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Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. PART VITAMINS 17 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. C H A P T E R 155 Inherited Disorders of Folate and Cobalamin Transport and Metabolism David S. Rosenblatt _ Wayne A. Fenton 1. Folate coenzymes participate in a number of critical single-carbon transfer reactions, including those involved in the biosynthesis of pyrimidines, purines, serine, and methionine and in the degradation of histidine and purines. 2. Five inherited disorders of folate transport and metabolism have been well substantiated: methylene-H4Folate reductase de®ciency (MIM 236250); functional methyltetrahydrofolate (methyl-H4Folate):homocysteine methyltransferase (methionine synthase) de®ciency caused by mutations in the gene for methionine synthase reductase (cblE) (MIM 236270) or mutations in the gene for methionine synthase itself (cblG) (MIM 250940); glutamate formiminotransferase de®ciency (MIM 229100); and hereditary folate malabsorption (MIM 229050). 3. Four putative inherited disorders in the literature cannot be considered to be well substantiated: dihydrofolate reductase de®ciency; methenyl-H4Folate cyclohydrolase de®ciency; cellular uptake defects; and the original description of primary methyl-H4Folate: homocysteine methyltransferase de®ciency from Japan. 4. Methylene-H4Folate reductase de®ciency, the most widely studied of the inherited disorders of folate metabolism, is a condition in which clinical severity correlates with the degree of enzyme de®ciency. The clinical symptoms vary, with developmental delay accompanied by motor and gait abnormalities, seizures, and psychiatric manifestations being described. The age of onset has ranged from the neonatal period to adulthood. The major biochemical ®ndings are moderate homocystinuria and hyperhomocystinemia with low or relatively normal levels of plasma methionine. Most severely affected patients have died. Pathologic ®ndings include vascular changes similar to those seen in classical homocystinuria and demyelination presumably due to low levels of neurotransmitters or methionine in the central nervous system. A variant form of methylene-H4Folate reductase de®ciency resulting in A list of standard abbreviations is located immediately preceding the index in each volume. Additional abbreviations used in this chapter include: Ado-B12 or AdoCbl 50 deoxyadenosylcobalamin; AICAR 5-phosphoribosyl-5-aminoimidazole-4-carboxamide; Cbl cobalamin; cbl cobalamin metabolism locus (cblA, cblB, etc.); CN-Cbl cyanocobalamin; FGAR a-N-formyl-glycinamide ribonucleotide; FIGLU formiminoglutamate; GAR 5-phosphoribosylglycinamide; GSCbl glutathionylcobalamin; H2PteGlu or H2Folate dihydrofolate; H4PteGlu, H4Folate, or THF tetrahydrofolate; IF intrinsic factor; methyl-B12, CH3-B12, or MeCbl methylcobalamin; methyl-H4Folate N 5-methyltetrahydrofolate; mut methylmalonyl CoA mutase locus; OH-B12 or OH-Cbl hydroxocobalamin; TC (I, II, or III) transcobalamin (I, II, or III). ``intermediate homocystinuria'' is associated with 50 percent residual activity and enzyme thermolability, and is suggested to be an inherited risk factor for coronary heart disease. In the majority of cases, this variant is due to homozygosity for a common polymorphism, 677C!T, in the methylene-H4Folate reductase gene. Severe methyleneH4Folate reductase de®ciency is resistant to treatment; folates, methionine, pyridoxine, cobalamin, and carnitine have all been used. Betaine has the theoretical advantage of both lowering homocysteine levels and supplementing methionine levels and has been the most promising therapeutic agent to date, particularly if started immediately after birth. Nevertheless, the prognosis is generally poor. 5. Functional methionine synthase de®ciency due to the cblE and cblG mutations is characterized by homocystinuria and defective biosynthesis of methionine. Most patients have presented in the ®rst few months of life with megaloblastic anemia and developmental delay. At least one patient presented in early adulthood with a misdiagnosis of multiple sclerosis. The distribution of cobalamin derivatives was altered in cultured cells, with decreased levels of MeCbl as compared with normal ®broblasts. The cblE mutation is associated with low methionine synthase activity when the assay is performed with low levels of thiol, whereas the cblG mutation is associated with low activity under all assay conditions. cblE and cblG represent distinct complementation classes. Both diseases respond to treatment with hydroxocobalamin (OH-Cbl). 6. Glutamate formiminotransferase de®ciency is a heterogeneous condition associated with elevated excretion of formiminoglutamic acid, 4-amino-5-imidazole-carboxamide, and hydantoin-5-propionate. Clinical ®ndings have varied from mental and physical retardation to massive excretion of formiminoglutamate in the absence of retardation. Therapy with folates and methionine has been described, but given that the correlation between symptoms and formiminoglutamate excretion remains uncertain, the basis for treating these patients is unclear. 7. Hereditary folate malabsorption is characterized by the early onset of failure to thrive and severe folate-responsive megaloblastic anemia. All patients have been severely restricted in their ability to absorb oral folic acid or oral reduced folates. Severe mental retardation may be a prominent feature if therapy does not succeed in maintaining adequate levels of folate in the cerebrospinal ¯uid. Two patients have shown increased susceptibility to 3897 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3898 PART 17 / VITAMINS 8. 9. 10. 11. 12. 13. 14. 15. 16. infection. This disorder provides the best evidence for the existence of a speci®c carrier for folate both at the level of the intestine and at the choroid plexus. Therapy has been attempted with large doses of oral or systemic folates. All of the clearly delineated disorders of folate metabolism appear to be inherited as autosomal recessive traits. Heterozygotes for methylene-H4Folate reductase de®ciency show decreased enzyme levels in somatic cells. A difference in folate absorption in the heterozygote has been suggested in at least one family with hereditary folate malabsorption. Prenatal diagnosis has been successfully performed for methylene-H4Folate reductase de®ciency, methionine synthase reductase de®ciency (cblE), and methionine synthase (cblG) de®ciency using cultured amniotic cells. Cobalamins (Cbls) are complex organometallic substances consisting of a corrin ring, a central cobalt atom, and various axial ligands. The basic structure, known as vitamin B12, is synthesized exclusively by microorganisms, but most higher animals are capable of converting the vitamin into the two required coenzyme forms, adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). Dietary Cbl is acquired mostly from animal sources, including meat and milk, and is absorbed in a series of steps that includes proteolytic release from its associated proteins, binding to a gastric secretory protein known as intrinsic factor (IF), recognition of the IF-Cbl complex by cubilin, a receptor on ileal mucosal cells, transport across those cells, and release into the portal circulation bound to transcobalamin II (TC II), the serum protein that carries newly absorbed Cbl throughout the body. The cellular metabolism by which the coenzymes are formed involves receptor-mediated binding of the TC IICbl complex to the cell surface, adsorptive endocytosis of the complex, intralysosomal degradation of the TC II, release of Cbl into the cytoplasm, enzyme-mediated reduction of the central cobalt atom, and cytosolic methylation to form MeCbl or mitochondrial adenosylation to form AdoCbl. Only two enzymes in mammalian cells are known to depend on cobalamin coenzymes: methylmalonyl CoA mutase, which requires AdoCbl; and methionine synthase (also known as N5-methyltetrahydrofolate:homocysteine methyltransferase), which requires MeCbl. Ten different inherited defects are known to impair the pathways of Cbl transport and metabolism in humans (see Fig. 155-12). Three affect absorption and transport; the other seven alter cellular utilization and coenzyme production. The defects affecting Cbl absorption and transport generally manifest themselves in infancy or early childhood as developmental delay with megaloblastic anemia. Serum Cbl levels may be reduced (in IF (MIM 261000) or cubilin-protein de®ciency (MIM 261100)) or near normal (in TC II de®ciency (MIM 275350)). Treatment with periodic injections of Cbl, with or without folate therapy, is generally effective in controlling these problems. The clinical manifestations of de®ciencies in cellular Cbl utilization and metabolism vary depending on whether one or both coenzymes are affected. Two abnormalities in AdoCbl synthesis only (designated cblA (MIM 251100) and cblB (MIM 251110) lead to impaired methylmalonyl CoA mutase activity and result in methylmalonic acidemia. In most, but not all, patients with these defects, pharmacologic supplements of Cbl (cyanocobalamin or hydroxocobalamin) produce distinct reductions in methylmalonate 17. 18. 19. 20. accumulation and offer a valuable therapeutic adjunct to dietary protein limitation. Oral antibiotic therapy may be useful to reduce propionate production by gut bacteria. The defect in cblA is unknown, while the defect in cblB patients is in cob(I)alamin adenosyltransferase, the ®nal step of AdoCbl biosynthesis. Three distinct mutations, designated cblC (MIM 277400), cblD (MIM 277410), and cblF (MIM 277380), lead to impaired synthesis of both AdoCbl and MeCbl and, accordingly, to de®cient activity of both methylmalonyl CoA mutase and methionine synthase. Children from these groups have methylmalonic aciduria and homocystinuria. Children with the cblC mutation appear to be more severely affected clinically than the two known sibs in the cblD group or those in the cblF group. Major clinical problems in cblC patients include failure to thrive, developmental retardation, and such hematologic abnormalities as megaloblastic anemia and macrocytosis. Treatment requires a combination of the therapies for the individual coenzyme de®ciencies: protein restriction and pharmacologic doses of hydroxocobalamin, possibly in combination with oral antibiotics and betaine supplements. The precise defects in the cblC and cblD patients are not yet known, but they must involve early steps in the intracellular metabolism of cobalamins, possibly cytosolic Cbl reduction. The defect in cblF appears to be in the transport mechanism by which Cbl is released from lysosomes. The discriminating biochemical features of the inherited defects in Cbl transport and metabolism are shown in Table 155-5. All the disorders of Cbl metabolism for which there are adequate data are inherited as autosomal recessive traits. Heterozygotes can be detected only for cblB. Genetic complementation analyses with somatic-cell heterokaryons have been particularly useful in demonstrating genetic heterogeneity and in con®rming the existence of autosomal recessive inheritance among defects in cellular Cbl utilization and metabolism. Prenatal detection of fetuses with defects in the complementation groups cblA, cblB, cblC, and cblF has been accomplished using cultured amniotic cells and chemical determinations on amniotic ¯uid or maternal urine. In several cases, in utero Cbl therapy was done with apparent success. FOLATE The chemistry, biochemistry, and physiology of folic acid and its derivatives have been extensively reviewed in earlier editions of this book,1,2 as well as in several excellent monographs.3,4 A detailed review by Erbe gives a case-by-case analysis in tabular form of each patient who had been reported up to 1986 with veri®ed methylenetetrahydrofolate reductase de®ciency or glutamate formiminotransferase de®ciency.5 Other reviews are also available.6± 16 The pteridine compounds referred to as ``folates'' participate as coenzymes in a number of critical 1-carbon transfer reactions, including those involved in the biosynthesis of purines, pyrimidines (dTMP), serine, and methionine, and in the degradation of histidine. In the 1930s, at about the same time that pteridine pigments of butter¯y wings were being isolated and characterized, Wills and her colleagues determined that the absence of folate from the diet resulted in a macrocytic megaloblastic anemia.17,18 The structural determination and synthesis of the parent compound were accomplished in the subsequent decade.19 ``Folic acid'' and ``folate'' are the preferred synonyms for pteroylglutamic acid Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM ``leucovorin,'' or ``citrovorum factor,'' is a reduced folate that has been used therapeutically because of its chemical stability. Folate Transport Two distinct systems have been described for the transport of folates and folate antagonists (antifolates) across mammalian cell membranes.20,21 One, the reduced folate carrier (RFC), encoded on chromosome 21q22.2-22.3 ([SLC19A1], NM_003056, MIM 600424), has been studied mostly in cancer cells and mediates a low af®nity, high-capacity system for the uptake of reduced folates and methotrexate at high (mM) concentrations.21±25 It shows considerable transcript heterogeneity;25,26 the putative intestinal folate transporter has an identical cDNA.27 The second system, a family of membrane-associated folate-binding proteins (FBP) or folate receptors (FR), is coded for by genes on chromosome 11 (q13.3-13.5).21,28 These glycoproteins mediate a high af®nity, lowcapacity system and operate at low (nM) concentrations of exogenous folate. The FR-a ([FOLR1], NM_000802, MIM 136430) and FR-b ([FOLR2], NM_000803, MIM 136425) genes have similar structures, but differ in their 50 -untranslated regions and in their transcription regulatory elements. Both FR-a and FR-b are attached to the cell membrane by a glycosylphosphatidylinositol anchor, and there is evidence for receptor-mediated internalization (potocytosis).29± 31 The role of nonclathrin-coated invaginations in the plasma membrane (caveola), the process of ``potocytosis,'' and the linkage of FR and RFC in this process remain debated.21,32,33 In addition to the above systems, there is evidence that passive diffusion may work together with folate receptors in transplacental folate transport.34 Folate Polyglutamates Fig. 155-1 Structure of folic acid and its derivatives. (Modi®ed from Rowe.1 Used with permission.) (PteGlu) and pteroylglutamate, respectively (Fig. 155-1). The term folate is also used in the generic sense to designate a member of the family of pteroylglutamates, each having a different level of reduction of the pteridine ring, 1-carbon substitution, and number of glutamate residues. In the folate compounds, pteroic acid is conjugated with one or more molecules of L-glutamate, each linked by amide bonds to the preceding molecule of glutamate through the g-carboxyl group. The terms pteroylpolyglutamate and folate polyglutamate apply to folate compounds with more than one glutamate residue. The biologically active folates are substituted derivatives of 5,6,7,8-tetrahydrofolic acid (H4Folate) (Fig. 155-2). As summarized in Fig. 155-1, there are at least three stages of reduction of the pyrazine ring of the pteridine moiety; at least six different 1-carbon groups substituted at positions N 5, N 10, or both; and g-glutamyl peptide chains of varying length. 5-MethylH4Folate is the predominant form of folate in serum and in many tissues. 5-Formyl-H4Folate, also known as ``folinic acid,'' Human cells need a critical concentration of intracellular folate to allow activity of folate-dependent enzymes. The amount required to maintain an optimal rate of growth in culture varies from about 50 nM in human ®broblasts to about 1 mM in human lymphocytes and certain tumor cells.35 Although the Kms for monoglutamate folates of many of the folate-dependent enzymes are greater than 1 mM, those for polyglutamate folates of appropriate chain length are generally much lower, allowing folate metabolism to progress at the concentration of folates present in cells. Both a cytoplasmic and a mitochondrial folylpolyglutamate synthase add glutamate residues to selected folate molecules. A single gene on chromosome 9cen-q34 ([FPGS], NM_004957, MIM 136510) with alternative splice sites codes for the two folylpolyglutamate synthase proteins.36± 38 These enzymes form a peptide bond between the g-carboxyl of the glutamate already present and the a-amino group of the glutamate to be added. Folylpolyglutamate synthase adds glutamate residues one at a time, requires ATP for its reaction, utilizes H4Folate and other folates as well as antifolates as substrates with different af®nities, and reacts poorly with folic acid and 5-methyl-H4Folate. There is a unidirectional ¯ow of triglutamate forms from the mitochondria to the cytoplasm, but longer forms cannot exit the mitochondria. Speci®c instances of channeling of polyglutamate intermediates between active sites of multifunctional proteins have been demonstrated. Thus, they may play a role in maintaining speci®c protein-protein Fig. 155-2 Structure of 5,6,7,8-tetrahydrofolic acid (THF). (Reprouced from Rowe.1 Used with permission.) 3899 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3900 PART 17 / VITAMINS interactions.39 Cell lines defective in folate polyglutamate formation have been reported. A mutant Chinese hamster cell line is auxotrophic for glycine, adenosine, and thymidine, apparently because reactions generating these within the cell require folate polyglutamates.40,41 A human breast carcinoma cell line is defective in the synthesis of methotrexate polyglutamates and, consequently, is resistant to methotrexate. Folate polyglutamates must be hydrolyzed in the intestine prior to absorption, and monoglutamates are released into the circulation.42,43 The g-glutamyl chain is resistant to digestion by the common proteolytic enzymes and is hydrolyzed by speci®c pteroylpolyglutamate hydrolase (conjugase) enzymes. Two distinct forms of human conjugase have been described, one in the intestinal brush border, which acts at neutral pH, and another within lysosomes. The lysosomal enzyme may play a role in regulating intracellular polyglutamate levels. Both human ([GGH], NM_003878, MIM 601509) and rat lysosomal conjugases have been cloned.44,45 Prostate-speci®c membrane antigen (PSMA) has conjugase activity.46 The major metabolic pathways of the folates are shown in Fig. 155-3. In most cells, because serine and glycine are the major sources of 1-carbon units, entry into the active 1-carbon pool of intermediates is by way of 5,10-methylene-H4Folate. This compound is used unchanged for the synthesis of thymidylate (Fig. 155-3, reaction 4). 5,10-Methylene-H4Folate is reduced to 5-methyl-H4Folate for the biosynthesis of methionine (Fig. 155-3, reaction 1), or is oxidized to 10-formyl-H4Folate for use in purine synthesis47 (Fig. 155-3, reactions 6 and 7). All the interconversions of folates involve exchange of side chains between tetrahydrofolates, except for the formation of thymidylate by thymidylate synthase (Fig. 155-3, reaction 4), which results in the oxidation of the folate moiety to dihydrofolate (H2Folate). Folic acid, a synthetic vitamin not found in nature, and H2Folate are reduced by dihydrofolate reductase (Fig. 155-3, reaction 5) to H4Folate. Dihydrofolate reductase has long been known to be the primary site of action of the chemotherapeutic drug, methotrexate, an antifolate. Unstable gene ampli®cation resulting in resistance to methotrexate is associated with doubleminute chromosomes, while in stably ampli®ed cells that are resistant to methotrexate, the ampli®ed genes are associated with elongated chromosomes.48 The gene for dihydrofolate reductase has been assigned to chromosome 5q11.1-q13.2 ([DHFR], NM_000791, MIM 126060).49,50 The major source of single-carbon units in most organisms is carbon 3 of serine, which is derived from glycolytic intermediates. Serine hydroxymethyltransferase catalyzes the cleavage of serine to glycine and 5,10-methylene-H4Folate (Fig. 155-3, reaction 3). In mitochondria, glycine is also metabolized to 5,10-methyleneH4Folate, plus carbon dioxide and ammonia, by the glycine cleavage system16 (also see Chap. 90). There are two separate serine hydroxymethyltransferases, a cytoplasmic form ([SHMT1], cSHMT, NM_004169, MIM 182144) and a mitochondrial form ([SHMT2], mSHMT, NM_005412, MIM 138450). Both have been cloned: cSHMT is on chromosome 17p11.2 (NM_004169), and mSHMT is on chromosome 12q13 (NM_005412).51± 53 The cytosolic form has been crystallized, and its structure solved.54 A mutant Chinese hamster ovary cell line de®cient in the mitochondrial serine hydroxymethyltransferase is auxotrophic for glycine,55 indicating that the cytoplasmic enzyme cannot take over all the functions of the mitochondrial enzyme. By catalyzing the conversion of glycine in the diet to serine, which then can form pyruvate, cSHMT may also play a role in gluconeogenesis.16 Both forms of serine hydroxymethyltransferase are capable of catalyzing the hydrolysis of 5,10-methenylH4Folate to 5-formyl-H4Folate.56 Two enzyme systems carry out folate interconversions in mammals.57 A trifunctional polypeptide bears activities of NADPdependent methylene-H4Folate dehydrogenase, methenylH4Folate cyclohydrolase, and 10-formyl-H4Folate synthase (Fig. 155-3, reactions 6, 7, and 8; [MTHFD], NM_005956).58 The interconversion of 5,10-methylene-H4Folate, 5,10-methenylH4Folate, and 10-formyl-H4Folate links the major source of Fig. 155-3 Scheme of folate-mediated 1-carbon transfer reactions: 1. Methionine synthase (methyl-H4Folate:homocysteine methyltransferase); 2. Methylene-H4PtGlu reductase; 3. Serine hydroxymethyltransferase; 4. Thymidylate synthase; 5. Dihydrofolate reductase; 6. Methylene-H4Folate dehydrogenase (NAD and NADP-dependent forms have been described); 7. Methenyl-H4Folate cyclohydrolase; 8. 10-Formyl-H4Folate synthase; 9. GAR (5-phosphoribosylglycineamide) transformylase; 10. AICAR (5-phosphoribosyl-5-aminoimidazole-4-carboxamide)transformylase; 11. Glutamate formiminotransferase; 12. Formimino-H4Folate cycloderaminase; 13. 5,10-MethenylH4Folate synthetase; 14. 10-Formyl-H4Folate dehydrogenase; 15. Glycine cleavage pathway. Metabolic Pathways and Enzymes Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM single-carbon units, as methylene-H4Folate, with synthesis of thymidylate (thymidylate synthase [Fig. 155-3, reaction 4]) or purine (GAR and AICAR transformylase [Fig. 155-3, reactions 9 and 10]). The trifunctional polypeptide also permits either the release of single carbons from folate as formate or, more probably, the scavenging of potentially toxic formate (Fig. 155-3, reaction 8). The trifunctional enzyme is found only in the cytosol,59 and is encoded by a gene on chromosome 14q24.60 A separate bifunctional NAD-dependent methylene-H4Folate dehydrogenase-cyclohydrolase, without synthase activity, also exists ([MTHFD2], NM_006636). This bifunctional enzyme is not detected in normal adult tissue but has been found to be expressed in tissues which contain undifferentiated cells and in transformed mammalian cells.57,61 It is encoded by a nuclear gene but is found predominantly in the mitochondria of transformed cells.59 The crystal structure of the dehydrogenase/cyclohydrolase domain of the human trifunctional enzyme has been determined in the presence of NADP,62 as has that of the bifunctional bacterial enzyme.63 While the NADP binding site is clear, the folate binding site(s) are only predicted by modeling, and the nature of the cyclohydrolase active site is not apparent.62 10-Formyl-H4Folate dehydrogenase ([Fig. 155-3, reaction 14)]; [10-FTHFDH], AF052732) releases excess active single-carbon fragments from the folate pool and generates carbon dioxide. Its activity is restricted to the liver64 and serves to maintain suf®cient H4Folate to permit acceptance of single carbons in folatedependent reactions. 5,10-Methenyl-H4Folate synthase is an ATP-dependent enzyme ([Fig. 155-3, reaction 13]; [MTHFS], NM_006441)65± 67 which converts 5-formyl-H4Folate (folinic acid) to 5,10-methenylH4Folate. Thus, this enzyme is important in supporting the clinical use of folinic acid for preventing methotrexate toxicity. 5,10-Methylene-H4Folate reductase ([Fig. 155-3, reaction 2]; [MTHFR], AJ237672, MIM 236250)68±70 converts 5,10-methylene-H4Folate to 5-methyl-H4Folate and probably uses only polyglutamates as substrates within the cell. The human enzyme binds FAD, uses NADPH as electron donor, and functions as a dimer of 77 kDa subunits.71,72 It is inhibited by adenosylmethionine, which is bound by the C-terminal regulatory region.72 The reaction is bidirectional in vitro, but in vivo, it is essentially unidirectional toward 5-methyl-H4Folate. It is usually assayed in the reverse direction in vitro, using menadione as electron acceptor, but it can be assayed in the physiological direction as well.68 Under the latter conditions, the concentration of adenosylmethionine required for inhibition is considerably smaller than that required for inhibition of the reverse reaction.69 The human gene has been cloned and localized to chromosome 1p36.3 (AJ237672) and consists of 11 exons.73,74 The homologous enzyme from E. coli is considerably smaller (33 kDa) by virtue of having no adenosylmethionine-binding regulatory domain. Its crystal structure has been solved.75 Methionine synthase, also known as 5-methyl-H4Folate:Lhomocysteine methyltransferase ([MTR], NM_000254, MIM 156570), is a cobalamin-dependent enzyme that catalyzes the transfer of a methyl group from methyl-H4Folate (or adenosylmethionine) to homocysteine to form methionine (Fig. 155-3, reaction 1). In the complete reaction, the methyl group from methyl-H4Folate is transferred to enzyme-bound cob(I)alamin to form methylcobalamin. The methyl group is then transferred to homocysteine, producing methionine and regenerating cob(I)alamin. After a number of cycles, the enzyme-bound cob(I)alamin oxidizes spontaneously to inactive, enzyme-bound cob(II)alamin, and a reducing system and adenosylmethionine are required to reform methylcobalamin and reactivate the enzyme.76±83 Mammalian methionine synthase is an 85-kDa cytoplasmic enzyme that functions as a monomer. Using the binding of cobalamin to methionine synthase in extracts of human-hamster hybrid cell lines as a marker, methionine synthase was assigned to human chromosome 1.84 The cloning of the gene for human methionine synthase has con®rmed this assignment at 1q43.85± 88 The predicted sequence of the human enzyme is 55 percent identical to the cobalamin-dependent methionine synthase from E. coli86 (bacteria also have a noncobalamin-requiring methionine synthase). The bacterial enzyme has been extensively studied, and the structures of its cobalamin-binding and adenosylmethioninebinding domains have been determined by x-ray crystallography.89,90 The structure of the cobalamin-binding domain is homologous to that of the C-terminal cobalamin-binding domain of methylmalonyl CoA mutase, the other cobalamin-requiring mammalian enzyme (see Chap. 94). Because the circulating form of folate in humans is methylH4Folate monoglutamate and because the methylene-H4Folate reductase reaction is essentially irreversible in the cell, folate entering cells must pass through the methionine synthase reaction in order to generate tetrahydrofolate and the other folate cofactors.91±95 In cobalamin de®ciency (acquired or inherited, see below), or when cobalamin is irreversibly oxidized by nitrous oxide,96 methionine synthase activity decreases or is absent, methyl-H4Folate and homocysteine accumulate, and methionine and, especially, adenosylmethionine are reduced. In addition to the folate being ``trapped'' as methyl-H4Folate, most of it remains as the monoglutamate because methyl-H4Folate is a poor substrate for the folylpolyglutamate synthase enzyme. Folic acid or folinic acid can bypass this block until methyl-H4Folate again accumulates as a result of methylene-H4Folate reductase activity. In E. coli, the reductive activation system that maintains the active form of the cobalamin cofactor on methionine synthase is a two-component ¯avoprotein system97,98 consisting of ¯avodoxin99 and ¯avodoxin reductase.100 A similar system had been postulated for eukaryotes. Recently, based on the hypothesis that the mammalian enzyme would be a multifunctional protein incorporating both reductase activities, Gravel and colleagues cloned an enzyme called methionine synthase reductase (MSR), which is at least one component of this system.81,83 MSR is a unique member of the ferredoxin-NADP reductase family of electron transferases, combining binding sites for FMN and FAD, along with NADPH. The biochemical details of the reactivation reaction are unknown, and it remains unclear whether another protein may be involved. MSR has a predicted molecular size of 77 kDa and is encoded by a gene on chromosome 5p15.2-15.3 ([MTRR], NM_002454, MIM 602568).83 During the catabolism of histidine, a formimino group is transferred to H4Folate, which transfer is followed by the release of ammonia and generation of 5,10-methenyl-H4Folate. The two enzyme activities, glutamate formiminotransferase (Fig. 155-3, reaction 11) and formimino-H4Folate cyclodeaminase (Fig. 155-3, reaction 12), share a single polypeptide which channels folate polyglutamate molecules from one reaction to the next.101,102 The pathway represents only a minor source of single-carbon folates and may exist only in liver and kidney; the enzymes seem to be absent from ®broblasts and blood cells. Disorders of Folate Nutrition, Transport, and Metabolism Nutritional Disorders. Although a number of children born to mothers with a diet de®cient in cobalamin have shown evidence of cobalamin de®ciency (see ``Cobalamin (Vitamin B12)'' below), folate de®ciency in the infant secondary to de®ciency in the mother is unusual.15 In nutritional folate de®ciency in adults, as described in Herbert's classic self-study,103 the peripheral blood and bone marrow changes that occurred after 4 months were preceded by a much-earlier fall in serum folate and a rise in urinary FIGLU levels. Psychologic and mental changes followed, but were rapidly reversed by folic acid supplementation. Red blood cell folate levels fall in folate de®ciency signi®cantly later than do serum folate levels. On the other hand, there are some situations in which there are no defects in folate metabolism per se, but in which folate therapy has been suggested. These include supplements of folic acid given to pregnant women to produce an increase in the mean birth weight of infants104 and, particularly, 3901 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3902 PART 17 / VITAMINS Fig. 155-4 Processes and reactions affected by inherited disorders of folate transport and metabolism; 1. Methylene-H4Folate reductase de®ciency; 2. and 3. Functional methionine synthase de®ciency (cblE, methionine synthase reductase de®ciency; cblG, methionine synthase de®ciency); see text; 4. Glutamate formiminotransferase de®ciency; 5. Hereditary folate malabsorption Ð A, dihydrofolate reductase de®ciency; B, methenyl-H4Folate cyclohydrolase de®ciency; C, cellular uptake defect of folate; and D, methyl-H4Folate:homocysteine methyltransferase de®ciency (original report from Japan9). Disorders involving folate transport are indicated by a broken line, whereas those involved in folate metabolism are indicated by a solid line. The numbered steps show the sites of well-characterized inherited disorders of folate transport or metabolism. Steps are the diseases that have been presented in the literature; those that remain in dispute are indicated with letters. AdoMet 5 adenosylmethionine; H2folate 5 dihydrofolate; H4Folate 5 tetrahydrofolate; methyl-B12 5 methylcobalamin; GAR 5 5-phosphoribosylglycinamide; FGAR 5 a-N-Formyl-glycinamide ribonucleotide; AICAR 5 5-phosphoribosyl-5-aminoimadazole-4-carboxamide; C2, C8 5 carbons number 2 and 8 or of purine ring. supplements given prior to conception to women at risk for bearing a child with neural tube defects to reduce the frequency of these disorders.105 It has also been suggested that increased folate intake may serve to reduce serum homocysteine concentration, a likely risk factor in peripheral vascular disease (see Chap. 88).106±108 The processes and reactions affected by inherited disorders of folate transport and metabolism are shown in Fig. 155-4. Those that are discussed in some detail below include hereditary folate malabsorption (reaction 5); glutamate formiminotransferase de®ciency (reaction 4); methylene-H4Folate reductase de®ciency (reaction 1); and functional methionine synthase de®ciency (reactions 2 and 3). with severe bilateral pneumonia. He was one of seven siblings, two of whom had died in the ®rst year of life without de®nitive diagnosis. In contrast to other cases, there was no sign of mental retardation, and correction of the serum folate levels did result in correction of the levels of folate in the cerebrospinal ¯uid (CSF). There is evidence for parental consanguinity in four families.112,114,116,121 The common clinical presentation in hereditary folate malabsorption is megaloblastic anemia in the ®rst few months of life with low serum folates. Laboratory ®ndings may include urinary excretion of formiminoglutamic acid (FIGLU) and orotic acid.12,120 All patients were severely restricted in their ability to absorb oral folic acid or oral reduced folates. Large doses of oral folates did cause a hematologic response in some patients.111,112,120 Parenteral therapy with folates has been effective in correcting anemia, but has been of limited effectiveness in correcting the levels of folate in the CSF. Other studies have suggested that folinic acid118,119 or methyl-H4Folate is more effective in increasing CSF. There is signi®cant clinical heterogeneity among patients. In some patients, seizures were ameliorated by folate therapy, while in others, they were exacerbated by it. It has been noted125 that the presence of seizures, with or without cerebral calci®cations, is coincident with the ability to respond hematologically to large doses of oral folinic or folic acid; the reason for this is not known. One of the patients120 had additional ®ndings, including a relative inability to retain plasma folate after parenteral folate administration, a ®nding also seen in another patient;114 high levels of folate in the red blood cells following folate therapy; low normal plasma levels of methionine; the presence of cystathionine Hereditary Folate Malabsorption (MIM 229050) Clinical and Laboratory Findings. This disorder [Fig. 155-4 (5)], which has also been called congenital malabsorption of folate because of its early clinical presentation, has been described in fewer than 20 patients, mostly females.109±123 The disease is characterized by severe megaloblastic anemia. Diarrhea, mouth ulcers, and failure to thrive are common, and most patients showed progressive neurologic deterioration. Folinic acid-responsive peripheral neuropathy has been described.115,123 Among the patients were two pairs of sisters,111,124 and there may have been additional unrecognized affected patients in these families, because one patient had a sib who died at age 3 months119 whose sex was not reported. Another patient, who was one of nine children, had sisters who died shortly after birth; in addition, she had a brother who died at the age of 13 years, but no further clinical details were provided.120 A report from Israel121 described a boy with this disorder, an infant who presented at age 4 months Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM in the CSF and a response of the patient to methionine therapy; and increased susceptibility to infections associated with low levels of serum IgM and IgA. One of the affected boys121 had a partial de®ciency in both humoral (surface Ig and response to pokeweed mitogen) and cellular (E-rosette forming and response to hemagglutinin and concanavalin A) immunity. These patients with hereditary folate malabsorption provide the best evidence for the existence of a speci®c carrier for folate both at the level of the intestine and the choroid plexus. Oxidized and reduced folates must share this system, because the absorption of both is effectively blocked in these patients. The same gene product must mediate both intestinal transport and transport of folates into the brain because, except in the two affected males,121,123 levels in the CSF remained low when blood folate levels were raised suf®ciently to correct the anemia. As mentioned earlier, a cDNA for the putative intestinal transporter has been cloned, and it is identical to that for the reduced folate carrier.27 It is likely that uptake of folates into other cells of the body is normal in patients with hereditary folate malabsorption, because a hematologic response occurs in the presence of relatively low blood folate levels. In addition, the content and distribution of folates were normal in cultured ®broblasts from the one patient studied.120 Thus, it will be interesting whether mutations in the putative gene for intestinal transport will be found in patients with hereditary folate malabsorption. Treatment. Cooper has stressed15 that it is essential to maintain folate levels in the serum, red blood cells, and CSF above levels associated with folate de®ciency (4, 150, and 15 ng/ml, respectively). As mentioned above, some patients may respond to large oral doses of folic acid, folinic acid, or methyltetrahydrofolic acid. Oral doses may be increased to 100 mg/day or more if necessary.15 If oral therapy does not work, systemic therapy must be instituted with daily injections (subcutaneous, intramuscular, or intravenous) of folinic acid.126 If CSF folate levels cannot be normalized, periodic intrathecal injections should be considered.15 Genetics. The occurrence of at least one sibship with hereditary folate malabsorption and the documented cases of consanguinity suggest inheritance as an autosomal recessive disorder. All but four of the documented cases121,123,126 have been female, although in one of the families, there is the suggestion of another possibly affected male.120 In the father of one patient, the absorption of oral folate was seen to be intermediate,114 again suggestive of autosomal recessive inheritance. Cellular Uptake Defects. These disorders (Fig. 155-4 (C)) appear in a group of reported patients with varied clinical ®ndings, some of which were associated with serious hematologic disease. Although the individual abnormalities of folate uptake are well characterized, it remains unclear whether these disorders represent primary inherited abnormalities. Branda et al. reported a patient with severe aplastic anemia that responded to high doses of folate therapy.127 The patient was part of a large kindred in which there was a high incidence of severe hematologic disease, including anemia, pancytopenia, and leukemia. These diseases were found in 34 individuals in four generations, resulting in the death of 18. The proband showed a marked reduction of the uptake of methyl-H4Folate in stimulated lymphocytes despite a normal uptake of folic acid. Among eight healthy family members, including three of the proband's children, four were found to have a similar abnormality. In addition, there was a less marked reduction in the uptake of methyl-H4Folate by bone marrow cells from the proband and his son. Of particular interest, however, was the ®nding that one son showed initially normal folate uptake, but neutropenia subsequently developed, and then the abnormality was exhibited. This observation has been taken to suggest that this disorder may not be a primary defect in folate uptake.125 Folate uptake by erythrocytes and the intestinal absorption of folate were found to be normal. Since the original report, the patient died at age 41 due to respiratory failure secondary to pleural effusion and ascites.128 Three children in the family had an increased incidence of sister chromatid exchange. An additional family was described with a transport defect which affected red cells and bone marrow, but not lymphocytes.129 The proband and his daughter had dyserythropoiesis without anemia; three brothers were normal. Erythrocytes from the patient showed abnormalities in the Vmax and total uptake of methylH4Folate, whereas folic acid uptake was normal; the daughter showed only a possible elevation in the Km for methyl-H4Folate, while the three clinically normal brothers resembled the proband kinetically. The status of both of these disorders of cellular uptake remains to be clari®ed. An 18-year-old male with progressive neurologic disease, which included sensorineural hearing loss, a cerebellar syndrome, distal spinal muscular atrophy, and pyramidal tract dysfunction, had an isolated folate de®ciency in the CSF and normal serum and red blood cell folate levels.126,130,131 The defect may lie in the isolated transport of folate into the CSF and may turn out to be a variant of hereditary folate malabsorption. Dihydrofolate Reductase De®ciency Ð Suspect Disorder. There are two published reports describing three cases of putative dihydrofolate reductase de®ciency132,133 [Fig. 155-4 (A)]. Megaloblastic anemia developed in these patients soon after birth and showed a better clinical response to folinic acid (5-formylH4Folate), a reduced folate, than to folic acid, an oxidized folate. In all three patients, dihydrofolate reductase activity was decreased in liver biopsies. The original patient132 had a reduction in dihydrofolate reductase activity in the liver to 35 percent of control values (more than 2 SD lower than autopsy liver samples in seven control subjects). This male had anemia at 6 weeks of age, which subsequently became megaloblastic. Oral doses of 50 to 500 mg/ day of folic acid did not produce a clinical response; 5 mg/day of oral folic acid resulted in a sustained 3-year remission. When folate therapy was discontinued, the patient relapsed. Small doses of folinic acid were effective in producing a remission. At age 19 years,5 he was not grossly mentally retarded but had manifested ``sociopathic and frankly criminal behavior that resulted in repeated incarcerations.''5 Although he was still folate-dependent, extracts of cultured ®broblasts showed normal total activity, kinetics, and heat stability of dihydrofolate reductase. Two unrelated patients were later reported with neonatal megaloblastic anemia that was attributed to dihydrofolate reductase de®ciency.133 Activity in a liver biopsy was not detectable in the routine assay in the ®rst case, but normal levels (1.0 to 1.7 nM of dihydrofolate reduced per min per mg of protein) were found in the presence of 0.6 M potassium chloride. At age 3 years, her bone marrow showed dihydrofolate reductase activity that was 10 percent of control levels and a heat-labile enzyme with a molecular size of 58,000 daltons, considerably higher than that of the normal enzyme.134 At age 9 years,1 the child was severely mentally retarded and still showed folate-dependent macrocytic anemia. We have shown that the correct diagnosis in this child is methionine synthase reductase de®ciency (cblE complementation group, see below). The second child was ®rst seen at age 26 days because of oral and anal moniliasis and poor feeding. Low neutrophil and platelet counts were seen, and over the next 2 weeks a megaloblastic anemia developed. The serum folate level at 9.5 ng/ml was borderline normal for his age,1 and the serum cobalamin level was normal. Dihydrofolate reductase activity in a liver biopsy specimen was 20 percent of the normal median value and was activated about twofold by 0.6 M potassium chloride, similar to the control liver samples. Subsequent study revealed that the patient was de®cient in functional transcobalamin II135 (see cobalamin section below). There was absent unsaturated serum cobalamin-binding capacity, although immunoassay did show transcobalamin II 3903 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3904 PART 17 / VITAMINS protein levels at 39 percent of the normal mean. There was no cobalamin-binding protein corresponding to transcobalamin II on Sephadex-gel chromatography. The patient was reinvestigated because of the development of mental retardation and severe neuropathy after 2 years of treatment.136 It was concluded that this patient had functionally inactive transcobalamin II of the type described by Seligman.137 No additional patients have been described. Although at least two of the reported children had inborn errors of cobalamin metabolism, which were not initially recognized, the low liver values of dihydrofolate reductase remain dif®cult to explain. Of interest, urinary amino acids were reported to show a normal pattern, and no FIGLU was detected in the urine of the two patients who were reported in the most detail.133 Thus, although the possibility of dihydrofolate reductase de®ciency in an infant with severe megaloblastic anemia must be considered, all other known causes must be ruled out before this diagnosis can be con®rmed. Methenyltetrahydrofolate Cyclohydrolase De®ciency Ð Suspect Disorder. As previously discussed, methenyl-H4Folate cyclohydrolase (Fig. 155-3, reaction 7) is part of a trifunctional protein that contains the activities of methylene-H4Folate dehydrogenase, methenyl-H4Folate cyclohydrolase, and 10-formyl-H4Folate synthase.138,139 Methenyl-H4Folate cyclohydrolase de®ciency [Fig. 155-4 (B)] was proposed in three children who had 44 percent of control enzyme activity on liver biopsy and levels of 58 percent, 36 percent, and 43 percent of control values in erythrocytes.127 Clinically, the patients had mental retardation, microcephaly, ventricular dilatation, and abnormal electroencephalograms. A later report from the same laboratory9 essentially retracted the diagnosis, and no additional cases have been reported. Glutamate Formiminotransferase De®ciency (MIM 229100). As a result of the catabolism of histidine, a formimino group is transferred to tetrahydrofolate, followed by the release of ammonia and the formation of 5,10-methenyl-H4Folate. The two enzyme activities involved in these steps, glutamate formiminotransferase (EC 2.1.2.5) (Fig. 155-3, reaction 11) and formiminoH4Folate cyclodeaminase (EC 4.3.1.4) (Fig. 155-3, reaction 12), share a single polypeptide, which forms an octameric enzyme101,102 that channels polyglutamate folates from one reaction to the next. This pathway represents a minor source of single-carbon units and may be present only in liver and kidney. Clinical and Laboratory Presentation. Reports on fewer than 20 patients have been published, and it is not clear whether this enzyme de®ciency is associated with a disease state or whether the association of clinical ®ndings with FIGLU excretion is a result of bias of ascertainment.5,126,131,140 Individuals with glutamate formiminotransferase de®ciency [Fig. 155-4 (4)] have been described with two distinct phenotypes. In one type, there is mental and physical retardation, cortical atrophy with dilatation of cerebral ventricles, and abnormal electroencephalograms. The second type shows no mental retardation but massive excretion of FIGLU. It has been postulated that the severe form is associated with a major block in the cyclodeaminase activity and the mild form with a block in the formiminotransferase activity,1 but no direct enzyme measurements have been presented to support this hypothesis. Diagnosis of these diseases is hampered by the absence of enzyme activity from cultured human cells,12 and there is dispute as to whether the de®ciency can be diagnosed using red blood cells.5,141 Indeed, in most cases in which the liver has been examined, enzyme activities were higher than would have been expected for a complete block resulting in disease.9 Erbe5 has summarized and tabulated most of the known patients with glutamate formiminotransferase de®ciency.124,142±155 The patients have come to medical attention from 3 months to 42 years of age. Three patients presented with delayed speech, two had mental retardation, and two presented with seizures. Two were studied because they were sibs of known cases. Mental retardation was described in most of the original Japanese patients,9 whereas only three of the eight remaining patients were reported to show evidence of mental retardation.149,151,152,154 Abnormal electroencephalograms and hypotonia have been described frequently. Several patients showed hematologic ®ndings, including hypersegmentation of neutrophils and macrocytosis. The reported biochemical ®ndings include: increased urinary, as well as serum, FIGLU, especially after a histidine load; normal to high serum folate levels with normal cobalamin levels; hyperhistidinemia; hypomethioninemia; and histidinuria. In several of the Japanese patients, FIGLU excretion was elevated only after histidine loading. Amino acid levels in plasma, including histidine, were usually normal, but occasionally low methionine levels were seen.124,154 Urinary excretion of 4-amino5-imidazolecarboxamide,149,156 an intermediate metabolite in purine synthesis, has been reported, as has excretion of hydantoin-5-propionate, the stable oxidation product of the FIGLU precursor, 4-imidazolone-5-propionate.15,154,155 Three patients of 12 months, 3.3 years, and 5.5 years, with a neuroblastoma, a germ cell tumor, and a ®bromatous sarcoma, respectively, were found to have increased excretion of FIGLU and hydantoin propionic acid.140 High levels persisted after treatment, and it was concluded that the patients had glutamate formiminotransferase de®ciency. Enzyme Activity. Enzyme activity was measured in the livers of ®ve patients and ranged from 14 to 54 percent of the activity in control livers; what these values signify is not yet known. In three families, the level of enzyme activity was said to be low in erythrocytes; on the other hand, several laboratories have been unable to detect enzyme activity in erythrocytes, even in controls.5 Treatment. Response to therapy has been judged on the basis of decreased urinary excretion of FIGLU. Two patients in one family responded to treatment with folates;150 six others did not.5 One of two patients152,154 responded to methionine supplementation. Given that the correlation between clinical phenotype and FIGLU excretion remains uncertain, the basis for treating these patients is unclear. Genetics. Glutamate formiminotransferase de®ciency has been found in both male and female offspring of unaffected parents. No consanguinity has been described. The de®ciency is presumed to be inherited as an autosomal recessive. In the absence of detectable enzyme activity in cultured cells, de®nitive resolution of the inheritance of this disorder awaits the cloning of the human gene and the localization of the primary defect, because it is likely that the primary defect could then be detected in DNA from patients. DNA from putative patients should be put aside to await molecular diagnosis. Differential Diagnosis. The major dif®culty in the diagnosis of this disorder lies in the lack of expression of enzyme activity outside of the liver. Aside from FIGLU excretion in the urine and assay of enzyme activity in liver biopsy, which in reported cases has shown unusually high residual activities,9 de®nitive diagnosis is dif®cult. In addition, FIGLU excretion may be caused by other defects in folate or cobalamin metabolism. Indeed, ®broblasts of one patient, who had megaloblastic anemia and folate-responsive homocystinuria,141 were examined further. This patient has low methionine biosynthesis, low methionine synthase activity, and low MeCbl, and, indeed, has methionine synthase de®ciency (cblG complementation group, see below). Thus, it is appropriate to study ®broblasts from all patients who show evidence of hypomethioninemia for evidence of a functional de®ciency in methionine synthase. Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Methylenetetrahydrofolate Reductase De®ciency (MIM 236250). Methylene-H4Folate reductase (EC 1.5.1.20) is a cytoplasmic enzyme that catalyzes the NADPH-linked reduction of methylene-H4Folate to methyl-H4Folate (Fig. 155-3, reaction 2). Methyl-H4Folate serves as the methyl donor for the methylation of homocysteine in the reaction catalyzed by methionine synthase (5-methyl-H4Folate:homocysteine methyltransferase [Fig. 155-3, reaction 1]). The combined action of methylene-H4Folatereductase and methionine synthase supplies single-carbon units for methylation reactions that use adenosylmethionine. The reaction catalyzed by methylene-H4Folate reductase is essentially irreversible under physiological conditions, and enzyme activity is regulated by levels of adenosylmethionine, which is an inhibitor.69,157,158 Clinical and Laboratory Findings. Since the ®rst reports of methylene-H4Folate reductase de®ciency in 1972159,160 [see Fig. 155-4 (1)], more than 40 cases have been reported.131,161±197 The major biochemical ®ndings have been moderate homocystinuria and hyperhomocystinemia with low or relatively normal levels of plasma methionine. The clinical severity of this disorder varies greatly from case to case, with most patients being symptomatic in infancy or early childhood, but the age of diagnosis has ranged from before birth to adulthood.159,182,193,198. An infant showed extreme progressive brain atrophy and demyelinization on MRI.197 A 10-year-old male exhibited a developmental history and physical signs compatible with Angelman syndrome.199 In a family with six sibs, three patients had severe recurrent strokes in their early 20s, resulting in the death of two of them 1 year after clinical onset.193 Two of these patients were noted to have a marfanoid habitus, although this is not a frequently reported ®nding. In another family, a younger brother developed limb weakness, incoordination, paresthesias, and memory lapses at age 15 years and was wheelchair-bound by his early 20s, whereas his older brother was asymptomatic at age 37 years.194 The most common clinical manifestation in methyleneH4Folate reductase de®ciency is developmental delay. Motor and gait abnormalities, seizures, and psychiatric manifestations have been reported.195,200,201 In Erbe's 1986 clinical review,5 about half of the patients were microcephalic; EEG abnormalities were present in most; some abnormalities of gait were described in almost all patients who were old enough to walk. Homocystinuria was present in all patients, with a reported range of 15 to 667 mM/ 24 h and a mean of 130 mM/24 h. Homocystine, not normally detected in urine or free in plasma, was found in the plasma: mean value 57 mM (range: 12 to 233 mM). Although data on total plasma or serum homocysteine (tHcy) are scarce, levels of 60 to 184 mM (controls: 4 to 14 mM) have been reported.194,202±204 Plasma methionine levels were low in all patients, ranging from 0 to 18 mM, with a mean of 12 mM; normal is 23 to 35 mM,5 although values vary among laboratories. Although homocystinuria was consistently seen in all patients, and indeed is the clinical clue by which the diagnosis of methylene-H4Folate reductase de®ciency is made, the excretion of homocystine in urine is much less than that found in homocystinuria due to cystathionine synthase de®ciency (see Chap. 88). Indeed, it may not be detected on spot testing, which, therefore, should not be used in isolation to diagnose the disease.205 The methionine levels in methylene-H4Folate reductase de®ciency are always low-normal or low. This, again, distinguishes these patients from those with cystathionine synthase de®ciency, who generally have hypermethioninemia. In contrast to patients who are functionally de®cient in methionine biosynthesis because of abnormalities in methylcobalamin formation (complementation groups cblC, cblD, cblE, cblF, and cblG; see below), patients with methylene-H4Folate reductase de®ciency do not have megaloblastic anemia. In addition, in contrast to patients with the cblC, cblD, and cblF disorders, these patients have no methylmalonic aciduria. Although serum folate levels were not always low, many of the patients with methylene-H4Folate reductase de®ciency had serum folate levels that were low on at least one determination. In contrast, serum cobalamin levels were almost always normal. Although the levels of neurotransmitters in the cerebrospinal ®eld have been measured in only a minority of patients, they have usually been low.5,200 Studies on Cultured Cells. A de®ciency of methylene-H4Folate reductase has been con®rmed on studies of liver, leukocytes, and cultured ®broblasts and lymphoblasts. The enzyme assay routinely used for these studies measures the activity in the nonphysiological direction, using radioactive methyl-H4Folate. Activity is extremely sensitive to the stage of the culture cycle of ®broblasts, with the speci®c activity in control cells being highest in con¯uent cultures.158 This variability is suf®ciently great to allow for the misclassi®cation of controls and heterozygotes if not taken into account. In general, there is rough correlation between residual enzyme activity and the clinical severity. Both the measurement of the proportion of folate present in cultured cells as methylH4Folate171 and the synthesis of methionine from labeled formate178,206 provide a better correlation with clinical severity. Studies on cultured ®broblasts164,171 and liver177,186 determined the levels and distribution of folate derivatives. In both control and mutant ®broblasts, most of the folates present were polyglutamates, and the proportion of polyglutamates relative to folate monoglutamate was similar; a direct relationship was found in cultured ®broblasts between the proportion of cellular folate which was methyl-H4Folate and both the clinical severity and the residual enzyme activity, indicating that the distribution of the different folates may be an important control of intracellular folate metabolism.171 Control cultured ®broblasts can grow when homocysteine, along with folate and cobalamin, is substituted in the culture medium for methionine, an essential amino acid for these cells. In contrast, ®broblasts from patients with methylene-H4Folate de®ciency do not grow on homocysteine.160,165 This inability to grow on homocysteine is shared by ®broblasts from patients who are functionally de®cient in methionine synthase (cblC, cblD, cblE, cblF, and cblG; see cobalamin section below).207 A differential microbiologic assay that makes use of the fact that Lactobacillus casei can utilize methyl-H4Folate for growth but that Pediococcus cerevisiae cannot is a useful screening test for methylene-H4Folate de®ciency, as analysis requires only small numbers of cultured ®broblasts.164 Genetic heterogeneity in the severe form of this disorder was suggested by the fact that ®broblast extracts from two of the original families showed differential heat inactivation at 55 C.165 Although several of the later onset patients had a thermolabile reductase under these conditions, thermolability was also found in patients with early onset disease.208 In some patients, this was shown to be due to the presence of severe methylene-H4Folate reductase mutations in combination with the common 677C ! T mutation that is responsible for the majority of enzyme thermolability in the general population.209,210 Kang and his colleagues originally suggested that thermolability of reductase activity at 46 C for 5 min in lymphocyte extracts of adults may be associated with ``intermediate homocystinemia'' and an increased risk for vascular disease in adult life.211±214 Because of the dif®culties in performing the assay for methylene-H4Folate reductase on small cell numbers, there were not many studies designed to test this hypothesis. It is now clear that hyperhomocystinemia is a risk factor for vascular disease.215±219 The ability to test the role of methylene-H4Folate reductase thermolability as a contributor to the pathology was greatly aided by the cloning of the gene220 and the discovery that a common mutation, 677C ! T, which converts an evolutionarily conserved alanine at amino acid residue 222 to valine (A222V), is responsible for the thermolability.209 The T allele was found to have a frequency of 35 to 40 percent in French-Canadians and other North Americans, but the frequency may vary in different ethnic groups.72,209,221,222 In particular, it was very low in samples from Africa and parts of Asia.222 The association between 3905 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3906 PART 17 / VITAMINS homozygosity for the T allele and plasma homocysteine levels in a population was found to be related to the folate status of the population, with elevations of homocysteine being dependent on the presence of lower plasma folate levels.223± 225. The role of the 677C ! T polymorphism as a risk factor for vascular disease and for neural tube defects226 remains a subject of great interest and debate.221,225± 231 Interestingly, the 677C ! T polymorphism was found to be associated with a decreased risk of colon cancer.232 Another polymorphism, 1298A ! C, which converts glutamate to alanine at amino acid residue 429 (E429A), is also associated with decreased enzyme activity.233±235 A silent genetic variant, 1317T ! C in the same exon, is common in Africans and may interfere with detection of the 1298A ! C polymorphism;235 677C ! T and 1298A ! C have not been found together in doubly homozygous form.234,235 Pathophysiology. The prominent biochemical manifestations of methylene-H4Folate reductase de®ciency include: (a) homocystinuria and homocystinemia; (b) hypomethioninemia; (c) decreased proportion of intracellular folate as methyl-H4Folate; and (d) decreased neurotransmitter levels. Patients with this disease rarely have megaloblastic anemia, suggesting that there is not a folate-related defect in purine and pyrimidine biosynthesis. The relative importance of homocysteine excess and methionine de®ciency in these patients remains a matter of conjecture. The neurologic ®ndings in monkeys treated with nitrous oxide, an agent that inactivates methionine synthase, are reported to be similar to that caused by cobalamin de®ciency;91±93 this effect is reversed by methionine therapy. Patients with disorders of cobalamin metabolism,236 who also have a block in methionine biosynthesis, may have neurologic deterioration, but they also have hematologic abnormalities that are absent in methylene-H4Folate reductase de®ciency. The pathologic changes5,163,166,168,184± 186,189,192 in the patients with methyleneH4Folate reductase de®ciency include dilated cerebral ventricles, internal hydrocephalus, microgyria, and low brain weight. Also seen in the brain are perivascular changes, demyelination, macrophage in®ltration, gliosis, and astrocytosis. Other major pathologic ®ndings are thromboses of arteries and cerebral veins; these appear to have been major factors in the death of these patients. These thromboses are the only pathologic ®ndings shared with cystathionine synthase de®ciency. It has been suggested that the combination of methylene-H4Folate reductase de®ciency and Factor V Leiden may contribute to the vascular pathology in some patients.237,238 One patient with methylene-H4Folate reductase de®ciency had a ®brosarcoma.192 It has been pointed out192 that the neuropathologic vascular ®ndings in methylene-H4Folate reductase de®ciency are similar to those seen in classical homocystinuria due to cystathionine synthase de®ciency. However, in methylene-H4Folate reductase de®ciency, it is necessary to explain the demyelination, astrogliosis, and lipid-®lled macrophages, which are associated in many patients with a progressive course of seizures, microcephaly, and severe psychomotor retardation. Two reports189,192 have described classical ®ndings of subacute combined degeneration of the cord similar to that observed in patients with untreated cobalamin de®ciency in patients dying with methylene-H4Folate reductase de®ciency. It has been proposed that methionine de®ciency causes demyelination, presumably by interfering with methylation. Methylene-H4Folate reductase is present in mammalian brain.239,240 Because several authors have suggested that only methyl-H4Folate among the natural folates can cross the bloodbrain barrier,172,241 methylene-H4Folate reductase de®ciency may result in functionally low folate levels in the brain. Because neurologic symptoms may be observed in patients without very low methionine levels, it has been suggested77 that the neurologic dysfunction may occur as a result of impaired purine and pyrimidine synthesis in the brain, as opposed to low levels of adenosylmethionine. The relative importance of low folate levels, low methionine levels, and low levels of neurotransmitters in the pathology of methylene-H4Folate reductase de®ciency is uncertain.242 Differences seen between functional methionine synthase de®ciency236,243 (cblC, cblD, cblE, cblF, and cblG) and methylene-H4Folate reductase de®ciency should be useful in sorting out the relative importance of low levels of reduced folates, other than methyleneH4Folate, and low levels of methionine. These comparisons have the potential of being made more dif®cult by developmental and tissue differences in the distribution of these enzyme activities.244,245 The most important ®nding in the clinical differential diagnosis is the absence of megaloblastic anemia in patients with methyleneH4Folate reductase de®ciency as compared to patients with functional methionine synthase de®ciency (cblC, cblD, cblE, cblF, and cblG complementation groups), and the absence of methylmalonic aciduria as compared to patients with cblC, cblD, and cblF disease (see below). It has been shown that levels of methylcobalamin and of methionine synthase may be low in ®broblasts from some patients with methylene-H4Folate reductase de®ciency and that this could lead to the incorrect diagnosis of methionine synthase de®ciency (cblE or cblG).208 Treatment. Methylene-H4Folate reductase de®ciency is very resistant to treatment but betaine has improved the overall prognosis.5,15,126,131,195 The rationale for therapy includes: (a) folates, such as folic acid or folinic acid, in an attempt to maximize any residual enzyme activity; (b) methyl-H4Folate to replace the missing product; (c) methionine to correct the cellular methionine de®ciency; (d) pyridoxine to lower homocysteine levels, because of its role as a cofactor for cystathionine synthase; (e) cobalamin, because of its role as a cofactor for methionine synthase; (f ) carnitine, because its synthesis requires S-adenosylmethionine; (g) betaine,176 because it is a substrate for betaine:homocysteine methyltransferase,245 a liver-speci®c enzyme that converts homocysteine to methionine; and (h) ribo¯avin, because of the ¯avin requirement of methylene-H4Folate reductase. Criteria for the success of treatment5 have included reduction of the plasma homocysteine levels with elevation of plasma methionine levels to normal, along with improvement in the clinical picture. In most cases, several of the agents mentioned above have been used in combination, and it is somewhat dif®cult to assess the ef®ciency of a single one. Cooper15 suggested a therapeutic regimen consisting of oral betaine, folinic acid, and methionine, with additional vitamin B6 and cobalamin. Cooper recommended cobalamin because of the observations of subacute combined degeneration of the cord189 in a child treated with methyl-H4Folate alone. Interestingly, therapy with methionine alone or with methyl-H4Folate is not particularly effective in most cases, even though adenosylmethionine de®ciency in the central nervous system appears to be playing a major role in the pathogenesis of this disease.242 Fowler reported that one patient responded to ribo¯avin.131 Supplementation with pyridoxine also has been suggested in order to enhance the transsulfuration pathway.195 Therapeutic successes include a patient who was treated with a combination of methionine, oral folinic acid and vitamin B6, and cobalamin,179,180 and several patients in whom betaine was included in the regimen.5,181,182,190 One patient who responded to betaine at doses of 20 g/day had not responded to other treatments, including folates and methionine. Cobalamin had not been used in this patient. The two patients who were treated from the ®rst month of life190 with folic acid and betaine had normal psychomotor testing at around the age of 5 years. Ronge and Kjellman described a 7.5-year-old female, with slight microcephaly, impaired vision, and moderate developmental delay, who was treated from infancy with 3 to 6 g of betaine daily. She developed an unexplained increase in appetite and weight gain from age 4 years. With treatment, her previously undetectable plasma methionine levels normalized, but total plasma homocysteine levels remained elevated.202 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Thus, betaine181,190,202,246 appears to be the most promising agent for therapy of methylene-H4Folate reductase de®ciency, although, as mentioned above, some of the other therapies have been partially successful. There is not a great deal of data on the optimum dose of betaine in these patients, but Ronge and Kjellman suggested a dose of 6 g/day (3gb.i.d.) but indicated that they intended to increase the dose to 12 g/day in their patient.202 Ogier de Baulny and colleagues suggested a dose of 2 to 3 g/day in young infants and 6 to 9 g/day in children and adults.195 Kakura and colleagues studied the relationship of serum total homocysteine and betaine levels during treatment of a patient with oral betaine in doses of between 20 and 120 mg/kg.204 They found that serum levels of total homocysteine decreased proportionally until betaine levels reached 400 mM, and suggested that this is the therapeutic threshold for serum betaine. Many authors5,12,196,202 have stressed the importance of early diagnosis and therapy because of the poor prognosis in this disorder once there is evidence of neurologic involvement. Even with early diagnosis, it is not clear that any of the therapeutic regimens are universally successful, and it is possible that genetic heterogeneity in the disease itself is responsible for some of the variability in clinical response to therapy. Genetics. Autosomal recessive inheritance of methylene-H4Folate reductase de®ciency was supported clinically by the occurrence of more than one case in several families, by the presence of both males and females with the disease within the same family, and by the decreased activity of the enzyme in the ®broblasts165 and lymphocytes167 of obligate heterozygotes. Consanguinity has been reported.5,169 The gene is on chromosome 1p36.3 and has 11 exons.74 Although nonsense and splice-site mutations have been reported in patients with methylene-H4Folate reductase de®ciency, most mutations have been missense, and each has been reported in only one or two families with severe de®ciency.203,210,220,247 Over 20 different mutations causing severe disease are known, in addition to the polymorphisms described above, which may contribute to disease in the general population; they are shown in Fig. 155-5 in a representation of the methylene-H4Folate reductase protein. The crystal structure of the E. coli enzyme, which is Fig. 155-5 The structures, domains, and mutations of the MTHFR polypeptide. Amino acid changes are indicated above the protein; base pair position is indicated below. An area of the polypeptide is considerably smaller than the mammalian one because it lacks the C-terminal adenosylmethionine-binding regulatory domain, has been reported.75 Based on this, the possible effect of the common A222V polymorphism on the quaternary structural stability of the enzyme and the role of folates in stabilizing this structure were rationalized.75 The effects of severe, disease-causing mutations have not yet been accounted for in this model system. Methylene-H4Folate reductase de®ciency has been diagnosed or excluded prenatally by enzyme assay or by measurement of the incorporation of labeled formate into methionine by cultured amniotic ¯uid cells.182,188,190,205,248,249 Enzyme activity is detectable in normal chorionic villi.182,188 If both mutations segregating in a family are known, molecular analysis allows for early prenatal diagnosis. Functional Methionine Synthase De®ciency Three metabolic pathways intersect at methionine synthase: those of folate, cobalamin (Cbl), and sulfur-containing amino acids. Because de®ciency of the activity of this enzyme results in diminished or absent methylcobalamin (MeCbl) synthesis, disorders affecting it have been considered and designated genetically as Cbl metabolism defects (symbolized as cbl ), along with others affecting both MeCbl and adenosylcobalamin (AdoCbl) synthesis or AdoCbl synthesis alone (see cobalamin section below). These designations have been reinforced by the use, in differential diagnosis, of genetic complementation analysis between cell lines from patients with defects in all aspects of Cbl metabolism. Nevertheless, because methyl-H4Folate participates in the methionine synthase reaction and because methionine synthase de®ciency signi®cantly affects folate metabolism, these defects can be considered inborn errors of folate metabolism as well. Here, we discuss the two that directly involve methionine synthase (complementation groups cblE (MIM 236270) and cblG (MIM 250940). In the subsequent sections on Cbl metabolism, the others indirectly affecting methionine synthase activity (cblC, cblD, and cblF ) will be considered in detail. For a full discussion of sulfur amino acid metabolism and transsulfuration pathways, see Chap. 88. Clinical and Laboratory Findings. Patients from the cblE and cblG complementation groups are very similar, both clinically and enlarged in order to show all the amino acid changes. (Courtesy of R. Rozen, McGill University ) 3907 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3908 PART 17 / VITAMINS Table 155-1 Clinical and Laboratory Features of Patient with Homocystinemia Due to Defects in MeCbl Synthesis* Mutant Class Finding cblE cblG Megaloblastic anemia Developmental retardation Cerebral atrophy Hypotonia Feeding dif®culty Lethargy Seizures Vision abnormalities Skeletal abnormalities 3/4 4/4 3/4 2/4 3/4 2/4 2/4 2/4 0/4 10/10 10/10 7/10 8/10 8/10 7/10 9/10 3/10 1/10 *Ratios denote the number of patients showing a particular finding/total nrmber of patients in each mutant class S O U RC E : Compiled from published summaries.243,259 biochemically. Most patients so far reported with these disorders presented in the ®rst few months of life with vomiting, poor feeding, and lethargy. Hypotonia, seizures, and developmental delay characterize their severe neurologic dysfunction. There are reports on at least 12 cblE and 20 cblG patients.83,243,250 ±261 Table 155-1 summarizes some of the clinical ®ndings available from the literature. The prevalence of neurologic signs and symptoms is striking. One patient in the cblG group presented as an adult with progressively impaired sensory responses and gait disturbances and was initially diagnosed as having multiple sclerosis.250 Megaloblastic anemia and homocystinuria or homocystinemia are generally present, and hypomethioninemia is often found. Serum Cbl and folate concentrations are normal or elevated, and methylmalonic aciduria is absent, except in one patient, in whom it was a transient ®nding.262 Localization of Defects. The constellation of homocystinuria and hypomethioninemia without methylmalonic aciduria suggested strongly that these patients had isolated de®ciencies in the activity of methionine synthase, either primary or secondary to abnormal synthesis or utilization of MeCbl, its cofactor (see Fig. 155-10). Studies of ®broblasts derived from several of these patients have con®rmed this hypothesis. Incorporation of [14C]propionate into macromolecules was normal, while incorporation of [14C]methylH4Folate was reduced to 5 to 35 percent of control (average: 15 percent),243 a value similar to that reported for patients from the cblC group (see below). Genetic complementation analysis based on [14C]methyl-H4Folate incorporation distinguished two complementation groups:263 cblE (index patient reported in reference 264) and cblG (index patient reported in reference 252). Accumulation of Cbl by ®broblasts was normal or increased in these groups, as was the fraction recovered as AdoCbl.243 In contrast, the fraction identi®ed as MeCbl was much reduced.243 cblE. When methionine synthase activities were determined under standard conditions in extracts of ®broblasts from cblE patients, both holoenzyme and total enzyme were normal or slightly reduced.243 Under suboptimal assay conditions, namely in the presence of lower concentrations of reducing agents, methionine synthase activities were less than those in controls.265 These ®ndings have led to the hypothesis that the cblE group has defects in an enzyme required either to reduce Cbl so that it can participate in the methionine synthase reaction or to maintain it in its active reduced form [cob(I)alamin] on the methionine synthase.265 For example, bacterial methionine synthase has accessory reductase proteins266 that perform these functions. Based on the sequences of these bacterial proteins, Gravel and colleagues cloned a cDNA for a multifunctional human protein called methionine synthase reductase. Sequence analysis of cDNAs for this enzyme from patients in the cblE group has revealed a number of likely deleterious mutations in this gene.83,257 Figure 155-6 is a linear representation of the protein, its cofactor-binding regions, and the localization of the known mutations. cblG. In the cblG complementation group, methionine synthase activities are reduced even under optimal assay conditions,243 although some heterogeneity has been noted.255 This ®nding suggests that patients in this group have primary defects in the catalytic subunit of methionine synthase itself.263 Moreover, the cblG group shows biochemical heterogeneity with respect to binding cellular Cbl to methionine synthase. In extracts of cell lines from most patients, about 75 percent of cellular Cbl migrated at the position of methionine synthase during gel electrophoresis, even though little of it was MeCbl. In a few cell lines (referred to as cblG variants), however, no Cbl of any form migrated at this position.256 This was highly suggestive of mutations in the Cblbinding domain of methionine synthase or absent methionine synthase protein in these patients, strengthening the possibility that the cblG group re¯ected primary de®ciencies in the methionine synthase apoenzyme. Human methionine synthase has now been cloned, and mutations likely to be deleterious have been found by sequence analysis of cDNAs from patients in the cblG group.85,86,267 Based on the crystal structures of the Cbl-binding and adenosylmethionine-binding domains of E. coli methionine synthase,89,90 one of the missense changes uncovered (P1173L) appears to disrupt the adenosylmethionine binding site,267 while two others, I881D267 and H920D,85 may affect the pocket in the Cbl-binding domain that accommodates the dimethylbenzimidazole moiety of the cofactor (see cobalamin section). Interestingly, Fig. 155-6 Mutations in the MSR polypeptide. Their relation to the FMN, FAD and NADPH binding sites are shown. (Modi®ed from Wilson et al.257 Used with permission.) Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM the cblG variants so far examined all appear to be effectively null for the methionine synthase protein, rather than Cbl-binding mutants.260 Pathophysiology and Genetics. The association of isolated functional methionine synthase de®ciency with megaloblastic anemia and neurologic defects in these patients provides further strong evidence for the hypothesis outlined in Chap. 94, that these clinical signs are sequelae of defects in the MeCbl-methionine synthase branch of Cbl metabolism, rather than the AdoCblmethylmalonyl CoA mutase one. Hall268 and Shevell and Rosenblatt269 have discussed in detail the relationships between the biochemical defects and their pathophysiologic consequences. As Hall has pointed out, there are three levels at which the impact of these disorders is felt: hematologic, short-term neurologic, and long-term neurodevelopmental. Effects at each of these may result from a different aspect of functional methionine synthase de®ciency, and the response of each to treatment may likewise be distinctive. The hematologic problems may re¯ect disturbed DNA synthesis, while the short-term neurologic symptoms are likely due to either acute toxic effects or aberrant neurotransmitter metabolism.268 The long-term developmental effects of these disorders appear to be related to defects in myelination in the central nervous system. Abnormal CT scans have been reported for most of the patients on whom the test was performed, with apparent atrophy or hypoplasia of the brain. MRI has been done for only a few patients; in two, myelination was delayed, even after a year of steady clinical improvement with Cbl therapy.268 Hall has suggested a wide range of possibilities for the disruption of function in the nervous system. They include toxicity of methylH4Folate or homocysteine, the classic folate trap hypothesis or variants of it (discussed above), and reduced methylation of proteins and neurotransmitters due to de®ciency in S-adenosylmethionine synthesis. DNA methylation, either from S-adenosylmethionine or directly from MeCbl,270 may also play a role. A more complete understanding of the impact of de®ciencies in this complex system of interrelated pathways on hematologic and neurologic development requires further study, both of the enzymes involved and of the patients with these and related disorders. Each of these diseases is inherited as an autosomal recessive trait, with about equal numbers of male and female patients reported.243,257,268 Both defects act as recessives in complementation analysis in culture.263 So far, heterozygote detection is possible only by DNA-based techniques. Diagnosis, Treatment, and Prognosis. The clinical hallmarks of these disorders appear to be developmental delay and megaloblastic anemia, with homocystinuria and without methylmalonic aciduria. Although most patients were diagnosed early in life, one (a cblG) did not come to medical attention until age 21.250 Differentiation from other possible diagnoses such as TC II de®ciency (see below), other folate transport or metabolism defects, or cystathionine b-synthase de®ciency (Chap. 88) can be accomplished by studies of cultured cells, particularly by incorporation of 14C from [14C]methyl-H4Folate and complementation analysis. These two assays are especially important because de®ciency of methylene-H4Folate reductase has a similar clinical presentation and may result in decreased cellular MeCbl accumulation and even decreased methionine synthase activity.208 Prenatal diagnosis using amniotic ¯uid cells is possible and has been performed for both the cblE and cblG disorders. With the cloning of both methionine synthase reductase and methionine synthase, molecular diagnosis may be possible in families where the mutations are known. Because the patients reported to date have responded to hydroxocobalamin (OH-Cbl) therapy with normalization of their biochemical parameters and at least partial resolution of their clinical symptoms236,243,255,268 and because it seems likely, as in cblC patients (below), that delays in treatment may result in incompletely reversible developmental delays or neurologic de®cits,243,250,268 institution of OH-Cbl administration should occur as soon as the diagnosis is made. Dosages of 1 mg OH-Cbl per day (intramuscular injection) have been used initially, then tapered to 1 mg, one to three times a week. Biochemical improvement has been rapid on this regimen, and most clinical symptoms have resolved in a few weeks. In some patients, macrocytic anemia has responded to folinic acid therapy.259 In general, neurologic symptoms and developmental problems have been slower to improve, sometimes requiring 3 or 4 months of therapy before consistent gains are apparent.243,268 On the other hand, the cblE patient diagnosed prenatally and treated with OHCbl both in utero and postnatally has developed normally with only minor clinical symptoms,269,271 suggesting that prenatal therapy may be warranted in these disorders. As in the case of some cblC patients (below), a variety of adjuncts to Cbl therapy have been tried, including supplementation with betaine, methionine, carnitine, and pyridoxine,269 with variable and poorly documented results. Of these, betaine supplementation to normalize the serum methionine:homocysteine ratio further, beyond what is achieved with OH-Cbl alone, may be justi®ed to avoid the vascular injury and thromboembolism associated with homocystinemia (see Chap. 88). One patient in the cblE group (diagnosed postmortem) died at 5 years of age from bilateral renal artery thrombosis and had arteriosclerotic changes elsewhere at autopsy,243 emphasizing the potentially serious consequences of untreated or poorly controlled homocystinemia. Because patients with these disorders have been described relatively recently, the long-term prognosis in these conditions remains unknown. The index cblE patient is 18 years old and thriving, although he is mildly developmentally delayed,236 while his prenatally diagnosed and treated brother (14 years old) appears normal, except for a slight speech impediment.269 In contrast, the index cblG patient, although clinically well, remains signi®cantly retarded with major visual defects.252 It seems likely that patients in the cblE and cblG groups will show a range of clinical outcomes,243,268 similar to those of patients in the cblC group (below), because the majority of the symptoms of all of these patients arises from the same cause, that is, functional methionine synthase de®ciency. Likewise, early diagnosis and treatment may be the only way to avoid permanent neurologic damage and its consequences.243,268 Differential Diagnosis of Folate Disorders A guide to the differential diagnosis of the well-characterized disorders of folate metabolism is shown in Table 155-2. Many of these disorders are associated with normal serum and red blood cell folate levels. Hereditary folate malabsorption is always, and methylene-H4Folate reductase de®ciency is usually, associated with low serum folate levels. Serum folate levels were reported as elevated in most of the original Japanese patients (but none of the subsequent ones) with glutamate formiminotransferase de®ciency. Homocystinuria has been described in methylene-H4Folate reductase de®ciency and in the cblE and cblG disorders. Megaloblastic anemia is seen in hereditary folate malabsorption and in cblE and cblG patients, but not in glutamate formiminotransferase de®ciency, except in the original Japanese patients, and only rarely in methylene-H4Folate reductase de®ciency. Defects detectable in cultured cells include a decreased incorporation of label from methyl-H4Folate into protein or from formate into methionine in cblE and cblG disease, and a decreased content of methyl-H4Folate in ®broblasts from patients with methylene-H4Folate reductase de®ciency. Cells from patients with cblE and cblG show decreased levels of MeCbl, as may cells from some patients with methylene-H4Folate reductase de®ciency.208 In cell extracts from cultured ®broblasts, activity of methyleneH4Folate reductase is decreased in methylene-H4Folate reductase de®ciency. In extracts of cblE and cblG cell lines, abnormalities in methionine synthase activity can be detected. Abnormalities of 3909 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3910 PART 17 / VITAMINS Table 155-2 Inherited Defects of Folate Metabolism Clinical ®ndings Prevalence Megaloblastic anemia Developmental delay Seizures Speech abnormalities Gait abnormalities Peripheral neuropathy Apnea Biochemical ®ndings Homocystinuria/hyperhomocysteinemia Hypomethioninemia Formiminoglutamic aciduria Folate absorption Serum Cbl Serum folate Red blood cell folate Defects detectable in cultured whole cells ®broblasts Methyl-H4Folate ®xation Methyl-H4Folate content MeCbl content Extracts-speci®c activity Methionine synthase holoenzyme Glutamate formiminotransferase Methylene-H4Folate reductase Treatment Hereditary Folate Malabsorption MethyleneH4Floate Reductase De®ciency Glutamate Formiminotransferase De®ciency Methionine Synthase Reductase De®ciency (cblE) Methionine Synthase De®ciency (cblG) < 20 cases A A A N N N* N > 40 cases N A A N A A A < 20 cases N* N* N* A* N* N* N* 12 cases A A A N N N N* 20 cases A A A N A* A* N N N A* A N A A A A N N N A A* N N A N N* N* N* A A N N N N N A A N* N N N N N N N N A N* N N N A N A A N A N N* N N** A Activity undetectable in cultured cells ? Abnormal in liver and erythrocytes N Folic acid, reduced folate A Betaine, folate, methionine N ?Folate N N OH-Cbl, betaine, reduced folate * exceptions described in some cases. ** abnormal activity with low concentrations of reducing agent in assay. N normal; A abnormal (i.e., clinical findings or laboratory findings present). glutamate formiminotransferase have not been detected in any cultured cell system. COBALAMIN (VITAMIN B12) The structure and function of cobalamins have intrigued students of human biology since Minot and Murphy demonstrated that oral administration of crude liver extract was effective in the treatment of pernicious anemia in 1926.272 In 1948, this ``antipernicious anemia factor'' was isolated from liver and kidney273,274 and was named ``vitamin B12.'' De®ciency of the vitamin leads to an alteration of function or morphology of several organ systems: megaloblastic anemia and defective granulocyte and immune system function; abnormal intestinal function; and neurologic disease, including neurologic degeneration and dementia. Administration of as little as 1 mg of the vitamin daily was shown to prevent relapse of pernicious anemia. Although the vitamin is widely distributed in animal tissues, there is strong evidence that it is synthesized only by microorganisms found in soil and water or in the rumen and intestine of animals. See Dolphin275 and Banerjee276 for comprehensive reviews of cobalamin structure, biosynthesis, and chemistry. Structural Features The isolation of vitamin B12 culminated in the elucidation of its three-dimensional structure by Hodgkin and coworkers using x-ray crystallographic techniques.277 Cobalamin (Cbl), as it is now of®cially designated, is composed of a central cobalt atom surrounded by a planar corrin ring, which has a complex side chain extending down from the corrin plane consisting of a phosphoribo-5,6-dimethylbenzimidazolyl group (Fig. 155-7). One of the nitrogens of the benzimidazole group is linked to the cobalt atom by coordination in the ``bottom''(a) axial position. The molecule is completed by coordination in the ``upper''(b) axial position of several different radicals. Thus, cyanocobalamin (CNCbl) (more strictly, a-(5,6-dimethylbenzimidazolyl)-cobamide cyanide) is formed by the complexing of a cyanide ion to the cobalt atom. Although this compound is the most common commercial form of the vitamin, it is an artifact of isolation and does not occur naturally in microorganisms, plants, or animal tissues. Many other Cbls have been formed with other ligands, but only four have been routinely isolated from mammalian tissue: hydroxocobalamin (OH-Cbl), the ``natural'' form of the vitamin, glutathionylcobalamin (GSCbl), methylcobalamin (MeCbl), and adenosylcobalamin (AdoCbl). Complexes of Cbl with other sulfhydryl compounds have also been reported. MeCbl and AdoCbl are unique for two reasons. They are the only two compounds in nature known to have a direct covalent carboncobalt bond, and they are the only two forms of Cbl known to act as speci®c coenzymes in mammalian systems. Oxidation and reduction of the cobalt atom further complicate the structure and nomenclature of the Cbls. In OH-Cbl, the cobalt Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM methyltetrahydrofolate (methyl-H4Folate), as well as methionine synthase and MeCbl.286,287 It is relevant to the manifestations of Cbl de®ciency and to the interrelationships between folate and Cbl, and is discussed in detail in the folate section above. The conversion of methylmalonyl CoA to succinyl CoA and the methylation of homocysteine to methionine are the only Cbldependent reactions that have been demonstrated conclusively in mammalian systems. Poston reported that AdoCbl acts as a cofactor in the enzymatic reaction by which a-leucine is isomerized to b-leucine,288 but this has not been con®rmed in other laboratories. In microorganisms, several other enzymes require AdoCbl289,290 such as: glutamate mutase, glycerol dehydratase, ethanolamine ammonia-lyase, and ribonucleotide reductase. In addition, MeCbl participates in the formation of methane and acetic acid and in the fermentation of lysine in bacteria. Cobalamin Absorption and Distribution Fig. 155-7 The structure of cobalamin. R 5 2CH2CONH2; R0 5 2CH2CH2CONH2; X 5 2OH (hydroxocobalamin), 2CN (cyanocobalamin), CH3 (methylcobalamin), or 50 -deoxy-50 -adenosyl (adenosylcobalamin). (Reproduced from Fenton and Rosenberg.343 Used with permission of the publisher.) atom is trivalent (cob(III)alamin), and this compound has been called ``vitamin B12a.'' When the cobalt is reduced to a divalent state (cob(II)alamin), it is called ``vitamin B12r,'' and in the monovalent state (cob(I)alamin), it is called ``vitamin B12s.'' These oxidation-reduction states are important because the cobalt atom must be reduced to its monovalent state prior to formation of MeCbl or AdoCbl, apparently by speci®c reductase enzymes that sequentially convert cob(III)alamin to cob(I)alamin, with cob(II) alamin as an intermediate.278 Cobalamin Coenzymes In 1958, Barker and his colleagues demonstrated that the glutamate mutase reaction in Clostridium tetanomorphum required vitamin B12,279 and, more speci®cally, that the active coenzyme form of the vitamin was AdoCbl.280,281 One year later, Smith and Monty reported that the analogous isomerization of methylmalonyl CoA to succinyl CoA was defective in the liver of Cbl-de®cient rats.282 They suggested that Cbl was a cofactor for the latter isomerization system, a thesis borne out by Gurnani et al.283 and Stern and Friedmann,284 who showed in vitro that the activity of methylmalonyl CoA mutase in liver from Cbl-de®cient animals could be restored to normal by addition of AdoCbl, but not by CNCbl or other vitamin B12 analogues. For several years, because AdoCbl was the only known coenzyme form of vitamin B12, it was designated ``coenzyme B12.'' In 1966, Weissbach and colleagues285 demonstrated that MeCbl is a cofactor in the complex reaction by which homocysteine is methylated to form methionine (Fig. 155-8). This reaction requires S-adenosylmethionine and N 5- Cbls have a unique and highly specialized mechanism of intestinal absorption that has been reviewed in detail.236,291,292 The ability to transport physiological quantities of vitamin depends on the combined action of gastric, ileal, and pancreatic components (Fig. 155-9). The gastric substance, called ``intrinsic factor''(IF) by Castle,293 who ®rst demonstrated its existence, is a glycoprotein that binds Cbl in the intestinal lumen. IF, which has been isolated, characterized extensively,291 and cloned,294 is synthesized by gastric parietal cells. Evidence obtained in vitro295,296 and in vivo297 suggests that three events precede the formation of IF-Cbl in the gut lumen. First, Cbls are released from dietary protein in the acid environment of the stomach. Second, Cbls bind to ``Rbinders,'' or haptocorrins, which are proteins of salivary and gastric origin; these R-binders are members of a family of glycoproteins with a high af®nity for Cbl. Third, pancreatic proteases digest the R-binders, thereby liberating Cbls in the upper small intestine, where they form complexes with IF. Subsequently, the IF-Cbl complex interacts through its protein moiety with a speci®c ileal receptor protein, called cubilin, in the presence of calcium ions. The IF-Cbl-cubilin complex is recognized by megalin, a general transport receptor, and transported into the enterocyte by an endocytic mechanism; the complex is dissociated; and the vitamin is transported across the basal membrane into the portal blood, bound to transcobalamin II (TC II), the transport protein for newly absorbed vitamin.292 Evidence from cultured adenocarcinoma cells, which behave like polarized intestinal epithelial cells,298,299 suggests that these latter steps re¯ect true apical-to-basal transcytosis in which the Cbl is bound to newly synthesized TC II when it is released from the basolateral membrane.299 When labeled Cbl is administered intravenously, most of the labeled vitamin is immediately bound to TC II and disappears from the plasma in a few hours.300,301 Only a small fraction binds to transcobalamin I (TC I) or transcobalamin III (TC III) serum glycoproteins of the R-binder family, even though they carry the majority of the steady state serum Cbl.302 The Cbl bound to the Rbinders turns over very slowly, and its physiological role is still unclear. MeCbl is the major circulating Cbl species, accounting for 60 to 80 percent of total plasma Cbl; OH-Cbl and AdoCbl make up the remainder.303 Because > 90 percent of total plasma Cbl is bound to TC I, it is clear that most of the circulating MeCbl travels with this R-binder. This Cbl distribution pattern is puzzling, particularly in the face of evidence indicating that AdoCbl accounts for 70 percent of total hepatic Cbl, whereas MeCbl constitutes a mere 1 to 3 percent.303 This preponderance of AdoCbl is also present in other tissues, such as erythrocytes, kidney, and brain. The physiological signi®cance of these widely different fractional Cbl distributions in extracellular and intracellular compartments remains obscure. TC II facilitates Cbl uptake by mammalian tissues. Finkler and Hall304 showed that CN-Cbl bound to TC II was accumulated by HeLa cells much more rapidly than free CN-Cbl or CN-Cbl bound to TC I, IF, or other binding proteins. Such TC II-mediated uptake 3911 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3912 PART 17 / VITAMINS Fig. 155-8 Reactions catalyzed by cobalamin coenzymes in mammalian tissues. Note the speci®city of adenosylcobalamin for the isomerization of methylmalonyl CoA and of methylcobalamin for the methylation of homocysteine. Me-H4Folate 5 N 5-methyltetrahydrofolate; H4Folate 5 tetrahydrofolate. was subsequently con®rmed in a variety of cell types, both in vivo and in culture (liver; kidney; heart; spleen; lung; small intestine; cultured ®broblasts; Chinese hamster ovary cells; mouse L cells; lymphoma cells; and phytohemagglutinin-stimulated lympho- Fig. 155-9 Pathway for the gastrointestinal absorption of dietary cobalamin. Cbl 5 cobalamin; HC 5 haptocorrin (transcobalamin I/ III); HC-Cbl 5 haptocorrin-bound cobalamin; degHC 5 degraded haptocorrin; IF 5 intrinsic factor; IF-Cbl 5 intrinsic factor-bound cobalamin; -IF-Cbl 5 intrinsic factor-bound cobalamin attached to cubilin, the ileal receptor; TCII-Cbl 5 cobalamin bound to transcobalamin II. (Reproduced from Rosenblatt and Fenton.477 Used with permission.) cytes) (see reference 292 for review). These ®ndings, coupled with the observations in vivo that TC II disappeared from plasma as TC II-Cbl was absorbed305 and appeared in lysosomal fractions of hepatic306 and kidney cells,307 led to the proposal that the circulating TC II-Cbl complex is recognized by a speci®c, widely distributed plasma membrane receptor. This hypothesis has been supported by considerable experimental evidence. Using 125Ilabeled TC II-Cbl complexes, Youngdahl-Turner and associates308 showed that the complex binds to a speci®c, high-af®nity (Ka 1010 M 1) cell-surface receptor on cultured skin ®broblasts through a membrane site that recognizes TC II and by a mechanism dependent on Ca2. They showed further that the TC II-Cbl complex is then internalized intact via adsorptive endocytosis309 and that the degradation of TC II and release of Cbl from the complex occur as a result of lysosomal protease activity.308,309 Cbl then exits from the lysosome by a mediated process,310 and is either converted to MeCbl bound to the methionine synthase in the cytosol or enters the mitochondrion, where, after reduction and adenosylation to AdoCbl, it is bound to methylmalonyl CoA mutase.311,312 The intricate process just described is the most widely distributed physiological means by which mammalian cells obtain Cbl, but it is not the only one. Hepatocytes, for instance, contain a surface receptor for asialoglycoproteins, and this receptor interacts with TCI-Cbl (and perhaps TC III-Cbl) complexes, thereby providing a second potential means by which this particular tissue obtains Cbl.313 There is also evidence that at least some tissues are capable of taking up free (unbound) Cbl, if the unbound vitamin is raised to suf®ciently high concentrations. In cultured ®broblasts, this uptake process for free Cbl is saturable, Ca2-independent, and sensitive to inhibitors of protein synthesis and sulfhydryl reagents.314 Its functional role, under most circumstances, is probably negligible. Coenzyme Biosynthesis and Compartmentation Because methylmalonyl CoA mutase, the mammalian enzyme dependent on AdoCbl, is a mitochondrial protein,315 whereas the MeCbl-dependent methionine synthase is cytoplasmic,316 it is important to relate the cellular biology of the vitamin to its cellular and molecular chemistry. The chemical pathway of AdoCbl Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Fig. 155-10 General pathway of the cellular uptake and subcellular compartmentation of cobalamins, and of the intracellular distribution and enzymatic synthesis of cobalamin coenzymes. TC II 5 transcobalamin II; OH-Cbl 5 hydroxocobalamin; MeCbl 5 methylcobalamin; AdoCbl 5 adenosylcobalamin; CblIII, CblII, CblI, 5 cobalamins with cobalt valences of 13, 12, and 11, respectively. synthesis was de®ned initially in bacteria.278,317 Three enzymes are required for coenzyme synthesis, two reductases and an adenosyltransferase. The reductases are ¯avoproteins that require NAD as a cofactor. The ®rst (EC 1.6.99.8) is responsible for converting cob(III)alamin, for example, OH-Cbl, to cob(II)alamin, and the second (EC 1.6.99.9) is responsible for catalyzing the further reduction to cob(I)alamin. The latter compound and ATP are substrates for an adenosyltransferase (EC 2.5.1.17) that completes the synthesis of AdoCbl. Neither of the reductases has been puri®ed extensively, but the adenosyltransferase has. It has a pH optimum of 8, requires Mn2, and has a Km of 110 5 M for cob(I)alamin and 1.610 5 M for ATP.317 The biosynthetic steps leading to de novo MeCbl formation are not as clear. Because maintenance of MeCbl on methionine synthase requires a reductase system to generate cob(I)alamin on methionine synthase itself, this reduction-methylation sequence seems likely for de novo MeCbl synthesis as well.318,319 Accumulated evidence indicates that mammalian cell metabolism of Cbl proceeds by a very similar set of reactions (Fig. 155-10). In 1964, Pawalkiewicz et al.320 showed that human liver and kidney homogenates could convert CN-Cbl to AdoCbl. Several years later, AdoCbl synthesis from OH-Cbl was observed in HeLa cell extracts incubated with ATP and a reducing system that presumably bypassed the enzymatic reduction of OH-Cbl (cob(III)alamin) to cob(I)alamin.321 Subsequently, Mahoney and Rosenberg322 demonstrated the synthesis of both AdoCbl and MeCbl by intact human ®broblasts growing in a tissue culture medium containing OH-[57Co]Cbl. This system was subsequently characterized in cell extracts.323,324 As with the HeLa cell system, chemical reductants were employed to bypass both cobalamin reductases.324 Such extracts synthesized AdoCbl, thereby demon- strating that a homologue of the adenosyltransferase found in bacteria also exists in normal human cells. These experiments also revealed that the adenosyltransferase was mitochondrial in location, implying that both the synthesis and cofactor activity of AdoCbl take place in this organelle. The reductive steps in mammalian systems are still poorly understood. Pezacka and colleagues have suggested that GSCbl may be an intermediate in these reactions.325 Watanabe et al. demonstrated both microsomal and mitochondrial reductase activities,326 but suggested that one or more of these may be nonspeci®c activities of other enzymes, such as the cytochrome b5-cytochrome b5 reductase complex.327 It seems certain, as shown in Fig. 155-10, that MeCbl synthesis takes place in the cytosol in conjunction with the methionine synthase and methionine synthase reductase (see ``Folate'' section above). Metabolic Abnormalities in Cobalamin De®ciency The biochemical abnormalities in plasma and urine of patients with Cbl de®ciency re¯ect the dysfunction of the enzymes dependent on Cbl coenzymes. The ®rst relevant observation in this context was Cox and White328 and Barness and his colleagues,329 demonstration that methylmalonic acid excretion in the urine was distinctly increased in Cbl-de®cient patients with classic pernicious anemia. The methylmalonic aciduria in these patients was reversed rapidly by administration of physiologic doses of Cbl, indicating that repletion of Cbl restored the methylmalonyl CoA mutase reaction to normal. Later, Cox et al. reported that patients with Cbl de®ciency also had distinctly increased amounts of propionic acid in the urine, this abnormality again being reversed by treatment.330 Interestingly, they also found excessive amounts of acetic acid in the urine of Cbl-de®cient subjects. The mechanism leading to this abnormality is not clear 3913 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3914 PART 17 / VITAMINS because acetate does not participate in the major pathway of propionate catabolism. The ®nding could, of course, re¯ect increased utilization of the alternative pathways of propionate metabolism in the face of a block in the major pathway, because each alternative route leads eventually to the formation of acetyl CoA (see Chap. 94). Excessive excretion of homocystine has also been documented in Cbl-de®cient patients,331,332 as has combined methylmalonic aciduria and homocystinuria. Allen and his colleagues examined a large series of patients with suspected or proven Cbl de®ciency333,334 and showed that 95 percent of them have methylmalonic acidemia, homocystinemia, or both, often without hematologic signs. A number of reports now document the occurrence of Cbl de®ciency in vegetarian and macrobiotic communities, with accompanying metabolic derangements.335,336 In some cases, clinical symptoms have been observed as well, particularly in breast-fed offspring of strict vegetarian mothers, who are themselves de®cient in the vitamin.337,338 Cobalamins and Folic Acid An interesting, important, and still puzzling aspect of Cbl function concerns its relationship to folic acid.339 Several lines of evidence bear out this relationship: the appearance of megaloblastic anemia in either Cbl or folate de®ciency; the reversal of megaloblastic anemia in Cbl de®ciency by large doses of folate; the amelioration of megaloblastic changes in folic acid de®ciency by pharmacologic doses of CN-Cbl; the increased plasma concentrations of methyl-H4Folate in patients with cobalamin de®ciency; the excretion of excessive amounts of FIGLU after histidine loading in patients with either Cbl or folate de®ciency; and the reduced amounts of total Cbl in the liver of patients with folate de®ciency. A plausible explanation for most of these effects was proposed independently by Herbert,340 Noronha,341 and Larrabee342 and their colleagues, and has been referred to as the folate trap hypothesis. This thesis rests on the evidence that the conversion of methyl-H4Folate to H4Folate depends on the MeCbl-dependent reaction in which homocysteine is methylated to methionine. If methionine biosynthesis is the only quantitatively signi®cant reaction using methyl-H4Folate, Cbl de®ciency will interfere with the folate cycle and, barring other control mechanisms, will lead to the accumulation of methyl-H4Folate and the depletion of other folate derivatives. This depletion could become severe enough to interfere with other reactions requiring H4Folate, such as the synthesis of purines or pyrimidines and the conversion of FIGLU to glutamate. Under these circumstances, H4Folate de®ciency could be relieved by administration of either folates or Cbl, but only the latter would complete the folate cycle. This scheme, if totally correct, would obviate the need for additional Cbldependent mechanisms to explain the megaloblastic changes observed in Cbl de®ciency and would account for the speci®c disorders of folate metabolism observed in Cbl-de®cient human beings. It does not explain the low Cbl content of livers from folate-de®cient subjects or the hematologic response of folate-de®cient patients to Cbl (also see ``Folate'' section above). INBORN ERRORS OF COBALAMIN TRANSPORT AND METABOLISM Inherited disorders in the transport and metabolism of Cbl manifest themselves clinically in ways that re¯ect the underlying defect and, in particular, that depend on which coenzyme is de®cient and, hence, which of the two Cbl-dependent enzymes is reduced in activity (see Fig. 155-8). Defects that affect only AdoCbl biosynthesis generally lead to metabolic ketoacidosis in the newborn or infant period, and regularly result in methylmalonic acidemia and methylmalonic aciduria. MeCbl de®ciencies present as failure to thrive, megaloblastic changes, and neurologic signs, usually with homocystinuria and hypomethioninemia. De®ciencies of both coenzymes produce a variable combination of these signs and symptoms. Combined AdoCbl and MeCbl De®ciency Cbl was ®rst described as ``extrinsic factor,'' an antipernicious anemia factor found in aqueous extracts of raw liver, which combined with ``intrinsic factor'' (IF), a component of normal gastric secretions, to cure pernicious anemia, an acquired disease resulting from gastric insuf®ciency. It is recognized, however, that there are several inborn errors of metabolism with presentations similar to pernicious anemia that result from abnormal Cbl transport or from altered cellular Cbl metabolism. Although these diseases share the general clinical phenotype of failure to thrive, developmental delay, neurologic dysfunction, and megaloblastic anemia, the details of their presentations allow them to be differentiated. (For reviews, see references 236, 269, 292, 302, and 343). Transport Defects: Clinical and Laboratory Findings Food Cobalamin Malabsorption. Carmel and colleagues described a number of patients, mostly adults, with a condition that includes low serum Cbl concentrations in the face of a normal Schilling test.344 Neurologic manifestations, with or without megaloblastic changes, appear to be common.344 These individuals suffer from an inability to release Cbl from the proteinbound state in which it is normally encountered in foodstuffs, usually measured by the absorption of Cbl bound to egg yolk.345 Because this process requires both an acid gastric pH and peptic activity,346 any underlying factors that compromise gastric function, including atrophic gastritis or partial gastrectomy, can result in this disorder.347 However, a signi®cant fraction of patients have shown no evidence of impaired gastric function,344 suggesting that a more subtle mechanism, whose nature is currently unknown, may be responsible.348 Intrinsic Factor (IF) De®ciency. A number of children have been described with a juvenile form of pernicious anemia (see reference 349 for references to case reports). The clinical symptoms, which usually appear after the ®rst year and before the ®fth year of age, include developmental delay and the megaloblastic anemia characteristic of pernicious anemia.236,349 Serum levels of Cbl are markedly de®cient, but, in contrast to the adult disease, gastric function and morphology are normal, and serum autoantibodies to IF are absent. Cbl absorption is abnormal in these children, but is restored when the vitamin is mixed with normal human gastric juice as a source of IF. Further investigations of the gastric secretions of these patients have shown that, as expected, they suffer from one of several different classes of functional IF de®ciency. One results in failure to produce or secrete any immunologically recognizable IF,350,351 while another causes production of immunologically reactive protein that is inactive physiologically.349,352±354 The latter group includes patients whose IF has reduced af®nity for the ileal IF receptor,353 reduced af®nity for Cbl,355 or increased susceptibility to proteolysis.349 In a few cases with partial de®ciency, presentation was delayed into the second decade or later.352,356 Although a cDNA for human intrinsic factor has been characterized ([GIF], NM_005142, MIM 261000), no mutations have yet been described.357 Enterocyte Cbl Malabsorption (Selective Vitamin B12 Malabsorption, MGA1, Imerslund-GraÈsbeck Syndrome) (MIM 261100). More than 250 cases of a related disorder have been described with the clinical signs of juvenile pernicious anemia, but with normal IF and normal gastrointestinal function, except for speci®c intestinal Cbl malabsorption.358± 361 In addition to megaloblastic anemia and serum Cbl de®ciency, many of these patients have proteinuria. Similarly to IF de®ciency, patients usually present between 1 and 5 years of age, although some have been diagnosed much later.362 In contrast to IF de®ciency patients, however, these children's Cbl absorption defect is not corrected by providing normal human IF with the vitamin.236 There has been a decrease in the number of new cases in recent years, and it has Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM been suggested that there may be dietary or other factors modifying expression.363 Most patients are found in Norway, Finland, Saudi Arabia, and among Sephardic Jews. Using microsatellite markers in Finnish and Norwegian families, the disease gene was mapped to a 6-cM region on chromosome 10p12.1.363 An IF-Cbl-binding protein called cubilin ([CUBN], NM_001081, MIM 602997) was puri®ed from renal proximal tubule, cloned, and localized to the same region.364,365 This large (400 kDa) protein comprises a short N-terminal domain, eight EGF repeats, and 27 contiguous, 110amino acid CUB domains, ®rst identi®ed in certain developmental control proteins. Two mutations in the cubilin gene (CUBN) have been found segregating in Finnish families with this disorder.366 One, P1297L, is found in homozygous form in most Finnish patients (31 of 34 alleles). It changes a highly conserved proline residue, which is predicted by x-ray structural analysis of other CUB domains367 to be part of a ligand-binding site. Interestingly, this change is in CUB domain 8, suggested by deletion and expression studies to be part of the IF-Cbl binding site (domains 5 to 8).368 The other change, homozygous in one patient, is a point mutation in an intron in CUB domain 6, resulting in missplicing, insertions containing stop codons in mRNA, and truncated predicted gene products. No cubilin was detected on western blots of proteins from this patient's urine, establishing clearly that CUBN is the gene for the IF-Cbl receptor protein.368 Neither of these mutations has been found in non-Finnish families, and the mutations causing this disorder in other populations are being sought. Cubilin is a peripheral membrane protein with no clearly de®ned transmembrane domain. It copuri®es with megalin, an even larger receptor protein of the LDL receptor gene family, and colocalizes with it in both intestinal and renal epithelium.364,369 Megalin appears to be a transporter for many proteins370 and may be responsible for the endocytosis of the cubilin-IF-Cbl complex. Cubilin itself may have other functions, as suggested by its speci®c binding of receptor-associated protein368 and its function in apolipoprotein A-I endocytosis.371 Earlier experiments suggested that, in at least some patients, the ileal receptor for IF-Cbl was normal as measured by IF-Cbl binding to homogenates of ileal biopsy specimens.360 In others, a functional receptor appeared to be absent.372 Because the mechanism for Cbl transport across the enterocyte is complex, it seems possible that this syndrome encompasses defects at several points in this overall pathway, including cubilin itself (as shown in the Finnish pedigrees), cubilin internalization via megalin, or Cbl transfer to TC II within the enterocyte (also see discussion of cblF below). It remains to be seen how many patients with this phenotype will be found to have mutations in the cubilin gene itself. Transcobalamin II (TC II) De®ciency. At least 36 cases have been described of de®ciency of TC II, including both twins and sibs.236,373 In contrast to the previous two disorders, TC II de®ciency has generally presented within the ®rst or second month of life as failure to thrive, with such nonspeci®c symptoms as vomiting and weakness, accompanied by megaloblastic anemia and, eventually, immunologic de®ciency and neurologic disease.236,373 Because the patients may have immature white blood cell precursors in a marrow that is otherwise hypocellular, they have been misdiagnosed with leukemia. Neurologic disease is not present at the time of diagnosis, but may develop with an extended duration of illness, inadequate cobalamin treatment, or treatment of the anemia with folates and not cobalamin.373 Interestingly, serum Cbl levels are normal or nearly so in these patients, re¯ecting the fact that most serum Cbl is carried by TC I and other R-binders.302 It is essential to measure blood levels of TC II before the patient has been started on cobalamin therapy. Intestinal Cbl absorption has been abnormal in some patients, but not in others.236 Most patients have had no immunologically detectable TC II in plasma,236 although a few had detectable protein,374,375 and at least one produced a TC II that was able to bind Cbl, although apparently without function.374 In those patients who do not synthesize TC II, both the diagnosis and prenatal diagnosis can be performed by studying the ability of cultured ®broblasts or amniocytes to synthesize TC II.376 Treatment of TC II de®ciency requires that serum cobalamin levels be kept very high. It is important to monitor levels carefully and to ensure that the patient is compliant, particularly if oral treatment is used. Serum levels ranging from 1000 to 10,000 pg/ ml have been required; these levels have been achieved with oral OH-Cbl or CN-Cbl in doses of 0.5 to 1.0 mg twice weekly or by weekly doses of 1 mg OH-Cbl. Although folic acid or folinic acid can reverse the megaloblastic anemia, folate should never be given as the only therapy because of the danger of hematologic relapse and neurologic deterioration. The human TC II gene is on chromosome 22, a cDNA has been cloned, and the molecular basis of some variants determined ([TCN2], NM_000355, MIM 275350).377± 380 In a number of patients who have no TC II synthesis, deletions and nonsense mutations have been found.381,382 R-Binder De®ciency ([TC I], NM_001062, MIM 193090). Several individuals are known who have de®cient or absent R-binder (TC I) in plasma, saliva, and blood cells.236,383 Although these patients have serum Cbl values in the de®cient range, they show no signs of Cbl de®ciency, probably because their TC II-Cbl levels are normal. Although several of these patients have had a myelopathy not attributable to other causes,383,384 the etiology of these symptoms remains unclear, emphasizing our lack of understanding of the role of R-binders in normal Cbl metabolism and homeostasis. It should be noted that R-binders carry Cbl in mother's milk, so the potential exists for Cbl de®ciency in breastfed infants of mothers with this de®ciency. Chemical Abnormalities and Pathophysiology. While megaloblastic anemia is the hallmark of the Cbl transport disorders, the chemical abnormalities expected to accompany functional Cbl de®ciency have also been found in many cases. In theory, Cbl de®ciency should lead to de®cient synthesis of both AdoCbl and MeCbl and, thus, to decreased activities of their respective enzymes, resulting in methylmalonic acidemia(-uria) and homocystinemia(-uria). When examined carefully, most patients with each of the transport de®ciencies have these chemical symptoms, although the quantities of both methylmalonate and homocystine excreted have generally been much lower than in patients with abnormalities in cellular Cbl metabolism (see below). On the other hand, some patients do not have one or either of these chemical abnormalities.236 To a certain extent, these variable ®ndings, which do not appear to correlate well with the nature of the defect or the severity of the general symptoms and hematologic aberrations, may result from the fact that alternative pathways of Cbl transport exist that, although minor in normal individuals, may contribute signi®cantly in patients with these transport defects. For example, receptors for free Cbl have been found on HeLa cells,385 human ®broblasts,314 and adenocarcinoma cells,299 and may permit some Cbl transport even in the absence of one of the transport proteins. In addition, hepatocytes may be able to recover some Cbl from asialo-TC I-Cbl by means of the asialoglycoprotein receptor system.386 This could be particularly important in TC II de®ciency. One major clinical difference between the two intestinal transport defects and TC II de®ciency lies in the different age of onset of these conditions. While neither intestinal Cbl transport de®ciency manifests itself before 1 year of age, many TC IIde®cient patients are symptomatic within 1 or 2 months of birth, with some exceptions (see above). This appears to be due to two factors. First, the IF-dependent pathway for intestinal Cbl absorption may not become important until later in infancy, when the gastrointestinal tract switches from pinocytotic mechanisms of transport to receptor-mediated ones. IF-Cbl transport falls 3915 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3916 PART 17 / VITAMINS into the latter category. Interestingly, one report387 demonstrates that, in rats, expression of IF by the gastric mucosa increases abruptly from the low levels found in the newborn animal to adult levels at about the time of weaning (13 to 20 days), consistent with this hypothesis. Second, the body stores considerable amounts of Cbl beyond daily requirements in blood, liver, and other tissues. Thus, IF-de®cient patients and those with Imerslund-GraÈsbeck syndrome likely show no signs of de®ciency early in infancy both because the IF-dependent mechanism for Cbl transport is not yet operating and because they have acquired suf®cient Cbl through other mechanisms or prenatally to sustain themselves for a time after the developmental switch in intestinal absorption has occurred. Conversely, because TC II is presumably necessary for ef®cient Cbl transport into cells regardless of the mechanism by which it is acquired, TC II-de®cient patients have no symptomfree period and become ill as soon as their maternally derived stores of Cbl are exhausted. The observation that most TC IIde®cient patients have been normal at birth likely re¯ects the fact that fetal tissues concentrate Cbl relative to the maternal serum.388 It is not certain why the neurologic manifestations of TC II de®ciency are less severe than those found in the inborn errors of cobalamin metabolism that affect cofactor synthesis.373 The megaloblastic anemia characteristic of these disorders likely re¯ects a de®ciency in the activity of methionine synthase brought about by the absence of its cofactor, MeCbl. Because patients with isolated de®ciency of AdoCbl (see below) or its partner enzyme, methylmalonyl CoA mutase (see Chap. 94), are usually hematologically normal, this conclusion seems to be solid. Likewise, the severe neurologic manifestations,373 particularly in patients who are diagnosed long after the onset of their disease, appear more likely to be due to de®cient methionine synthase activity. Although isolated mutase de®ciency can produce central nervous system dysfunction (see Chap. 94), this is believed to be at least partially a consequence of the severe metabolic ketoacidosis experienced by these patients, a condition not generally present in patients with Cbl transport defects. The speci®c etiology of the hematologic and neurologic disturbances in these individuals is not completely understood, but clearly it must derive from the central role of methionine synthase in cellular 1-carbon metabolic pathways, both in terms of folate metabolism (see sections above) and homocysteinemethionine balance (see Chap. 88). Because the folate cycle in mammalian cells requires that methyl-H4Folate transfer its methyl group to homocysteine, via MeCbl, in order to regenerate tetrahydrofolate, it has been suggested that the accumulation of methyl folate in the absence of the Cbl coenzyme serves as a folate trap, which produces functional folate de®ciency intracellularly.340,342 The effects of this de®ciency on the important roles that folate metabolism plays in the synthesis of nucleotides and, hence, of RNA and DNA could easily account for the general disruptions in cellular homeostasis in rapidly dividing tissues, such as the hematopoietic system. Whether this folate trap hypothesis is equally applicable to explaining the neurologic dysfunction in these patients is not clear. An alternative explanation might involve disruption of the interconversion of homocysteine and methionine and of S-adenosylhomocysteine and S-adenosylmethionine and interference with the role these compounds play in methylation and enzyme regulation in the central nervous system.270,389 Until more is known about these pathways, however, either hypothesis will be dif®cult to establish. Genetics and Molecular Biology. Each of the genetic lesions in Cbl transport appears to be inherited as an autosomal recessive trait on the basis of classic genetic criteria.236 Because the Imerslund-GraÈsbeck syndrome may actually encompass de®ciencies in more than one protein (receptor) (see above), it remains possible that other modes of inheritance exist for a subset of families with this disorder. Rat and human IF cDNAs have been cloned,294,390 and the IF gene has been localized to human chromosome 11.294 There is suggestive evidence from biochem- ical analysis of some presumptive heterozygotes for IF de®ciency that they express both a normal and an abnormal allele for IF and for the idea that some patients with IF de®ciency express two different mutant alleles.349 The rat IF cDNA has been used to establish that only one cell type Ð the chief cell of the rat gastric mucosa Ð expresses IF in the adult animal,387 in keeping with immunochemical studies that indicate that the parietal cell is the only source of IF in human beings and other mammals.291 As mentioned above, both cubilin and megalin have been cloned, and deleterious mutations in cubilin have been described in Finnish Imerslund-GraÈsbeck patients. TC II has electrophoretic isoforms in normal individuals,391 and it has been suggested that some cases of TC II de®ciency manifest themselves as abnormal isoforms.392 The structural locus for TC II is on the long arm of chromosome 22,294,393 linked to the P blood group system locus. Although TC I has been cloned,394 mutations in it leading to R-binder de®ciency have not been described. Diagnosis and Treatment. The Cbl transport de®ciencies, except for food Cbl malabsorption, are usually diagnosed initially by the observation of the combination of macrocytic anemia with developmental delay or failure to thrive.236 Neurologic symptoms may be present at later times.373 Serum Cbl levels are low in food Cbl malabsorption, IF de®ciency, and Imerslund-GraÈsbeck syndrome, but usually normal in TC II de®ciency. Schilling tests are normal in the ®rst disorder, abnormal in the second two, and may also be abnormal in some cases of TC II de®ciency.236 The second two disorders can be differentiated by determining whether the Schilling test becomes normal when the test Cbl is incubated with normal human IF before it is administered. Only IF de®ciency patients show correction. Con®rmation of either of the intestinal malabsorption defects entails demonstration of normal gastric and ileal function other than the speci®c Cbl absorption de®ciency, the absence of antibodies to IF, and, in some cases of IF de®ciency, the absence of functional (i.e., Cbl-binding) or immunologically cross-reacting IF in the patient's gastric secretions.236,349 TC II de®ciency can sometimes be differentiated from the other two by an age of onset within the ®rst months (as opposed to years) of life. The diagnosis can often be established by measuring the unsaturated Cbl-binding capacity of the patient's serum; in normal individuals, this largely re¯ects the amount of TC II present. Gel®ltration chromatography can be used to separate TC II from serum R-binders and thus provides a more accurate assessment of Cbl-binding capacity. Unfortunately, both these tests can be compromised by previous Cbl therapy, possibly even by previous Schilling tests.395 Because TC II is synthesized by many cell types, including ®broblasts, and because ®broblasts from TC II-de®cient patients synthesize a defective protein or none at all,314 a more satisfactory approach may be to grow patient ®broblasts in medium without TC II and to then determine whether any functional TC II has been synthesized by incubating the cells with radiolabeled Cbl and measuring the extent to which TC II-Cbl accumulates in the medium or in the cells.314,376 In the case of sibs or other relatives in families in which one of these defects has been diagnosed, hematologic changes can provide an early sign of the presence of the disease, as can methylmalonic acidemia and homocystinemia. In the two intestinal transport disorders, Schilling tests may prove abnormal before the onset of clinical symptoms. In TC II de®ciency, because cord blood contains fetal, not maternal, TC II,396 it is possible to test immediately for the presence of functional TC II. Biochemical prenatal diagnosis is possible only for TC II de®ciency, based on the ability of normal amniocytes to synthesize functional TC II.376 Because no fetus at risk for TC II de®ciency has yet been tested by this method and predicted to be affected, its applicability remains hypothetical. The two intestinal malabsorption syndromes are not expressed in accessible fetal tissues (if, in fact, those proteins are expressed at all during fetal life) and, thus, cannot be diagnosed prenatally by a biochemical test. The cDNA cloning of IF294 and cubilin364,365 may make a DNA-based diagnostic procedure, such Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Table 155-3 Clinical and Laboratory Features of Patients with Methylmalonic Acidemia and Homocystinuria* Mutant Class Finding Clinical Sex (male/female) Failure to thrive Developmental retardation Seizures Feeding dif®culties Hypotonia Microcephaly Nystagmus Hydrocephalus Dementia Myelopathy Laboratory Normal serum cobalamin Hematologic abnormalities Acidosis Microthrombi Pigmentary retinopathy Decreased visual acuity cblC Early Onset cblC Late Onset 21/44 26/44 21/44 32/44 27/44 19/44 10/44 8/44 1/44 1/44 1/6 2/6 2/6 3/6 3/6 0/6 0/6 0/6 3/6 3/6 44/44 31/44 9/44 3/44 19/44 15/44 6/6 3/6 0/6 0/6 1/6 0/6 cblD cblF 2/0 0/2 1/2 0/2 0/2 2/4 5/6 5/6 1/6 3/6 3/6 2/2 0/2 0/6 1/2 3/6 3/6 *Ratios (except for sex) denote the number of patients showing a particular finding/total number of patients in each mutant class; empty cells indicate data were not available. S O U RC E : Information obtained from published surveys,420 case reports,413,417±419,425 and personal communications. as RFLP analysis, possible in at least some cases involving de®ciency of these proteins. The major treatment regimen for each of these disorders has been pharmacologic doses of Cbl, either CN-Cbl or OH-Cbl, usually administered by injection to avoid dependence on gastric factors and ileal uptake of free Cbl.236,395 Titration of the dosage used and frequency of therapy should be carried out to ensure resolution of all clinical abnormalities, particularly in TC IIde®cient patients with defective immune system function397,398 or neurologic disorders.373 The serum Cbl concentration at which patients become asymptomatic has varied widely, especially in TC II de®ciency, and should be used as a guide only after the patient has been stabilized. Folate has been administered to some TC IIde®cient patients with effective correction of the hematologic signs of the disease.236 At least one of these patients suffered a relapse, however, and the ability of folate to resolve other symptoms, particularly long-term neurologic dysfunction, has not been determined. Consequently, folate therapy should only accompany effective doses of Cbl.373 The prognosis in these diseases appears to be generally very good as long as serum Cbl levels are maintained appropriately.373,399 Interestingly, while Cbl therapy has been effective in normalizing the hematologic signs in Imerslund-GraÈsbeck patients, the proteinuria often observed in this disorder has been unchanged even by many years of therapy.399 Although one woman with TC II de®ciency has borne two normal children,375 it remains unclear whether Cbl therapy can achieve complete reversal of the neurologic damage and developmental delay that occur if patients remain undiagnosed for an extended period (or if adequate Cbl levels are not maintained) or whether some residual de®cit may persist in these cases.373 Defects in Cellular AdoCbl and MeCbl Synthesis Clinical and Laboratory Findings. In comparison to the Cbl transport defects described above, defects in the cellular metabolism of Cbl generally result in clinically more severe metabolic disease. As a consequence, patients with these disorders regularly show the metabolic disturbances that result from de®cient synthesis of both AdoCbl and MeCbl, namely methylmalonic acidemia and homocystinuria (Fig. 155-8). Because the amounts of these metabolites detected in these patients generally greatly exceed those found in patients with Cbl transport defects or Cbl de®ciency, their measurement has served to distinguish these groups of individuals clinically. We are aware of over 100 patients with inherited combined methylmalonic acidemia and homocystinuria. Many of the early patients were the subjects of individual case reports.400± 413 Cells from these children comprise three biochemically and genetically distinct complementation groups, designated cblC (MIM 277400), cblD (MIM 277410), and cblF (MIM 277380).323,414± 416 The cblC group is by far the largest (more than 100 patients), with the cblD represented by 2 sibs400 and the cblF group by 6 unrelated individuals.413,417±419 cblC. Clinical ®ndings have varied widely among patients in the cblC group; Table 155-3 presents a summary of the clinical and laboratory data from a survey of 50 such patients.420 Most of the early described patients presented in the ®rst few months of life because of failure to thrive, poor feeding, or lethargy. Subsequent reports have emphasized that some patients have a much delayed onset of symptoms: for example, a 4-year-old with fatigue, delirium, and spasticity,406 and a 14-year-old with the rather sudden onset of dementia and myelopathy.405 Thus, regardless of age, neurologic manifestations have been prominent. Most, but not all, of these patients have had hematologic abnormalities characterized by megaloblastoid and macrocytic anemia; hypersegmented polymorphonuclear leukocytes and thrombocytopenia have been observed less often. Several patients have had features of hemolytic-uremic syndrome.421 A few patients have had a characteristic pigmentary retinopathy with perimacular degeneration, as well as other ophthalmologic changes.404,406,409,412 Hydrocephalus, cor pulmonale, and other congenital malformations also have been seen.422± 424 Moderate to severe developmental delay has been common in the early onset patients, and about a third of early onset patients have died despite treatment.420 3917 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3918 PART 17 / VITAMINS In addition to the methylmalonic aciduria and homocystinuria that characterize this group of patients, they have hypomethioninemia and cystathioninuria. The methylmalonic aciduria in these children is distinctly less severe than that encountered in children with isolated mutase de®ciency (see Chap. 94), although much more severe than that reported for patients with Cbl transport defects. Moreover, neither hyperglycinemia nor hyperammonemia has been reported in any of the cblC (or cblD or cblF ) patients. Serum cobalamin and folate concentrations have been normal. cblD. In sharp contrast with this description, neither of the brothers in the cblD group400 had any clinically signi®cant problems until much later in life. The older brother came to medical attention because of severe behavioral pathology and moderate mental retardation at 14 years of age. He had, as well, a poorly de®ned neuromuscular problem involving his lower extremities. His then 2-year-old brother was asymptomatic, although biochemically affected. No hematologic abnormalities have been noted in either sib. cblF. There have been six unrelated patients reported in the cblF group.413,417± 419,425 The ®rst two girls presented during the ®rst weeks of life with stomatitis and hypotonia, together with minor facial anomalies. The ®rst patient, who was not diagnosed until 8 months, developed poorly and was clearly delayed.413 No hematologic abnormalities were found in the ®rst patient, while the second showed macrocytosis and hypersegmented polymorphonuclear neutrophils.425 The second patient died suddenly at home. Other cblF patients have had pancytopenia, neutropenia, or thrombocytopenia.417,418 Although the ®rst patient had no detectable homocystinuria despite cellular de®cits in methionine synthase activity and in MeCbl synthesis, all the others have shown both methylmalonic aciduria and homocystinuria. A male, diagnosed at 11 years of age, had a grossly abnormal Schilling test and low serum Cbl.417 He had recurrent stomatitis in infancy, arthritis at age 4 years, and confusion and disorientation at age 10 years. He also had a pigmentary skin abnormality. Localization of Cellular Metabolism Defects cblC and cblD. It is clear that patients in the cblC and cblD groups have a defect in cellular metabolism of Cbl based on several criteria: total Cbl content of liver, kidney, and cultured ®broblasts is markedly reduced;401,426±428 the ability of cultured cells to retain [57Co]-labeled CN-Cbl429 or to convert [57Co]labeled CN-Cbl or OH-Cbl to AdoCbl and MeCbl is markedly impaired;430 activities of methylmalonyl CoA mutase and methionine synthase in cultured cells are de®cient, such de®ciency being partially reversed by supplementation of the growth medium with OH-Cbl;415,431,432 and the mutase and the methionine synthase apoenzymes in cells from affected patients appear to be normal.84,400,415,431 Because these mutant cells demonstrate normal receptormediated adsorptive endocytosis of the TC II-Cbl complex and normal intralysosomal hydrolysis of TC II,274,309,415,433 perusal of Fig. 155-10 makes it clear that the defects in the cblC and cblD cells must affect some step or steps subsequent to cellular uptake, common to the synthesis of both coenzymes, and prior to the binding of the Cbl coenzymes to their respective apoproteins. Signi®cantly, cblC (and, to a lesser extent, cblD) cells use CN-Cbl less well than OH-Cbl432,434 and are unable to convert CN-Cbl to OH-Cbl, a step shown in normal cells to be a metabolic prerequisite for the synthesis of both AdoCbl and MeCbl.434 The latter results have been interpreted as evidence for a defect in a cytosolic cob(III)alamin reductase, which is required for reducing the trivalent cobalt prior to alkylation.434 Partial de®ciencies of CN-Cbl b-ligand transferase and of microsomal cob(III)alamin reductase have been described in cblC and cblD ®broblasts.435,436 A partial de®ciency of mitochondrial NADH-linked aquacobalamin reductase was described by Watanabe and his coworkers in two cblC ®broblast extracts.437 The suggestion by Pezacka and her colleagues that GSCbl may be the product of an intermediate step in this process325 provides another potential site for the mutation in one of these groups. Finally, it should be mentioned that the distinction between the cblC and cblD classes is based ®rst and foremost on complementation studies that de®ne the two classes as unique.415 Their biochemical differences appear to be quantitative rather than qualitative, with the cblC group having more severe metabolic derangements than the siblings designated cblD. Therefore, it remains possible that the cblD mutation is allelic to cblC and shows interallelic complementation. cblF. Studies using cultured ®broblasts from two patients in the cblF group413,416,425 are of particular interest. As with cells from cblC and cblD patients, both mutase and methionine synthase activities were impaired, and AdoCbl and MeCbl contents were reduced. In contrast to the cblC and cblD mutants, however, the cblF cells accumulated unmetabolized, nonprotein-bound CN-Cbl in lysosomes.438,439 These ®ndings indicate that cblF cells are de®cient in the mediated process by which Cbl exits from lysosomes after being taken up by receptor-mediated endocytosis.310 Two brief reports further indicate that two cblF patients had abnormal Schilling tests with both free and IF-bound Cbl,417,440 suggesting that the putative lysosomal defect affects ileal Cbl transcytosis as well (see Cbl transport section above). Pathophysiology. The megaloblastic anemia so commonly observed in the cblC patients almost surely re¯ects the disturbance of methionine synthase activity. This can be stated with some assurance because patients with isolated methylmalonyl CoA mutase de®ciency (see Chap. 94) more severe than that encountered in the cblC patients exhibit no megaloblastic anemia. The early and severe central nervous system abnormalities encountered in the cblC group probably re¯ect the methionine synthase abnormality as well, in that such patients generally do not experience the severe metabolic ketoacidosis that probably accounts for the central nervous system problems in patients with mutase de®ciency only. Thus, patients with severe, inherited dysfunction in the synthesis of both Cbl coenzymes resemble closely patients with exogenous Cbl de®ciency Ð both groups having prominent hematologic and neurologic manifestations resulting from the blocked methionine synthase system. Genetic Considerations. Because equal numbers of affected males and affected females exist in the cblC group, because females have been as seriously affected as males, and because cells from affected patients behave as recessives in complementation studies,414 it seems safe to predict that this disorder is inherited as an autosomal recessive trait. The mode of inheritance of the cblD and the cblF mutations cannot yet be de®ned, because of the paucity of known patients (both affected cblD patients in the only family yet described are male); both males and females have been identi®ed in the cblF group. Identi®cation of heterozygotes for the cblC, cblD, or cblF group has not yet been accomplished. Diagnosis, Treatment, and Prognosis. The combination of methylmalonic aciduria and homocystinuria with normal serum Cbl concentrations and normal TC II is the set of biochemical parameters needed to distinguish patients in the cblC, cblD, and, probably, cblF groups from those with methylmalonic acidemia caused by isolated methylmalonyl CoA mutase de®ciency (see Chap. 94); from those with homocystinuria due to cystathionine synthase de®ciency (see Chap. 88), or methylene-H4Folate reductase de®ciency, or isolated methionine synthase de®ciency (see ``Folate'' section above); and from those with Cbl transport defects or exogenous Cbl de®ciency (see above). It should be noted that one cblF patient had low serum Cbl when diagnosed.417 Because each of the cellular metabolic defects is expressed in cultured cells from affected individuals, the diagnosis should be con®rmed by genetic complementation analysis between patient ®broblasts and ®broblasts from patients whose complementation Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Table 155-4 Salient Biochemical Features of Cultured Fibroblasts from Patients with Various Defects in Cellular Cbl Metabolism* Mutant Class Biochemical Parameter Studies with intact cells [14C]propionate oxidation [14C]Methyl-H4Folate ®xation MeCbl synthesis AdoCbl synthesis Conversion of CN-Cbl to OH-Cbl Lysosomal ef¯ux of free Cbl Enzyme activities in cell extracts{ Mutase holoenzyme Mutase total enzyme Methionine synthase holoenzyme Methionine synthase total enzyme Methionine synthase reductase Cob(I)alamin adenosyltransferase cblA cblB cblC cblD cblE cblF cblG ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± nt nt ** ** ± nt nt nt ± nt nt ± ± nt * normal; ± markedly deficient or undetectable; partially deficient; nt not tested. {Holoenzyme is defined as that enzyme activity measured in the absence of added cofactor; total enzyme is that activity measured in the presence of saturating concentrations of cofactor. **Activity dependent on reducing conditions used (see folate section). Abbreviations: Methyl-H4Folate N 5-methyltetrahydrofolate; MeCbl methylcobalamin; AdoCbl adenosylcobalamin; CN-Cbl cyanocobalamin; OH-Cbl hydroxocobalamin. groups have been determined previously. This technique also allows the cblC, cblD, and cblF groups to be distinguished from each other. Biochemical studies on cultured cells, such as Cbl uptake, lysosomal Cbl ef¯ux, or AdoCbl and MeCbl synthesis, and direct measurement of mutase and methionine synthase activities in cell extracts can be performed to provide further con®rmation (see Table 155-4). Because normal amniotic ¯uid cells appear to carry out all the steps of Cbl metabolism observed in cultured ®broblasts, it is be possible to detect each of these defects prenatally by assaying any of these parameters in cultured amniocytes. This has been carried out successfully in both the cblC and cblF269 groups. The distinctions between these cellular metabolic defects and other related conditions are critically important, because appropriate therapy and prognosis depend on them. Whereas exogenous Cbl de®ciency responds dramatically to physiologic amounts of Cbl and transport defects to somewhat larger dosages, successful management of cblC, cblD, and cblF demands the administration of large amounts of OH-Cbl (up to 1 mg daily) by intramuscular injection.195,400,402,404,406,417,440 Such treatment has resulted in dramatic decreases in urinary methylmalonate and in less dramatic, but signi®cant, decreases in urinary homocystine in many patients who have received it. The form of Cbl administered is important, at least in cblC patients, because studies on cultured cells from this group have shown that supplementation in culture is much less ef®cient with CN-Cbl than with OH-Cbl in eliciting an increase in the activity of the affected enzymes.438 A recently published study of the effects of the chemical form of Cbl administered to two cblC patients supports the greater ef®cacy of OH-Cbl, both biochemically and clinically.441 A number of adjunctive therapies have been employed for cblC patients with variable success, including moderate protein restriction to reduce the load of metabolic end products and, hence, the amount of methylmalonate produced; carnitine supplementation, to improve organic acid excretion and relieve a postulated functional carnitine de®ciency (see Chap. 94); folic and folinic acid administration, to bypass the so-called methylfolate trap and restore hematologic function; and betaine administration, to provide substrate for betaine:homocysteine methyltransferase, which is not dependent on a Cbl coenzyme, and thus to return the serum methionine:homocysteine ratio toward normal. Few investigators have evaluated the ef®cacy of these treatments critically, however. Bartholomew and his colleagues attempted to determine the effects of OH-Cbl dosage schedule and of treatment with carnitine, folinic acid, and betaine on the clinical and biochemical status of two patients with the cblC defect.442 In each case, the OH-Cbl injection schedule could be titrated to control the patient's methylmalonic acidemia and homocystinuria. In addition, betaine administration (250 mg/ kg/day) appeared to act synergistically with the OH-Cbl to produce a further reduction in plasma homocystine. No speci®c clinical improvement accompanied the betaine therapy, however. Neither patient responded clinically or biochemically to folinic acid or carnitine treatment. The overall result in both patients was good metabolic control, as measured by reduced methylmalonic acidemia and normal serum homocysteine and methionine concentrations, and resolution of most of their clinical symptoms, such as lethargy, irritability, vomiting, and failure to thrive, with a treatment regimen of daily betaine administration and biweekly injections of OH-Cbl. Signi®cantly, both patients remained somewhat delayed developmentally, even after a year or more of therapy. In addition, the retinal degeneration present in these patients was not reversed by the therapy, although some improvement in cone response was noted in one of them. This report serves also to emphasize that early diagnosis and prompt institution of therapy with OH-Cbl (and possibly betaine) may be the only way to change the outcome of these patients, which, at least in the case of the cblC group, is dismal thus far (Fig. 155-11). Many have died despite intensive therapy. Severe hemolytic anemia is a major complication in the deceased cblC patients, as has congestive heart failure. Thromboemboli, so often encountered in patients with homocystinuria due to cystathionine synthase de®ciency, have, thus far, been documented in only a few cblC patients420 and in the older of the two cblD brothers, in whom this complication was not noted until he reached 18 years of age. Betaine treatment may reduce this risk by normalizing the serum methionine/homocysteine ratio, even when Cbl-responsiveness is incomplete.442 Surviving patients, even those under apparently good metabolic control, continue to show signs of neurologic dysfunction, including mild to moderate mental retardation and delayed development of motor skills,236,420 and, in some cases, the continued presence of abnormal ophthalmologic ®ndings.442 These problems could be the result of irreversible damage that 3919 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3920 PART 17 / VITAMINS Fig. 155-11 Outcome in cblC patients based on age-of-onset less than 1 year (early onset, shaded bars) or greater than 1 year (late onset; solid bars). Late onset patients presented between ages 4 and 14 years. Impairment is classi®ed in functional terms as severe, moderate, or mild according to the expectation for age. (Reproduced from Rosenblatt et al.420 Used with permission.) occurred prior to diagnosis and therapeutic intervention, or could re¯ect the impossibility of completely correcting the cellular lesion in Cbl metabolism in certain cells whose function is critical in neurologic development. Signi®cantly, patients with apparently later onset or milder disease have done considerably better (Fig. 155-11). Until a number of patients with these defects are diagnosed before birth or soon thereafter, and treated immediately, or even prenatally, with Cbl and betaine supplements, we will not know whether the poor outcome in this group can be modi®ed signi®cantly. Documentation of such experience is particularly important in assessing the clinician's ability to modify the natural history of these disorders. Clinical and Laboratory Presentation. As mentioned above, the clinical ®ndings in patients with methylmalonic acidemia due either to defective mutase enzyme (mut) or defective AdoCbl synthesis (cblA, cblB) are remarkable more for their similarities than for their differences. A survey of the natural history in 45 such patients has been reported:451 20 were mut; 14 were cblA, and 11 were cblB (also see Chap. 94). There were approximately equal numbers of males and females in each group. The most common signs and symptoms at the onset of clinical dif®culty were lethargy, failure to thrive, recurrent vomiting, dehydration, respiratory distress, and muscular hypotonia. Little interclass difference was observed for these major clinical manifestations or for such less common ones as developmental retardation, hepatomegaly, or coma. The only major clinical distinction between the mut group and the groups with defective AdoCbl synthesis was that most of the former group presented very early in life ( < 1 to 4 weeks), while 60 percent of the cblA group and 45 percent of the cblB group presented between 1 month and 1 year.451 The laboratory ®ndings in cblA and cblB patients at the time that methylmalonic acidemia was ®rst documented are shown in Table 155-5, with those in mut patients for comparison. As expected, serum cobalamin concentrations were routinely normal. Metabolic acidosis, with blood pH values as low as 6.9 and serum bicarbonate concentrations as low as 5 mEq/liter, was observed in the majority of patients. Ketonemia or ketonuria, hyperammonemia, and hyperglycinemia or hyperglycinuria were also observed in many affected patients. Leukopenia, thrombocytopenia, and anemia were the only other manifestations that were noted. Earlier case reports (reviewed in reference 452) found that hypoglycemia occurred in about 40 percent of affected patients. Signi®cantly, the megaloblastic anemia characteristic of functional Cbl de®ciency or the inherited disorders of MeCbl synthesis (cblC, cblD, cblE, cblF, and cblG) was not present in these patients. Defects in AdoCbl Synthesis In 1968, Rosenberg443,444 and Lindblad445,446 and their colleagues described infants with severe metabolic ketoacidosis and developmental delay who accumulated very large amounts of methylmalonate in blood and urine, similar to patients reported earlier by Oberholzer447 and Stokke448 and their coworkers. In contrast to the earlier patients, however, these infants responded dramatically to pharmacologic doses of CN-Cbl or AdoCbl with resolution of their clinical symptoms and major reductions in their excretion of methylmalonate. Further studies indicated that the methylmalonyl CoA mutase enzyme was normal in these patients, but that synthesis of AdoCbl was impaired.430,449 Somewhat later, Kaye et al.450 reported two patients with methylmalonic acidemia who were unresponsive in vivo to high doses of CN-Cbl but who also had apparently normal mutase enzyme and defective AdoCbl synthesis. Subsequent biochemical and genetic complementation analysis established that lesions at two genetically distinct loci can be responsible for defective AdoCbl synthesis; they are designated cblA (MIM 251100) and cblB (MIM 251110)323,414 Because both groups of patients with de®cient AdoCbl synthesis share many clinical features with those with primary defects in the methylmalonyl CoA mutase enzyme, the reader is also referred to Chap. 94 for a discussion of the latter group. Chemical Abnormalities In Vivo. Large amounts of methylmalonic acid have appeared in the urine or blood of all reported Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Table 155-5 Laboratory Findings in 45 Patients with Methylmalonic Acidemia* Mutant Class Finding at Clinical Onset cblA cblB mut Normal serum cobalamin Metabolic acidosis Ketonemia and/or ketonuria Hyperammonemia Hyperglycinemia and/or -glycinuria Leukopenia Anemia Thrombocytopenia 100 100 78 50 70 70 10 75 100 88 67 83 83 45 45 45 100 89 88 76 60 69 44 40 * Numerical values are the percentages of the patients in each group in whom the particular finding was made. S O U RC E : From Matsui et al.451 patients. Whereas normal children and adults excrete less than 0.04 mM (5 mg) methylmalonate daily, children with isolated methylmalonic acidemia have excreted from 2.1 to 49 mM (240 to 5700 mg) in a 24-h period. Their plasma concentrations of methylmalonate, almost undetectable in normal subjects, have ranged from 0.22 to 2.9 mM (2.6 to 34 mg/dl). In the few patients in whom it was measured, the CSF concentration of methylmalonate equaled that of plasma (see reference 392 for references to early case reports). No relationship between the quantities of methylmalonate accumulated in body ¯uids and the etiology of mutase de®ciency (i.e., apoenzyme vs. coenzyme synthesis de®ciency) has been reported. Methylmalonate is surely the major, but not the only, abnormal metabolite found in body ¯uids of these patients. Because propionyl CoA carboxylation is reversible, propionate and some of its precursors (butanone) or metabolites (b-hydroxypropionate and methylcitrate) also accumulate in blood and urine,443,453,454 their amounts being small compared to that of methylmalonate (see Chap. 94). Several groups have studied the relationship between protein or amino acid loading and methylmalonate accumulation in these patients. Without exception, administration of protein or amino acids known to be precursors of propionate and methylmalonate, such as methionine, threonine, valine, or isoleucine, has resulted in augmented methylmalonate accumulation and, in some instances, ketosis or acidosis.443,445,447,448 When Cbl-responsive patients are given supplements of this vitamin, such augmentation by methylmalonate precursors is lessened considerably.455 All these ®ndings suggest that patients with discrete defects at the mutase step have a major block in the utilization of methylmalonyl CoA, which is expressed as methylmalonate accumulation. Localization of Enzymatic Defects. Because the conversion of propionate to succinate is blocked in each of the methylmalonic acidemias, whether due to mutase defects or AdoCbl synthesis de®ciencies, an early screening test for these disorders measured the ability of intact peripheral blood leukocytes or cultured ®broblasts to oxidize [14C]propionate or [14C]methylmalonate to 14 CO2 and compared this with the oxidation of [14C]succinate to 14CO .444 More recently, incorporation of [14C]propionate into 2 trichloroacetic acid-precipitable material by intact cultured cells has replaced the more cumbersome 14CO2 evolution technique.456,457 Further discrimination among the methylmalonic acidemias has depended on studies of cobalamin uptake and AdoCbl formation by intact cultured ®broblasts, on assays of mutase activity in cell extracts, and on genetic complementation studies with cultured cells. cblA. A series of observations by Rosenberg449 and Mahoney430 and their colleagues on the ®broblasts of the index patient with Cbl-responsive methylmalonic acidemia led to the demonstration of a primary defect in AdoCbl synthesis. Such intact cells were unable to convert OH-[57Co]Cbl to Ado[57Co]Cbl, although they took up the labeled vitamin normally and had no abnormality in synthesizing the other cobalamin coenzyme, MeCbl.430 On the other hand, cell-free extracts from this line synthesized AdoCbl normally when incubated with OH-[57Co]Cbl, ATP, and a reducing system designed to bypass cob(III)alamin reductase and cob(II)alamin reductase and to measure only cob(I)alamin adenosyltransferase.323 Fibroblasts from other patients in this clinically de®ned group had identical ®ndings in similar studies.323 Genetic complementation analysis established unequivocally that all these patients belonged to a single complementation group, designated cblA, and thus presumably had defects in the same enzyme or protein.414 Because it has been shown that intact mammalian mitochondria can synthesize AdoCbl from OH-Cbl in vitro without prior reduction458 and because Cbl adenosyltransferase activity is normal in this group,323 it is presumed that the defect must lie in one of the early steps of mitochondrial Cbl metabolism, possibly in a mitochondrial Cbl reductase (see Fig. 155-10). So far, the lack of a speci®c assay for the reductive step(s) in AdoCbl synthesis326,327 has prevented a more precise localization of the defect in the cblA group. cblB. Another group of patients with defective AdoCbl synthesis was uncovered when Cbl metabolism was examined in a number of cell lines from patients with methylmalonic acidemia.323,459 Some of these ®broblasts showed a primary defect in AdoCbl synthesis similar to that described for the cblA class (above), except in one aspect. When cell-free extracts from these lines were incubated with OH-[57Co]Cbl, a reducing system, and ATP, no AdoCbl synthesis was detected,323 in contrast to the result in the cblA cell lines. Because this assay is speci®c for ATP:cob(I)alamin adenosyltransferase, the patients in this group must have defects in this enzyme.324 Complementation analysis indicates that a single locus, cblB, is involved in all these patients and that it is distinct from the cblA locus.414 Pathophysiology. All studies in vivo and in vitro in patients with methylmalonic acidemia due to methylmalonyl CoA mutase de®ciency, either primary or secondary to AdoCbl synthesis defects, indicate that the block in the conversion of methylmalonyl CoA to succinyl CoA explains fully the accumulation of methylmalonate in blood and urine; the augmentation of methylmalonate excretion and the precipitation of ketosis by protein, amino acids, or propionate; and the excretion of longchain ketones formed in the catabolism of branched chain amino acids. See Chap. 94 for a complete discussion of methylmalonic acidemia. By comparing and contrasting the ®ndings in patients with isolated mutase de®ciency, whether due to defects in mutase or in AdoCbl synthesis, with those in patients with functional Cbl de®ciency (as in classic pernicious anemia or the Cbl transport defects discussed above), it is possible to shed some light on the mechanism responsible for the hematologic and neurologic abnormalities in the latter disorders. Thus, the absence of megaloblastic anemia in any patient with isolated mutase de®ciency militates against any involvement of this enzyme in the typical megaloblastoid seen in Cbl de®ciency. Similarly, the cerebellar and posterior column abnormalities so often encountered in Cbl-de®cient patients have never been observed in patients with methylmalonic acidemia due to speci®c mutase dysfunction. Therefore, the notion that neurologic dysfunction in pernicious anemia re¯ects aberrant incorporation of odd-chain or branched chain fatty acids into myelin because of a block in the propionate pathway has little to recommend it. It appears likely, then, that abnormalities in Cbl-dependent methionine synthase account for the hematologic and neurologic abnormalities in Cbl-de®cient patients. Genetic Considerations. Both cblA and cblB are almost certainly inherited as autosomal recessive traits. This conclusion is based on 3921 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 3922 PART 17 / VITAMINS these observations: (a) approximately equal numbers of affected males and females have been reported in each group; (b) no instance of vertical transmission from affected parent to affected child has been reported; (c) each mutant class behaves as a recessive in culture in complementation experiments;414,415,431 and (d) cell lines from heterozygotes for the cblB mutation show partial adenosyltransferase de®ciency.324 Diagnosis, Treatment, and Prognosis. Because simple colorimetric assays for urinary methylmalonate and more complex gas chromatography-mass spectrometry assays for serum and urinary methylmalonate are available, it should not be dif®cult to make a diagnosis of methylmalonic acidemia, once this condition is considered. Other sources of neonatal or infantile ketoacidosis must be ruled out. The quantity of methylmalonate excreted, the absence of megaloblastic changes, and the normal amounts of serum homocysteine, methionine, and Cbl all serve to differentiate these diseases from others that may lead to methylmalonic aciduria. Distinguishing between primary mutase de®ciency and primary AdoCbl synthesis defects and between the two causes of the latter ultimately depends on studies with cultured cells B routinely, genetic complementation analysis.415 Prenatal detection of methylmalonic acidemia has been accomplished in two different ways: by measurement of methylmalonate in amniotic ¯uid and maternal urine at midtrimester460,461 and by studies of mutase activity and Cbl metabolism in cultured amniotic ¯uid cells.456,461,462 Assays of [14C]propionate utilization456 in uncultured chorionic villous biopsy specimens have proven unsatisfactory, however. AdoCbl synthesis defects of both complementation groups461,462 have been identi®ed in these ways. Two treatment regimens for children with methylmalonic acidemia exist and should be employed in tandem for patients with AdoCbl synthesis de®ciencies. A diet restricted in protein (or a special formula restricted in amino acid precursors of methylmalonate) should be instituted as soon as such life-threatening problems as ketoacidosis, hypoglycemia, or hyperammonemia have been addressed; and supplementary Cbl (1 to 2 mg OH-Cbl intramuscularly daily for several days) should be given as soon as the diagnosis of methylmalonic acidemia is made (or even seriously considered). Such measures should decrease the circulating concentrations of methylmalonate and propionate. Even Cbl-unresponsive children with delayed development improve markedly when treated with careful dietary protein restriction.463,464 In Cbl-responsive patients, titration of Cbl dosage schedules against methylmalonate excretion and clinical status is probably worthwhile. The methylmalonic aciduria is not completely eliminated in even the most responsive patients, even though clinical symptoms such as ketosis and acidosis are completely resolved. As discussed in Chap. 94, Roe and associates465±467 have pointed out that L-carnitine supplements may be a useful therapeutic adjunct in patients with methylmalonic acidemia, presumably by repleting intracellular and extracellular stores of free carnitine that are depleted in affected patients because of exchange with excess methylmalonyl CoA and propionyl CoA. No trial of this compound has been reported in cblA or cblB patients. As suggested in Chap. 94, oral antibiotic therapy may prove useful as well. Thompson and his colleagues reported that three Cbl-unresponsive patients showed subjective improvement in alertness and appetite following brief metronidazole therapy;468 longer treatment periods have resulted in signi®cant improvements in other patients, including decreased number and severity of acidotic episodes, increased appetite, decreased vomiting, growth acceleration, and improved behavior in a cblB patient.469 The previously mentioned survey451 suggested that both the response to Cbl supplements and the long-term outcome in affected patients depends considerably on the nature of the biochemical lesion. Whereas more than 90 percent of the cblA patients responded to Cbl supplements with a distinct fall in blood or urinary methylmalonate, only 40 percent of the cblB patients showed such a response. Presumably, the 60 percent of cblB patients unresponsive to Cbl supplements have such complete adenosyltransferase de®ciency that AdoCbl synthesis cannot be augmented by Cbl supplements, in distinction to the cblB patients with apparently ``leaky'' mutations that permit responsiveness in vivo. The uniform responsiveness of patients in the cblA group suggests either that the responsible mutations are generally leaky, thereby allowing mass action to result in more AdoCbl synthesis, or that alternative pathways of Cbl reduction, which require high substrate concentrations, exist in cells.326,327 As in the case of primary mutase de®ciency, it should be emphasized that clinical responsiveness in vivo does not require complete correction of the functional mutase de®ciency or complete normalization of biochemical parameters such as the methylmalonic acidemia (see Chap. 94). Some patients in the cblB group, unresponsive to CN-Cbl or OH-Cbl in vivo, might be expected to respond to AdoCbl itself, but published reports on two patients suggest that this logical alternative is ineffective.470,471 Unpublished experiments on cells in culture suggest that AdoCbl is largely converted back to OH-Cbl during transport. The long-term outlook for affected patients is revealing. The cblA patients (i.e., the group biochemically most responsive to Cbl supplements) had the best outcome according to the survey Ð 70 percent were alive and well at ages up to 14 years and presumably continue to be so. The cblB group had about equal fractions in the alive and well, the alive and impaired, and the deceased category. It is interesting, albeit anecdotal, that the index patient in the cblA group (now over 30 years old) discontinued Cbl supplements at age 9 years despite advice to the contrary. In the ensuing years, despite accumulation of very large amounts of methylmalonate in the blood and urine, his development and general health have remained excellent, with one exception (see below). Perhaps, as in some other inherited metabolic disorders, treatment of methylmalonic acidemia is most critical early in life. If this experience is borne out, it makes expert clinical management in the early weeks or months of life most important. There have been several reports of ``metabolic stroke'' in patients following episodes of metabolic decompensation.472± 474 Three of the patients472,473 belonged to the Cbl-responsive cblA group, but were not being treated at the time. Extrapyramidal signs, particularly dystonia, were accompanied by bilateral lucencies of the globus pallidus and persisted after the acute crisis had passed. In one case, the dystonia was gradually progressive over a period of 7 years without visible progression of the neurologic lesions.474 One complication of long-term survival of some methylmalonic acidemia patients may be chronic renal failure. One report has indicated that 8 of 12 nonresponsive patients (aged 1 to 9 years) had reduced GFR, with 5 severely affected.475 In one patient ``greatly improved metabolic control'' over a period of 18 months led to increased, but still impaired, renal function.475 Signi®cantly, the index cblA patient referred to above recently returned to attention following treatment for renal dysfunction due to biopsy-proven interstitial nephritis. The impact of better metabolic control and Cbl supplementation has not been explored in this case. Finally, the feasibility of prenatal therapy with Cbl supplements has been demonstrated. Ampola et al.461 showed that administration of Cbl supplements to a woman carrying an affected fetus of the cblA group resulted in signi®cant reduction in maternal excretion of methylmalonate and the presence of only moderate methylmalonic acidemia(-uria) in the newborn child. She was doing well at the time of the report (20 months) with moderate protein restriction and occasional Cbl therapy, whereas an undiagnosed affected sib had died at 3 months of age.461 A second, similar case has been reported.476 SUMMARY Tables 155-2 and 155-4 summarize the salient biochemical features of patients with defects in various aspects of folate and Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 155 / INHERITED DISORDERS OF FOLATE AND COBALAMIN TRANSPORT AND METABOLISM Fig. 155-12 Summary scheme of inherited defects of cobalamin metabolism. The circled numbers and their key signify the general sites at which abnormalities have been identi®ed and the Cbl transport and metabolism and their cultured cells. Figures 155-4 and 155-12 summarize the localization of these defects. ACKNOWLEDGMENTS The authors thank L.E. Rosenberg for his contributions to this chapter in previous editions and R. Rozen for the provision of Fig. 155-5. We thank the many clinicians who have provided clinical histories and ®broblasts for analysis. REFERENCES 1. Rowe PB: Inherited disorders of folate metabolism, in Stanbury JB, Wyngaarden JB, Frederickson DS, Goldstein JL, Brown MS (eds): The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1983, p 498. 2. 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