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STRUCTURE OF TETRAHYDROFOLATE

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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STRUCTURE OF TETRAHYDROFOLATE

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  1. STRUCTURE OF TETRAHYDROFOLATE

  2. STRUCTURE OF FOLIC ACID AND REDUCED FOLATES INVOLVED IN ONE-CARBON METABOLISM

  3. FOLATE PATHWAY

  4. 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)

  5. 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).

  6. HERDITARY FOLATE MALABSORPTION

  7. 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).

  8. 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

  9. 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

  10. 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).

  11. SEVERE METHYLENETE-TRAHYDROFOLATE (MTHFR) REDUCTASE DEFICIENCY

  12. 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

  13. COMMON POLYMORPHISMS IN MTHFR

  14. 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

  15. 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).

  16. 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.

  17. MTHFR 677CTDisease Associations (Incomplete) • Cardiovascular Disease • Alzheimer Disease • Colon Cancer • Diabetes Mellitus • Down Syndrome • Leukemia • Neural Tube Defects (NTD) • Pregnancy Complications

  18. 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

  19. GLUTAMATE FORMININOTRANSFERASE DEFICIENCY

  20. 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).

  21. 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.

  22. 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

  23. 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.

  24. 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)

  25. 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.

  26. 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.

  27. FTCD Assay

  28. FTCD Assay

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

  30. FUNCTIONAL METHIONINE SYNTASE DEFICIENCY Overlap in Folate and Cobalamin Metabolism: One phenotype Two Genotypes: cblE (Methionine synthase reductase deficiency) cblG (Methionine synthase deficiency)

  31. METHIONINE SYNTHASE REDUCTASE DEFICIENCY (cblE)

  32. 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

  33. 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”

  34. 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.

  35. 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

  36. 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

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