STRUCTURE OF TETRAHYDROFOLATE

STRUCTURE OF FOLIC ACID AND REDUCED FOLATES

INVOLVED IN ONE-CARBON 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)

HERDITARY 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 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

FOLATE PATHWAY

Formate + THF

Purine nucleotides

5, 10-Methenyl-THF

5-Formimino-THF

5-Formyl-THF

FormiminoglutamateHistidine

Glutamate formiminotransferase

Cyclodeaminase

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).

10-Formyl-THF

5, 10-Methylene-THF

THF

5-Methyl-THF DHF

Glycine

Serine

NADPH

dUMP

dTMP

Pyrimidine nucleotides

Methylene-THF reductase

Methioninesynthase

Homocysteine Methionine + THF

SAM MeCbl

NAD+ NADH

NADP+ NADPH

NADP+

NADPH

Transport across intestine + CP

1

2

2

3

54

SEVERE METHYLENETE-TRAHYDROFOLATE (MTHFR)

REDUCTASE DEFICIENCY

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: 677CT; 1298AC

COMMON POLYMORPHISMS IN MTHFR

MTHFR 677CT

• 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 677CT

• 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 677CT

• 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 677CTDisease Associations

(Incomplete)• Cardiovascular Disease • Alzheimer Disease• Colon Cancer• Diabetes Mellitus• Down Syndrome• Leukemia• Neural Tube Defects (NTD)• Pregnancy Complications

MTHFR 1298AC

• 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

GLUTAMATE FORMININOTRANSFERASE

DEFICIENCY

Mutations in the FTCD gene on chromosome 21 are the causeof glutamate formiminotransferase deficiency.

Rosenblatt DS1,2,3, Hilton JF3, Christensen K4, Hudson TJ1,2,5,Raby BA2,5, Estivil R6, de la Luna S6, MacKenzie RE4.Department of Human Genetics1, Medicine2, Biology3 andBiochemistry4, McGill University, Montreal Genome Centre5,Medical and Molecular Genetics Center, IRO, Hospital Duran iReynals, Barcelona (Spain)6.

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.

transferase cyclodeaminase

N-formiminoglutamate + THF 5-formiminoTHF NH3 + 5,10-methenylTHF

glutamate

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

Figure # 9:Genomic structure and location of PCR primers(numbers under primer indicate base pair location upstream or downstream from exon splice junction)

Overall Size of Genomic Sequence: 19 291 bp(F=forward primer, R=reverse primer)

1F 2F 3F 4-5F 6F 7F 8F 9F 10F 11-12F 13-14F 15F -92 -66 -83 -67 -80 -218 -124 -127 -29 -86 -73 -89

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 98 bp 183 bp 128 bp 88 bp 179 bp 137 bp 131 bp 61 bp 129 bp 161 bp 43 bp 138 bp 95 bp 88 bp 217 bp

10R +89 11-12R 13-14R 1R 2R 3R 4-5R 6R 7R 8R +77 +97 +61 +72 +84 +50 +86 +253 +99 9R +38 15R +81

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

• 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

MC

H2

4

WG

17

95

MC

H3

9

WG

11

91

MC

H2

4

MC

H2

4

MC

H3

9

MC

H3

9

WG

17

95

WG

17

95

WG

11

91

WG

11

91

HindIII BamHI Kpn I

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

175 kDa

83.0 kDa

62.0 kDa

47.5 kDa

32.5 kDa

25.0 kDa

16.5 kDa

FT

H6

R13

5C

R29

9P

FT

CD

H6

c103

3ins

G

CD

333H

6

S40

7L

A43

8E

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.

Figure # 4:Genomic structure and location of PCR primers(numbers under primer indicate base pair location upstream or downstream from exon splice junction)

WG 1758,1759 WG 1758,1759 WG 1795 c457CT c940G T c1033insG R135C R299P

1F 2F 3F 4-5F 6F 7F 8F 9F 10F 11-12F 13-14F 15F -92 -66 -83 -67 -80 -218 -124 -127 -29 -86 -73 -89

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 98 bp 183 bp 128 bp 88 bp 179 bp 137 bp 131 bp 61 bp 129 bp 161 bp 43 bp 138 bp 95 bp 88 bp 217 bp

10R +89 11-12R 13-14R 1R 2R 3R 4-5R 6R 7R 8R +77 +97 +61 +72 +84 +50 +86 +253 +99 9R +38 15R +81

The c457 C->T (R135C) and c940 C->G (R299P) mutations wereintroduced into an expression construct containing the porcine FTdomain cDNA with a C-terminal six histidine tag using in vitrooverlap PCR. The proteins were expressed in E. coli (BL21 DE3)and purified using a NiNTA column.

The c1033insG mutation was similarly introduced into the full-length porcine FTCD and expressed in E. coli. A western blot ofthe crude extract of the c1033insG mutation shows that the proteinis truncated by a premature stop codon, completely lacking thecyclodeaminase domain.

FTCD Assay

Crude Extract Ni-NTA Purified

mpleActivity(U/mL)

[Protein](mg/mL)

Specific Activity(U/mg)

Activity(U/mL)

[Protein](mg/mL)

Specific Activity(U/mg)

% wild typeActivity

TH6 1.31 3.09 0.42 2.45 0.240 10.2 10035C 0.48 3.11 0.16 0.98 0.157 6.2 6199P 0.46 4.05 0.11 0.67 0.116 5.8 57

CDH6 0.12 1.45 0.087 - - - -c1033insG 1.93 2.21 0.87 - - - -

333H6 0.23 3.62 0.064 0.29 0.12 2.37 10007L 0.17 2.96 0.057 0.80 0.11 1.84 7838E 0.26 3.17 0.083 0.32 0.12 2.61 110

U= mol/minActivites are averages of one or two triplicate determinations.Protein concentrations are the averages of one or two duplicate determinations.

FTCD AssayFormiminotransferase activity of mutations made in the formiminotransferase domain (+6-His) constructexpressed in BL 21 DE3 and partially purified using a Ni-NTA column:

Specific Activity(mol/min/mg)

% wild-type Activity

FTH6 (wild type) 10.2 100R135C 6.24 61.1R299P 5.80 56.8

Cyclodeaminase activity of mutations made in the cyclodeaminase domain (+6-His) construct expressed inBL 21 DE3 and partially purified using a Ni-NTA column:

Specific Activity(mol/min/mg)

% wild-type Activity

CD333H6 2.37 100S407L 1.84 77.5A438E 2.61 110

All activities are the averages of two separate experiments done in triplicate.

Assays of the c1033insG mutation for formiminotransferase activity confirm that the mutant is active.

Conclusions

• First mutations in Human FTCD in three patients with glutamate formiminotransferase 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 (cblE)

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: 66AG

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

• 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 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

Methionine

Homocysteine

Cob(I)alamin

Cob(I)alamin

Methylcobalamin

Cob(II)alamin

Cob(II)alamin

5-MethylTHF

5,10-methyleneTHF

THF

Cob(III)alamin

AdoCbl

Methylmalonyl-CoA

Succinyl-CoA

Methylmalonyl-CoAMutase

Methionine Synthase

Methionine SynthaseReductase

MTHFR

Mitochondrion

cblA

cblB

cblC

cblD

cblE

cblGcblG

cblH

mut

TCII

Cob(III)alamin

TCII-Cob(III)alamin

cblF

CytoplasmExtracellular Space

Lysosome

AdoMet

METHIONINE SYNTHASE DEFICIENCY (cblG)

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: 2756AG

SUMMARY OF MUTATIONS FOUND IN cblG PATIENTS

Cell Line Patient Gender Patient Race Age at Onset First Mutation Second Mutation

WG2292 Female Caucasian 2 weeks c.3518C T (P1173L) c.3613G T (E1204X)WG1975 Male Caucasian 1 month c.3518C T (P1173L) c.1753C T (R585X)WG1308 Male Caucasian 1.5 months c.3518C T (P1173L) IVS9 –2A CWG1505 Male Caucasian 2 months c.3518C T (P1173L) c.2411T C (I804T)WG1386 Female Caucasian 3 months c.3518C T (P1173L) c.1784A C (H595P)WG2507 Female Caucasian 6 months c.3518C T (P1173L) c.2796-2800delAAGTCWG2306 Female Caucasian 7 months c.3518C T (P1173L) c.1310C A (S437Y)WG1352 Female Caucasian 1 year c.3518C T (P1173L) c.1348-1349TC CA (S450H)WG1321 Male Caucasian 1.7 years c.3518C T (P1173L) c.2669-2670delTGWG1892 Male Caucasian 3.5 years c.3518C T (P1173L)1 c.2641-2643delATC 1,2

WG1408 Female Caucasian 21 years c.3518C T (P1173L) c.2101delTWG2009 Male Caucasian 38 years c.3518C T (P1173L) c.1228G C (A410P)WG1671 Male Black Neonatal IVS3 –166A G 3 c.2112-2113delTC 3

WG1670 Female Black 2 days IVS3 –166A G 3 c.2112-2113delTC 3

WG1655 Male Caucasian 1.7 months IVS6 G A 3 c.3378insA3

WG1223 Female Caucasian 5.5 months c.3518C T (P1173L)WG2989 Female Caucasian 1 year c.3518C T (P1173L)WG2867 Male Caucasian 13 years c.3518C T (P1173L)WG2724 Male Caucasian 31 years c.3518C T (P1173L)WG1765 Male Caucasian 2.5 months c.12-13delGCWG2725 Male Caucasian 2.5 months c.1348-1349TC CA (S450H)WG2290 Male Caucasian 2.7 months c.2758C G (H920D) 2

WG2829 Female Caucasian 5 months c.381delAWG2918 Female Black 3 weeks

EXON 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Homocysteine-binding Domain Methyltetrahydrofolate-binding Cobalamin-binding Activation Domain Domain Domain

c.12-13delGC

IVS3 –166A GIVS6 G A

c.1784A C(H595P)

c.2112-2113delTC

c.2669-2670delTG

c.2758C G(H920D)

c.2796-2800delAAGTC

c.3518C T(P1173L)

c.3613G T(E1204X)

c.381delA

c.1228G C(A410P)

c.1310C A(S437Y)

IVS9 –2A C

c.1348-1349TC CA(S450H)

c.2101delT

c.2641-2643delATC

c.2411C T(I804T)

c.1753C T(R585X)

c.3378insA

Table 155-2: Inherited Defects of Folate Metabolism

:

I-F-Cobalamin Receptor Deficiency

(Imerslund –Gräsbeck Syndrome) (MGA1)

Example of One Phenotype, 2 Genes

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 -

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.

Intracellular Cobalamin Metabolism:

Endocytosis

Reduction

Mitochondrial Transport & Adenosylation-AdoCbl

Methylation-MeCbl

Methionine

Homocysteine

Cob(I)alamin

Cob(I)alamin

Methylcobalamin

Cob(II)alamin

Cob(II)alamin

5-MethylTHF

5,10-methyleneTHF

THF

Cob(III)alamin

AdoCbl

Methylmalonyl-CoA

Succinyl-CoA

Methylmalonyl-CoAMutase

Methionine Synthase

Methionine SynthaseReductase

MTHFR

Mitochondrion

cblA

cblB

cblC

cblD

cblE

cblGcblG

cblH

mut

TCII

Cob(III)alamin

TCII-Cob(III)alamin

cblF

CytoplasmExtracellular Space

Lysosome

AdoMet

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

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.

MUT

Missense Mutations

Nonsense Mutations

Deletions and insertions

Splice Mutations

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

seen in more than one patient

seen in only one family

Cobalamin-responsive MMA

• Two genes cloned on the basis of homology:

• MMAA: cblA complementation group

• MMAB: cblB complementation group

MMAA

c.64C>T (R22X)

c.161G>A (W54X)

c.283C>T (Q95X)

c.260-267dupATAAACTT

c.266T>C (L89P )

c.387C>A (Y129X)

c.450_451insG

c.503delC

c.592_595delACTG

c.620A>G (Y207C)

c.653G>A (G218E)

c.733+1G>A Splice

c.742C>T (Q248X)

c.959G>A (W320X)

c.988C>T (R330X)

c.970-2A>T Splice

c.1076G>A (R359Q)

Exon 2 3 4 5 6 71

c.433C>T (R145X)

c.434G>A (R145Q)

c.439+1_4delGTCA Splice

c.440G>A (E147G) c.1089_1090delGA

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)

MMAB

MMAB

c.572_576 del GGGCC

c.654_657 del CTAT

c.700 C>T (Q234X)c.IVS2-1 G>T

c.IVS3-1 G>A

c.IVS7-2 A>C

c.403 G>A (A135T)

c.556 C>T (R186W)

c.571 C>T (R191W)

c.656 A>G (Y219C)c.569 G>A (R190H)

c.575 G>A (E193K)c.56-57 GC>AA (R19Q) c.716 T>A (M239K)

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

MMADHC-cblD variant=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

2 4 5 6 7 83

9 478 609 696 891154 372

cblD-MMA

cblD-HC+MMA

cblD-HC

L20fsX21

S228M

T152fsX162

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)

TC/Cbl CblTC

Lysosome

Co(III)bl

Mitochondrion

Cell membrane

MeCblHomocysteine Methionine

5-Methyl-THF

THF

Methionine synthase

MTRR

Co(I)bl

Co(II)bl

Co(II)bl

Co(I)bl

AdoCbl Methylmalonyl-CoA Succinyl-CoA

Methylmalonyl-CoA mutase

cblE

cblG

cblF cblC, cblD

cblB

cblA, cblH

mut

cblD variant 1

cblD variant 2

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

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• 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 cblC

• Special investigations: cultured fibroblasts– Incorporation of label from [14C]propionate and

5-[14C]methyltetrahydrofolate into cellular macromolecules

– Cbl distribution studies

– Complementation studies

c.271dupA

c.331C>T

c.394C>T

c.547_548delGT c.609G>A

c.440G>A c.3G>A

c.608G>A

•Homozygosity mapping and haplotype analysis

•MMACHC

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

•204 patients

•42 mutations

•c.271dupA: 40%

•c.331C>T: 9%

•c.394C>T: 8%

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• 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• 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

• 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 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 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 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

• 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

• 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

• 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 yearsBodamer 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

• 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.

• 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.

Moras et al., 2006

Cobalamin metabolism

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