7 Hereditary Folate Malabsorption Hereditary folate malabsorption (HFM) (OMIM ) 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
8 Hereditary Folate Malabsorption Qui A et al. Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption. Cell 127, , December 1, 2006Proton coupled, high affinity folate transporter operating at low pH.Loss of function mutations in HFMPCFT/HCP1
11 Glutamate formiminotransferase HistidineFormiminoglutamate2Glutamate formiminotransferase5-Formimino-THFFormate + THF2Cyclodeaminase5-Formyl-THFNAD NADH5, 10-Methenyl-THF10-Formyl-THFNADP+NADPHNADP NADPHPurine nucleotides5, 10-Methylene-THFMethylene-THF reductasedUMP3GlycinedTMP5-Methyl-THFDHFSerine1Pyrimidine nucleotidesTransport across intestine + CPTHFNADPHSAM45MeCblMethioninesynthaseHomocysteineMethionine + THFFigure 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).
12 SEVERE METHYLENETE-TRAHYDROFOLATE (MTHFR) REDUCTASE DEFICIENCY
13 Methylenetetrahydrofolate Reductase Deficiency (Severe) Hyperhomocysteinemia and homocystinuriaLow or normal plasma methionineNo 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) asymptomaticGene/location: MTHFR/ Chr. 1p36.3Common polymorphisms: 677CT; 1298AC
16 MTHFR 677CTOriginally discovered because specific activity of MTHFR in cell extracts was thermolabile50-60% decrease in specific activity of MTHFRFirst postulated association (Kang et al) was between thermolability of MTHFR and heart disease
17 MTHFR 677CTAfter 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).
18 MTHFR 677CTT allele is associated with elevated levels of total homocysteine (tHcy).Effect is much more prominent in TT individualsDietary folate (multivitamins, fortification of cereal grains) can mask the effect of the T allele.
20 MTHFR 1298AC Associated with 35% decrease in MTHFR specific activity Not associated with enzyme thermolabilityFrequency of C allele: 30% Western Europe and 18% in Asians1298C and 677T rarely found together in cisFewer studies have looked at this polymorphism
26 Human FTCDDiscovered by examination of EST’s on chromosome 21 as part of a study assessing the molecular basis of Down SyndromeEST compared to porcine FTCDHuman 21q22.315 exons541 amino acid residues with 84% homology to the pig.Five different transcripts
28 GFT PatientsSiblings: 1) Age 2 1/2 years - speech delay, some growth delay, hypotonia, increased FIGLU excretion 2) Age 8 years-hypotonia, abnormal EEG, increased FIGLU excretionTwo missense mutations: c457 c->T (R135C) and c940 C->G (R299P). Not found in 200 control alleles.
31 Third GFT Patient Apnea in the first year of life Recurrent infections At age 2, mild developmental delay, hypotonia, breathing difficultiesHypersegmented neutrophilsIncreased FIGLU excretionOne mutation: c1033 insG (not found in 200 control alleles)
33 Southern Blot HindIII BamHI Kpn I MCH24 WG1795 MCH39 WG1191 MCH24 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.
34 Western Blot c1033insG FTCDH6 CD333H6 S407L FTH6 R135C R299P A438E 175 kDa83.0 kDa62.0 kDa47.5 kDa32.5 kDa25.0 kDa16.5 kDa25 ? 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.
43 Methionine Synthase Reductase Deficiency-cblE Megaloblastic anemia, hyperhomocysteinemia and homocystinuriaLow plasma methionineCerebral atrophy, nystagmus, blindness, altered toneReduced methionine synthase activity in the absence of an exogenous reducing systemGene/ location: MTRR/ 5pPolymorphism: 66AG
44 Methylcobalamin-Dependent Methionine Synthase in E. Coli 2 component flavoprotein systemflavodoxinNADPH-ferredoxin (flavodoxin) oxidoreductase, a member of electron transferases termed the “FNR family”
45 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.
46 Methionine Synthase Reductase Localized to chromosome 5p15.2-p15.32094 bp amino acidsPredicted molecular mass 77,000 DaProminent RNA species of 3.6 kb with an additional smaller 3.1 kb species in brain38% identity (49% similarity) with human cytochrome P-450 reductase
57 I-F-Cobalamin Receptor Deficiency (Imerslund –Gräsbeck Syndrome) (MGA1)Example of One Phenotype, 2 Genes
58 I-F-Cobalamin Receptor Deficiency (Imerslund -Gräsbeck) (MGA1) Early onset megaloblastic anemia, low serum cobalamin levels, and proteinuriaHomocystinuria and methylmalonic aciduria may be found but are not prominentDecreased absorption of cobalamin in the presence of normal synthesis of intrinsic factorCommon in Finland, Norway and the Middle EastDefects in CUBN (cubilin) & AMN (amnionless)Genes/ Locations: Chrs. 10p12.1 & 14q32
59 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.
60 Intracellular Cobalamin Metabolism: EndocytosisReductionMitochondrial Transport & Adenosylation-AdoCblMethylation-MeCbl
62 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 PAImersund-Grasbeck (MGA1)-mutations in cublin or amnionless (Stephan Tanner-Ohio)Combined MMA and Homocystinuria (cblC, cblD, cblF)
64 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 phenotypesThere are a number of mutations that are more common in specific ethnic groups and a number of common mutations.
65 MUT Missense Mutations Nonsense Mutations Deletions and insertions seen in more than one patient seen in only one familyMissense Mutations Nonsense Mutations Deletions and insertions Splice Mutations
66 Cobalamin-responsive MMA Two genes cloned on the basis of homology:MMAA: cblA complementation groupMMAB: cblB complementation group
73 MMAB Mutations 22 mutations Identified Most predicted to affect the active site of the enzyme, identified from the crystal structure of is bacterial orthologC.556C>T (p.R186W) represents 33% of affected alleles.
75 MMADHC-cblD variant=cblH Associated with isolated MMADecreased propionate incorporationDecreased AdoCbl synthesisNovel gene MMADHC isolated by Brian Fowler in SwitzerlandIdentical to cblHMutations in N-terminal regions associated with isolated MMA
77 Genes Associated with Isolated MMA MUTMMAAMMABMMADHC (NEJM in press)MCEE-may not be related to clinicalSUCLA2-developmental delaySUCLG1-fatal infantile lactic acidosis (Ostergaard E et al. Am J Hum Genet 81:383, 2007)
79 cblC Most common inborn error of Vitamin B12 metabolism Early-onset: Feeding difficulties, hypotonia/hypertonia, lethargyAbnormal movement, seizuresMultisystemic involvementPancytopenia or megaloblastic anemiaSalt-and-pepper retinopathyModerate to severe cognitive disability
81 Diagnosis of cblC Clinical history, physical exam Laboratory investigations:CBC with smear, ± bone marrow biospyPlasma amino acids (elevated Hcy, low methionine)Urine organic acids (elevated MMA)Total plasma homocysteineOthers as clinically indicated (Normal serum cobalamin and folate levels).
82 Diagnosis of cblC Special investigations: cultured fibroblasts Incorporation of label from [14C]propionate and [14C]methyltetrahydrofolate into cellular macromoleculesCbl distribution studiesComplementation studies
88 Phenotype-Genotype Correlations: Seeking Answers in Case-Reports 37 previously published patients:25 early-onset cases12 late-onset cases:9: neurological phenotype3: renal phenotype
89 Early-Onset Cases 25 out of 37 patients 9/25: homozygous for c.271dupA 3/25: homozygous for c.331C>T5/25: c.271dupA / c.331C>T1/25: c.271dupA / c.394C>TRemaining 8 patients either:Compound heterozygous for different nonsense mutationsHomozygous for another nonsense mutation
90 Late-Onset Cases 12 of 37 patients 9/12: neurological phenotype 3/12: renal phenotypeNeurological phenotype:4/9: homozygous for c.394C>T2/9: c.271dupA and c.394C>T3/9: c.271dupA and a missense mutationRenal phenotype:3/3: c.271dupA and c.82-9_-12delTTTC
91 Observations on Ethnic Background Homozygosity for c.271dupA: 9 patients5 White1 Hispanic1 Iranian1 Middle Eastern1 ? EthnicityIn database: 44 other patients of various ethnic backgroundsTherefore, not specific to one ethnic group
92 Observations on Ethnic Background Homozygosity for c.331C>T: 3 patients“Cajun”3 unpublished French Canadian patients from Québec and New BrunswickCompound heterozygosity c.331C>T/c.271dupA:5 patients:1: White (USA, “French” background on pedigree in lab)1: French Canadian from Québec3: Louisiana, USA (New Orleans)In database: 5 additional patients of French-Canadian or Cajun backgroundSuggest possible founder effect/genetic drift
93 Observations on Ethnic Background Homozygosity for c.394C>T: 4 patients3: Asiatic-Indian (incl. 2 sibs)1: Middle EasternIn database: 9 other patients, all Asiatic-Indian, Pakistani or Middle EasternHeterozygosity c.394C>T / c.271dupA: 3 patients1: Greek1: Portuguese1: ? EthnicityMutational hot-spot: arose at least twice independently
94 Observations on Ethnic Background Homozygosity for c.440G>A: 2 patientsNative American (Southwestern)In database: 1 unpublished Native American patient of the same tribe
95 Compound heterozygosity: c.394C>T / c.271dupA 2 late-onset published cases:Ages 4.5 and 10 years1 early-onset published case:Age 6 monthsIntrafamilial 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):Late-onset AND early-onset in the same family!!!Interpretation of anticipated phenotype based on this genotype may be unreliable
96 Response to Cbl Supplementation Homozygosity c.271dupA:Tend to have progression of disease despite TxHomozygosity c.394C>T:Almost complete reversal of psychiatric and neurological symptomsCompound heterozygosity c.394C>T / c.271dupA
97 c.271dupA / missense mutations Late-onset neurological phenotype:c.271dupA / c.440G>C, 45 yearsPowers JM, Rosenblatt DS, Schmidt RE et al. Neurological and neuropathologic heterogeneity in two brothers with cobalamin C deficiency. Ann Neurol 2001; 49(3):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):1113c.271dupA / c.347T>C, 24 yearsRoze E, Gervais D, Demeret S et al. Neuropsychiatric disturbances in presumed late-onset cobalamin C disease. Arch Neurol 2003; 60(10):
102 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 prokaryotesMotifs were identified in MMACHC that are homologous to motifs in bacterial genes with vitamin B12-related functions.
105 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.