Survey
* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project
Download Slides for Biology 575
Document related concepts
no text concepts found
Transcript
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 • Methylenetetrahydrofolate Reductase Deficiency • Methionine Synthase Reductase Deficiency (cblE) • Methionine Syntase Deficiency (cblG) Histidine Formiminoglutamate 2 Glutamate formiminotransferase Formate + THF 5-Formimino-THF 5-Formyl-THF 2 Cyclodeaminase NAD+ NADH 10-Formyl-THF 5, 10-Methenyl-THF NADP+ NADP+ NADPH NADPH Purine nucleotides 5, 10-Methylene-THF dUMP Methylene-THF reductase 3 Glycine 5-Methyl-THF DHF Serine 1 Transport across intestine + CP SAM THF 4 5 MeCbl Methionine synthase Homocysteine dTMP Pyrimidine nucleotides NADPH 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 Histidine Formiminoglutamate 2 Glutamate formiminotransferase Formate + THF 5-Formimino-THF 5-Formyl-THF 2 Cyclodeaminase NAD+ NADH 10-Formyl-THF 5, 10-Methenyl-THF NADP+ NADP+ NADPH NADPH Purine nucleotides 5, 10-Methylene-THF dUMP Methylene-THF reductase 3 Glycine 5-Methyl-THF DHF Serine 1 Transport across intestine + CP SAM THF 4 5 MeCbl Methionine synthase Homocysteine dTMP Pyrimidine nucleotides NADPH 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 (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 Histidine Formiminoglutamate 2 Glutamate formiminotransferase Formate + THF 5-Formimino-THF 5-Formyl-THF 2 Cyclodeaminase NAD+ NADH 10-Formyl-THF 5, 10-Methenyl-THF NADP+ NADP+ NADPH NADPH Purine nucleotides 5, 10-Methylene-THF dUMP Methylene-THF reductase 3 Glycine 5-Methyl-THF DHF Serine 1 Transport across intestine + CP SAM THF 4 5 MeCbl Methionine synthase Homocysteine dTMP Pyrimidine nucleotides NADPH 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). SEVERE METHYLENETETRAHYDROFOLATE (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 677CT Disease 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 Histidine Formiminoglutamate 2 Glutamate formiminotransferase Formate + THF 5-Formimino-THF 5-Formyl-THF 2 Cyclodeaminase NAD+ NADH 10-Formyl-THF 5, 10-Methenyl-THF NADP+ NADP+ NADPH NADPH Purine nucleotides 5, 10-Methylene-THF dUMP Methylene-THF reductase 3 Glycine 5-Methyl-THF DHF Serine 1 Transport across intestine + CP SAM THF 4 5 MeCbl Methionine synthase Homocysteine dTMP Pyrimidine nucleotides NADPH 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). Mutations in the FTCD gene on chromosome 21 are the cause of 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 and Biochemistry4, McGill University, Montreal Genome Centre5, Medical and Molecular Genetics Center, IRO, Hospital Duran i Reynals, 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 N-formiminoglutamate + THF cyclodeaminase 5-formiminoTHF glutamate NH3 + 5,10-methenylTHF 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 -92 2F -66 1 98 bp 2 183 bp 3F -83 4-5F -67 3 4 128 bp 88 bp 6F -80 5 179 bp 7F -218 6 137 bp 8F -124 9F -127 7 8 131 bp 9 10F -29 11-12F -86 10 11 61 bp 129 bp 161 bp 13-14F -73 12 15F -89 13 43 bp 138 bp 95 bp 14 15 88 bp 217 bp 10R +89 1R +61 2R +72 3R +84 4-5R +50 6R +86 7R +253 11-12R +77 8R +99 9R +38 13-14R +97 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 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. WG1191 MCH39 WG1795 MCH24 Kpn I WG1191 MCH39 WG1795 MCH24 BamHI WG1191 MCH39 WG1795 MCH24 HindIII A438E S407L CD333H6 c1033insG FTCDH6 R299P R135C FTH6 Western Blot 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. 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 c457CT R135C 1F -92 2F -66 1 98 bp 2 183 bp 3F -83 4-5F -67 3 4 128 bp 88 bp WG 1758,1759 c940GT R299P 6F -80 5 179 bp 7F -218 6 137 bp WG 1795 c1033insG 8F -124 9F -127 7 8 131 bp 10F -29 9 11-12F -86 10 11 61 bp 129 bp 161 bp 13-14F -73 12 15F -89 13 43 bp 138 bp 95 bp 14 15 88 bp 217 bp 10R +89 1R +61 2R +72 3R +84 4-5R +50 6R +86 7R +253 11-12R +77 8R +99 9R +38 13-14R +97 15R +81 The c457 C->T (R135C) and c940 C->G (R299P) mutations were introduced into an expression construct containing the porcine FT domain cDNA with a C-terminal six histidine tag using in vitro overlap PCR. The proteins were expressed in E. coli (BL21 DE3) and purified using a NiNTA column. The c1033insG mutation was similarly introduced into the fulllength porcine FTCD and expressed in E. coli. A western blot of the crude extract of the c1033insG mutation shows that the protein is truncated by a premature stop codon, completely lacking the cyclodeaminase domain. FTCD Assay Crude Extract Ni-NTA Purified Activity [Protein] Specific Activity Activity [Protein] Specific Activity mple (U/mL) (mg/mL) (U/mg) (U/mL) (mg/mL) (U/mg) TH6 1.31 3.09 0.42 2.45 0.240 10.2 35C 0.48 3.11 0.16 0.98 0.157 6.2 99P 0.46 4.05 0.11 0.67 0.116 5.8 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 07L 0.17 2.96 0.057 0.80 0.11 1.84 38E 0.26 3.17 0.083 0.32 0.12 2.61 U= mol/min Activites are averages of one or two triplicate determinations. Protein concentrations are the averages of one or two duplicate determinations. % wild type Activity 100 61 57 100 78 110 FTCD Assay Formiminotransferase activity of mutations made in the formiminotransferase domain (+6-His) construct expressed in BL 21 DE3 and partially purified using a Ni-NTA column: FTH6 (wild type) R135C R299P Specific Activity (mol/min/mg) 10.2 6.24 5.80 % wild-type Activity 100 61.1 56.8 Cyclodeaminase activity of mutations made in the cyclodeaminase domain (+6-His) construct expressed in BL 21 DE3 and partially purified using a Ni-NTA column: CD333H6 S407L A438E Specific Activity (mol/min/mg) 2.37 1.84 2.61 % wild-type Activity 100 77.5 110 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 Lysosome Mitochondrion Methylmalonyl-CoA TCIICob(III)alamin mut cblB TCII Cob(I)alamin Cob(III)alamin AdoCbl Methylmalonyl-CoA Mutase cblF cblA Succinyl-CoA cblH Cob(III)alamin Cob(II)alamin cblC cblD Cob(I)alamin Methionine 5-MethylTHF MTHFR cblG Cob(II)alamin 5,10-methyleneTHF AdoMet Extracellular Space Cytoplasm THF cblG Methionine Synthase Methionine Synthase Reductase cblE Homocysteine Methylcobalamin METHIONINE SYNTHASE DEFICIENCY (cblG) Methionine Synthase 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: 2756AG SUMMARY OF MUTATIONS FOUND IN cblG PATIENTS Cell Line Patient Gender Patient Race Age at Onset First Mutation Second Mutation WG2292 WG1975 WG1308 WG1505 WG1386 WG2507 WG2306 WG1352 WG1321 WG1892 WG1408 WG2009 WG1671 WG1670 WG1655 WG1223 WG2989 WG2867 WG2724 WG1765 WG2725 WG2290 WG2829 WG2918 Female Male Male Male Female Female Female Female Male Male Female Male Male Female Male Female Female Male Male Male Male Male Female Female Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Black Black Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Black 2 weeks 1 month 1.5 months 2 months 3 months 6 months 7 months 1 year 1.7 years 3.5 years 21 years 38 years Neonatal 2 days 1.7 months 5.5 months 1 year 13 years 31 years 2.5 months 2.5 months 2.7 months 5 months 3 weeks c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) 1 c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) 3 IVS3 –166AG 3 IVS3 –166AG 3 IVS6 GA c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.3518CT (P1173L) c.12-13delGC c.1348-1349TCCA (S450H) 2 c.2758CG (H920D) c.381delA c.3613GT (E1204X) c.1753CT (R585X) IVS9 –2AC c.2411TC (I804T) c.1784AC (H595P) c.2796-2800delAAGTC c.1310CA (S437Y) c.1348-1349TCCA (S450H) c.2669-2670delTG 1,2 c.2641-2643delATC c.2101delT c.1228GC (A410P) 3 c.2112-2113delTC 3 c.2112-2113delTC 3 c.3378insA c.2641-2643delATC c.2669-2670delTG c.1228GC (A410P) c.1348-1349TCCA (S450H) c.2101delT c.3518CT (P1173L) c.381delA c.1310CA (S437Y) c.1753CT (R585X) c.2796-2800delAAGTC c.2411CT (I804T) IVS9 –2AC c.12-13delGC 2 c.2112-2113delTC IVS6 GA IVS3 –166AG EXON 1 c.1784AC (H595P) 3 4 5 6 7 8 9 10 11 12 13 14 15 Homocysteine-binding Domain c.2758CG (H920D) c.3378insA 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Methyltetrahydrofolate-binding Domain Cobalamin-binding Domain 30 Activation Domain c.3613GT (E1204X) 31 32 33 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 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 & AdenosylationAdoCbl Methylation-MeCbl Vitamin B12 Pathway Overview TCblR Transcobalamin Receptor Transcobalamin Receptor Jacobsen and Glushchenko, 2009 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 Receptor • Total Cobalamin Uptake Total Uptake (pg cobalamin/mg) 14.000 Control 12.000 10.000 8.000 Se 6.000 Se 4.000 WG3733 2.000 0.000 0 1 2 3 4 Time (hours) 5 6 7 Transcobalamin Receptor Uptake and accumulation of B12 in fibroblasts during six days in culture Transcobalamin Receptor Binding kinetics of TC-Cbl to normal and mutant fibroblasts Transcobalamin Receptor Amino acid sequence of TCblR (Deletion is shown in red and the polymorphisms in green) Transcobalamin Receptor Ribbon Diagram depicting the secondary structure of the wild type and mutant TCblR cblF • Combined homocystinuria and methylmalonic aciduria • Accumulation of free cobalamin in lysosomes • Postulated defect in efflux of cobalamin from lysosomes cblF • Microcell-mediated chromosome transfer – Transferred chromosome 2-7, 10, 12 and 16 into cblF patient fibroblasts. cblF Microcell-mediated chromosome transfer: Colonies tested Chromosome Recipient Line # colonies Correcting (%) Non-correcting (%) 2 PV014 3 0 (0) 3 (100) 3 PV014 0 0 (0) 0 (0) 4 PV014 5 0 (0) 5 (100) 5 PV014 5 0 (0) 5 (100) 6 PV014 9 8 (73) 3 (27) 7 PV014 5 0 (0) 5 (100) 10 PV014 2 0 (0) 2 (100) 12 PV014 0 0 (0) 0 (0) 16 PV014 1 0 (0) 1 (100) Cell lines ch r6 1 #1 #6 10 ch r6 #3 4 #1 3 #1 2 #1 0 #8 #1 ch r6 ch r6 ch r6 ch r6 ch r6 ch r6 #4 #2 #1 F 14 ch r6 ch r6 ch r6 cb l nt ro l Co nd ro u Ba ck g nmol propionate/mg protein/18h cblF Chromosome 6 Propionate Incorporation C-Propionate Incorporation 20 18 16 14 12 Normal 8 6 4 2 0 cblF • Mapping of a locus for cblF on chromosome 6q13 Rutsch et al, 2009 cblF • Mapping of a locus for cblF on chromosome 6q13 Rutsch et al, 2009 cblF • Localization of LMBD1 to lysosome 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 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 • 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.260-267dupATAAACTT c.266T>C (L89P ) c.283C>T (Q95X) c.387C>A (Y129X) c.433C>T (R145X) c.434G>A (R145Q) c.439+1_4delGTCA Splice Exon 1 2 3 4 c.742C>T (Q248X) c.959G>A (W320X) c.970-2A>T Splice 5 6 7 c.733+1G>A Splice c.620A>G (Y207C) c.653G>A (G218E) c.592_595delACTG c.503delC c.450_451insG c.440G>A (E147G) c.988C>T (R330X) c.1076G>A (R359Q) 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.56-57 GC>AA (R19Q) c.IVS2-1 G>T c.572_576 del GGGCC c.575 G>A (E193K) c.571 C>T (R191W) c.569 G>A (R190H) c.556 C>T (R186W) c.IVS7-2 A>C c.403 G>A (A135T) c.IVS3-1 G>A c.716 T>A (M239K) c.700 C>T (Q234X) c.656 A>G (Y219C) c.654_657 del CTAT 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 cblD-HC cblD-MMA L20fsX21 T152fsX162 2 3 9 4 154 5 372 478 6 609 S228M cblD-HC+MMA 7 696 8 891 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) THF 5-Methyl-THF cblG Methionine synthase Homocysteine MTRR cblE Cbl TC Lysosome MeCbl Co(I)bl cblC, cblD cblF TC/Cbl Methionine Co(III)bl cblD variant 1 Co(II)bl cblD variant 2 cblA, cblH Co(II)bl Co(I)bl cblB AdoCbl Cell membrane Methylmalonyl-CoA Succinyl-CoA Methylmalonyl-CoA mutase mut Mitochondrion 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.331C>T c.440G>A c.608G>A c.271dupA c.3G>A c.394C>T c.547_548delGT c.609G>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 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 • 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. Cobalamin metabolism Moras et al., 2006 :
Related documents