund gr sbeck mga1
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
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.

slide64
Intracellular Cobalamin Metabolism:

Endocytosis

Reduction

Mitochondrial Transport & Adenosylation-AdoCbl

Methylation-MeCbl

transcobalamin receptor67
Transcobalamin Receptor

Jacobsen and Glushchenko, 2009

transcobalamin receptor68
Transcobalamin Receptor
  • 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
Transcobalamin Receptor
  • Total Cobalamin Uptake

Control

WG3733

transcobalamin receptor70
Transcobalamin Receptor

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

transcobalamin receptor71
Transcobalamin Receptor

Binding kinetics of TC-Cbl to normal and mutant fibroblasts

transcobalamin receptor72
Transcobalamin Receptor

Amino acid sequence of TCblR

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

slide73

Transcobalamin Receptor

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

slide74
cblF
  • Combined homocystinuria and methylmalonic

aciduria

  • Accumulation of free cobalamin in lysosomes
  • Postulated defect in efflux of cobalamin from lysosomes
slide75

cblF

  • Microcell-mediated chromosome transfer
    • Transferred chromosome 2-7, 10, 12 and 16 into cblF patient fibroblasts.
slide77
cblF

Chromosome 6 Propionate Incorporation

Normal

slide78
cblF
  • Mapping of a locus for cblF on chromosome 6q13

Rutsch et al, 2009

slide79
cblF
  • Mapping of a locus for cblF on chromosome 6q13

Rutsch et al, 2009

slide80
cblF
  • Localization of LMBD1 to lysosome
slide81
MMA
  • 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)
mut mma
mut MMA
  • 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
MUT

 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
Cobalamin-responsive MMA
  • Two genes cloned on the basis of homology:
  • MMAA: cblA complementation group
  • MMAB: cblB complementation group
slide88

c.64C>T (R22X)

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
MMAA
  • 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)
slide91

MMAB

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
MMAB 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 cbld variant cblh
MMADHC-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

cblD-HC

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
Genes Associated with Isolated MMA
  • 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

5-Methyl-THF

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
cblC
  • 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
cblC
  • 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
Diagnosis of cblC
  • 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
Diagnosis of cblC
  • Special investigations: cultured fibroblasts
    • Incorporation of label from [14C]propionate and 5-[14C]methyltetrahydrofolate into cellular macromolecules
    • Cbl distribution studies
    • Complementation studies
slide104

c.331C>T

c.440G>A

c.271dupA

c.3G>A

c.394C>T

c.608G>A

c.547_548delGT

c.609G>A

slide105

Homozygosity mapping and haplotype analysis

  • MMACHC
  • Some degree of homology with TonB, a bacterial protein involved in energy transduction for vitamin B12-uptake
slide106

204 patients

  • 42 mutations
    • c.271dupA: 40%
    • c.331C>T: 9%
    • c.394C>T: 8%
phenotype genotype correlations seeking answers in case reports
Phenotype-Genotype Correlations:Seeking Answers in Case-Reports
  • 37 previously published patients:
    • 25 early-onset cases
    • 12 late-onset cases:
      • 9: neurological phenotype
      • 3: renal phenotype
early onset cases
Early-Onset Cases
  • 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
late onset cases
Late-Onset Cases
  • 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
observations on ethnic background
Observations on Ethnic Background
  • 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

observations on ethnic background111
Observations on Ethnic Background
  • 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

observations on ethnic background112
Observations on Ethnic Background
  • 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

observations on ethnic background113
Observations on Ethnic Background
  • Homozygosity for c.440G>A: 2 patients
    • Native American (Southwestern)
    • In database: 1 unpublished Native American patient of the same tribe
compound heterozygosity c 394c t c 271dupa
Compound heterozygosity: c.394C>T / c.271dupA
  • 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

response to cbl supplementation
Response to Cbl Supplementation
  • 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
c 271dupa missense mutations
c.271dupA / missense mutations
  • 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

slide121
The 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.
slide124
It is possible that the 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.
slide125

Cobalamin metabolism

Moras et al., 2006

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