Chapter 147. Vitamin B₁₂ and Folic Acid

Cobalamin (vitamin B12) is a complex organometallic molecule that is synthesized by many bacteria and is obtained in the human diet from meat, fish, and dairy products. It is not present in plant foods, so strict vegetarians are at risk for dietary deficiency. Derivatives of cobalamin are required for the activity of two enzymes: (1) methylcobalamin (MeCbl) is generated during the catalytic cycle of methionine synthase, a cytoplasmic enzyme that catalyzes methylation of homocysteine to methionine (see Chapter 138), and (2) 5′-deoxyadenosylcobalamin (AdoCbl) is required for the mitochondrial enzyme methylmalonyl-CoA mutase to catalyze the conversion of methylmalonyl-CoA, formed during catabolism of branched-chain amino acids and odd-chain fatty acids, to succinyl-CoA (see Chapter 137). Therefore, inborn errors of cobalamin metabolism result in isolated methylmalonic aciduria, isolated homocystinuria, or combined methylmalonic aciduria and homocystinuria, depending on which step in cobalamin metabolism is affected.1-4 In these disorders, hypomethioninemia usually occurs together with hyperhomocysteinemia. Elevated homocysteine levels are associated with an increased risk of thrombosis; decreased methionine is associated with abnormalities of the white matter of the nervous system. Elevated levels of methylmalonic acid are usually associated with metabolic acidosis.

Metabolism of Cobalamin

Cobalamin consists of a planar corrin ring with a central cobalt atom, a 5,6-dimethylbenzimidazole base, and an upper axial ligand attached to the cobalt atom that varies in different forms of cobalamin.5 Physiologically important cobalamins include hydroxycobalamin (OHCbl), methylcobalamin (MeCbl), and adenosylcobalamin (AdoCbl). The most common commercially available form of vitamin B12 contains a cyano group in the upper axial position (CNCbl). The central cobalt can exist in the oxidized Co3+ state (cob[III]alamin), the Co2+ state (cob[II]alamin), or the fully reduced Co1+ state (cob[I]alamin). Converting exogenous cobalamin, typically in the form of cob(III)alamin, to its biologically active coenzyme forms involves reducing the central cobalt to the cob(I)alamin form, then adding the appropriate upper axial ligand (eFig. 147.1).

Absorption of dietary cobalamin is a complex process that is dependent on a cobalamin-binding protein, intrinsic factor (IF), secreted by the parietal cells of the stomach.6 Cobalamin in food exists bound to proteins. After proteins are digested, cobalamin becomes bound in the stomach to haptocorrin, a cobalamin-binding protein secreted in the saliva. Haptocorrin is broken down in the intestine and cobalamin binds the IF. The IF-cobalamin complex is taken up by enterocytes in the distal ileum in a process mediated by a receptor, cubam, composed of the proteins cubilin and amnionless. After uptake by enterocytes, cobalamin is released into the circulation, bound to the transport protein transcobalamin (TC).

TC-bound cobalamin is available for uptake by most cell types. Following endocytosis, mediated by an as-yet uncharacterized cell-surface receptor, the cobalamin-TC complex dissociates in lysosomes, and free cobalamin is transferred to the cytoplasm. Subsequently, cobalamin may become associated with methionine synthase in the cytoplasm, or it may be transported to the mitochondria, where it is converted to adenosylcobalamin and becomes associated with methylmalonyl-CoA mutase.1 The early steps of cellular cobalamin metabolism common to synthesis of both coenzyme derivatives remain poorly understood. Two proteins that play a role in these early steps, the products of the MMACHC and MMADHC genes, have been identified on the basis of patients with inborn errors, but their functions are not known.

The catalytic cycle of methionine synthase involves transfer of a methyl group from 5-methyl-THF to an enzyme-bound cob(I)alamin molecule, forming methylcobalamin (MeCbl); the methyl group is then transferred from MeCbl to homocysteine, forming methionine and regenerating cob(I)alamin. Occasionally the cob(I)alamin is oxidized to cob(II)alamin; when this occurs, regeneration of MeCbl requires the activity of a second protein, methionine synthase reductase, which uses adenosylmethionine as its methyl group donor. Remethylation of homocysteine to form methionine thus requires the activity of both methionine synthase and methionine synthase reductase.7,8

The system by which cobalamin enters the mitochondria is unknown, as is the form of cobalamin that crosses the mitochondrial membrane. Mitochondrial reductases that can support reduction of cob(II)alamin to cob(I)alamin have been identified in vitro, but the enzyme that catalyzes this reaction in vivo has not been identified. Cob(I)alamin reacts with adenosine triphosphate (ATP) to form adenosylcobalamin (AdoCbl) in a reaction catalyzed by cob(I)alamin adenosyltransferase, encoded by the MMAB gene.9 This is a member of the PduO family of cobalamin adenosyltransferases, and it has been argued that, in addition to catalyzing adenosylation of cob(I)alamin, it also binds cobalamin in an activated base-off conformation (in which the dimethylbenzimidazole base no longer coordinates with the central cobalt atom) and directly transfers the activated AdoCbl product to methylmalonyl-CoA mutase.10 Another protein, the product of the MMAA gene, appears to play a role in supporting the formation of holo-methylmalonyl-CoA mutase and in maintaining the enzyme-bound cobalamin in its active form.11,12 Presence of methylmalonyl-CoA mutase, cobalamin adenosyltransferase, and the MMAA protein, possibly in association with a cob(II)alamin reductase, is required for conversion of methylmalonyl-CoA to succinyl-CoA in vivo.

Isolated Methylmalonic Aciduria

Metabolic Derangement

Isolated methylmalonic aciduria is seen in patients with mutations affecting methylmalonyl-CoA mutase itself (the mut disorder [OMIM 251000] due to mutations at the MUT locus on chromosome 6p21; see Chapter 137) or with mutations resulting in decreased synthesis of adenosylcobalamin (the cblA disorder [OMIM 251100], caused by mutations at the MMAA gene on chromosome 4q31.1-31.2, and the cblB disorder [OMIM 251110], caused by mutations in the MMAB gene on chromosome 12q24). The mut disorder has been subdivided into two classes: (1) in mut0 there is no stimulation mutase activity in cultured cells on incubation with hydroxycobalamin (OHCbl), and there is often no detectable mutase protein; (2) in mut–, mutase activity is stimulated by addition of OHCbl.1

Clinical Presentation

Patients with isolated methylmalonic aciduria are prone to episodes of life-threatening acidosis, often in response to infection or to increased protein intake. Initial presentation is most frequently during infancy, with vomiting, hypotonia, irritability, and lethargy, then progressing to coma and death if not treated. In the most severely affected individuals, there may be intractable acidosis during the neonatal period that leads to death, but in many patients the disorder is characterized by recurrent acidotic crises characterized by severe ketoacidosis, hyperammonemia and hyperglycemia, or hyperglycemia with anemia, neutropenia, thrombocytopenia or pancytopenia, failure to thrive, or metabolic stroke. Prolonged elevation of plasma methylmalonic acid may lead to end-stage renal failure necessitating transplantation. Studies of series of patients have shown that presentation is typically most severe in individuals with mut0, while cblB is less severe and cblA and mut– patients have the least severe presentation and the best prognosis.13,14

Diagnostic Tests

Organic acid analysis in these patients reveals elevated levels of methylmalonic acid and of metabolites such as methylcitric acid, 3-hydroxypropionic acid, and tiglylglycine. Tandem mass spectroscopy of acylcarnitines shows elevated propionyl carnitine. Elevated glycine may be present in blood and urine. Elevation of methylmalonic acid levels may also be seen in individuals with methylmalonyl-CoA epimerase (racemase) deficiency and in patients with mutations affecting either subunit of succinyl-CoA ligase; however, in these orders, the levels are lower than in mut, cblA, and cblB.

Differentiation between the different forms of methylmalonic aciduria has depended on studies of cultured fibroblasts. In all three groups, mutase function (assessed by measuring the incorporation of label from [14C]propionate into cellular macromolecules) is decreased in the presence of normal methionine synthase function (assessed by measuring the incorporation of label from [14C]methyl-THF or [14C]formate into cellular macromolecules). There is deficient conversion of exogenous [57Co]CNCbl into adenosylcobalamin (AdoCbl) in cblA and cblB fibroblasts in the presence of normal methylcobalamin synthesis; AdoCbl synthesis may be normal or reduced in mut fibroblasts. The cblA and cblB disorders can be differentiated on the ability of cell extracts to support conversion of [57Co]CNCbl to AdoCbl, but this test is rarely performed. Differentiation between the three classes is typically carried out by complementation analysis.1

Genetics

All three disorders are inherited as autosomal recessive traits. Over 180 mutations in the MUT gene have been identified in patients with mut, the most common of the disorders. The mutations in most families are private, but several mutations are relatively common within a specific ethnic group. These include a c.2150G→T (p.G717V) mutation in African patients, c.349GÆT (p.E117X) in Japanese patients, and c.323CÆT (p.R108C) in American Hispanic patients.15 Thirty MMAA mutations have been identified in cblA patients. The most common of these, c.433CÆT (p.R145X), represents 43% of mutant alleles in Western patients but is less frequent in Japanese patients, where a c.503delC predominates.16,17Twenty-three MMAB mutations have been identified in cblB patients. A c.556CÆT (p.R186W) mutation was the most common. In X-ray crystallographic studies, virtually all identified MMAB mutations clustered in an area of the gene product that is the active site of the enzyme.18

Isolated Homocystinuria

Metabolic Derangement

Homocystinuria in the absence of methylmalonic aciduria is seen in patients with the cblE (OMIM 250940) and cblG (OMIM 236270) disorders. In both disorders, methionine synthase function is impaired; in the cblG disorder, synthase-specific activity in cell extracts is decreased under all assay conditions, while in the cblE disorder, specific activity is decreased only in the presence of limiting concentrations of exogenous reducing agent.19 The cblG disorder is caused by mutations affecting the MTR gene on chromosome 1q43, which encodes methionine synthase.20,21 The cblE disorder is caused by mutations of the MTRR gene on chromosome 5p15.3-15.2, which encodes methionine synthase reductase.22

Clinical Presentation

Patients with these disorders are characterized by megaloblastic anemia and neurological problems, including developmental delay, cerebral atrophy, hypotonia, microcephaly, and seizures. Homocystinuria is less severe in patients with these disorders than in patients with homocystinuria due to cystathionine β-synthase deficiency. Presentation is typically within the first 2 years of life but can present in adulthood as well.23

Diagnostic Tests

Decreased methionine synthase activity results in increased serum total homocysteine and in homocystine in the urine, combined with reduced levels of methionine and elevated cystathionine. Serum cobalamin levels are normal. These disorders can be differentiated from homocystinuria due to cystathionine β-synthase deficiency, which presents with higher levels of homocysteine, elevated methionine, and decreased cystathionine levels (see Chapter 138, Table 138-1). Fibroblasts are characterized by decreased methyl-THF incorporation with normal propionate incorporation and by decreased methylcobalamin synthesis with normal adenosylcobalamin synthesis. The disorders can be differentiated by the response of methionine synthase–specific activity to the titration of a reducing agent but more typically are differentiated by complementation analysis.

Genetics

Both disorders are inherited as autosomal recessive traits. Eighteen MTRR mutations have been identified in cblE patients. The most common of these is an intronic c.903+469TÆC mutation that results in a 140 bp insertion of intronic sequence in mRNA; a c.1361CÆT (p.S454L) mutation is common among patients of Iberian origin and appears to be associated with a mild form of the disease.24,25 Twenty MTR mutations have been identified in cblG patients. The most common of these, c.3158CÆT (p.P1173L), has never been observed in homozygous form.26

Combined Methylmalonic Aciduria and Homocystinuria

Metabolic Derangement

Three inborn errors of metabolism that affect early steps in cobalamin metabolism result in combined methylmalonic aciduria and homocystinuria. The cblC disorder (OMIM 277400) is caused by mutations at the MMACHC locus on chromosome 1p34.1,27 and the cblD disorder (OMIM 277410) is caused by mutations at the MMADHC locus on chromosome 2q23.2.28 The function of neither of these gene products is understood. Although the original patients with the cblD disorder had combined methylmalonic acid and homocystinuria, additional patients with isolated homocystinuria (cblD variant 1) and isolated methylmalonic aciduria (cblD variant 2) have subsequently been recognized.29 The cblF disorder results in inability to transfer endocytosed cobalamin from the lysosome to the cytoplasm.30 The gene underlying this disorder has not yet been identified.

Clinical Presentation

The large majority of patients with combined methylmalonic aciduria and homocystinuria belong to the cblC class, with over 300 patients identified. Individuals with this disorder typically present during the first year of life with megaloblastic anemia, failure to thrive, developmental delay, hypotonia, seizures, macrocephaly, and cerebral atrophy. Despite the presence of methylmalonic aciduria, metabolic decompensation is infrequent. Other patients present later in life or in adulthood with ataxia, dementia, or psychosis. A distinctive salt-and-pepper retinopathy has been described in some cblC patients. Presentation in classic cblD disease is similar to that of cblC. Presentation of the cblF disease is variable. Frequent findings have included feeding difficulties, failure to thrive, growth retardation, and persistent stomatitis.

Diagnostic Tests

Patients with these disorders have the biochemical characteristics of both methylmalonic aciduria and homocystinuria, although methylmalonic acid levels are generally lower than in patients with isolated methylmalonic aciduria. Fibroblasts show decreased function of both methylmalonyl-CoA mutase and methionine synthase, and decreased synthesis of both adenosylcobalamin and methylcobalamin (Table 147-1). Fibroblasts from patients with the cblC or cblD disorder are unable to accumulate cobalamin in cells, reflecting the inability of cells to retain cobalamin that is not bound to one of the cobalamin-dependent enzymes. In cblF disorder, fibroblasts accumulate large amounts of cobalamin, but most of this is unmetabolized cobalamin that is trapped within the lysosomes.

Genetics

Both disorders are inherited as recessive autosomal traits. Over 40 mutations in the MMACHC gene have been identified. The most common of these is a c.271dupA mutation that represents 40% of identified mutant alleles, primarily in patients of European or western Asian origin. It is associated with early onset severe disease when homozygous. Several other mutations show evidence of clustering in specific ethnic groups, and genotype-phenotype correlations have been observed.27,31 A small number of mutations in the MMADHC gene have been identified in patients with the much rarer cblD disorder. The classical cblD disorder is associated with truncating mutations, while mutations at the C-terminal domain are associated with isolated homocystinuria (variant 1), and mutations in the N-terminal domain are associated with isolated methylmalonic aciduria (variant 2).28 The gene underlying the cblF disorder remains unknown.

Treatment

Patients with inborn errors of cobalamin are treated with large doses (up to 1 mg per day) of cobalamin administered intramuscularly. In the cblC disorder, hydroxycobalamin is more effective than CNCbl, the standard form of pharmacological vitamin B12)32; this may be true for other disorders as well. In patients with methylmalonic aciduria, protein restriction represents an important aspect of treatment. Supplementation with carnitine and with antibiotics (to reduce propionate production by gut bacteria) has also been used. Supplementation with betaine, which supports conversion of homocysteine to methionine by the liver enzyme betaine-homocysteine methyltransferase, has proved effective in patients with homocystinuria.

Inborn Errors of Cobalamin Uptake and Transport

Combined methylmalonic aciduria and homocystinuria is also seen in patients with defects affecting intestinal uptake of cobalamin and its transport in the serum. Decreased uptake occurs in individuals with intrinsic factor deficiency (OMIM 261000), caused by mutations at the GIF gene on chromosome 11q13,33,34 and in those with Imerslund-Gräsbeck syndrome (IGS; OMIM 261100). The latter disorder is the result of dysfunction of the cubam receptor in the distal ileum, which can be caused by mutations at the CUBN gene on chromosome 10p12.1 or the AMN gene on chromosome 14q32, which encode the cubilin and amnionless components of cubam; additional IGS families are not linked to either chromosome and represent further genetic heterogeneity in this disorder.35,36 Patients usually present between 1 and 5 years of age with decreased serum cobalamin levels, megaloblastic anemia, and neurological impairment. IGS patients often have proteinuria as well.37 Fewer than 30 patients with intrinsic factor deficiency have been identified, and approximately 300 patients with IGS have been identified. Clusters of IGS have been identified in Finland (CUBN mutations), Norway (AMN mutations), and in the eastern Mediterranean (mutations in both genes). In the absence of readily available clinical tests to assess cobalamin uptake, specific diagnosis in patients with disorders of cobalamin uptake depends on sequencing of the GIF, CUBN, and AMN genes.

Patients with transcobalamin deficiency (OMIM 275350) usually present in the first months of life with megaloblastic anemia; failure to thrive; weakness; and, frequently, immunologic dysfunction; without treatment, neurological impairment develops.1 Because a majority of cobalamin circulates bound to haptocorrin (which is not available for uptake by most types of cells) rather than to transcobalamin, circulating cobalamin levels may be normal in this disorder. Mutations in the TCN2 gene on chromosome 22q11.2 have been identified in a small number of patients.36 Treatment involves maintaining very high serum levels of cobalamin by either oral or intramuscular route.

The folates are a class of molecules comprising folic acid (pteroylglutamic acid) and its derivatives. Folic acid consists of a pteroic acid (a pteridine ring attached to para-aminobenzoic acid) with an attached glutamate residue. Biologically active folates differ from folic acid by reduction of the pterin ring to its tetrahydrofolate (THF) derivative, by the attachment of a variety of one-carbon groups to N5 and N10 of the pterin ring, and by addition of up to six additional glutamate residues by γ-peptide linkage. Folate is synthesized by many types of bacteria and is obtained in the diet from both plant and animal foods.

Within cells, folates accept one-carbon units from various sources, primarily serine (via the reaction catalyzed by serine hydroxymethyltransferase) but also from glycine (the glycine cleavage system), formate (formyl-THF synthetase), and histidine (breakdown mediated by glutamate formiminotransferase and formiminotetrahydrofolate cyclodeaminase). Several reactions utilize one-carbon units derived from folates (Fig. 147-1). Methyl-THF is required for methylation of homocysteine to form methionine, catalyzed by methionine synthase (described in the section on vitamin B12;see also Chapter 138). Catalytic conversion of uridylate to thymidylate requires 5,10 methylene THF for the activity of thymidylate synthase. Carbons 2 and 8 of the purine ring are added by the folate-dependent enzymes GAR transformylase and AICAR transformylase, both of which use 10-formyl-THF as a one-carbon donor. Thus, synthesis of both purines and one of two pyrimidines required for DNA synthesis are dependent on folates.3,4,38

Interconversion of one-carbon substituted folates is an important aspect of cellular metabolism. Methylene-tetrahydrofolate (THF) can be reduced to methyl-THF by the enzyme methylene-THF reductase (MTHFR). Alternatively, it can be oxidized to form 10-formyl-THF by sequential reactions catalyzed by methylene-THF dehydrogenase and methenyl-THF cyclohydrolase (the two activities are catalyzed by a single multifunctional protein). The reaction catalyzed by MTHFR is irreversible under physiological conditions; this means that when activity of methionine synthase, the only reaction that utilizes methyl-THF, is impaired (as in cobalamin deficiency or in the cblC, cblD, cblF, cblE, and cblG disorders), cellular folate is trapped as methyl-THF, resulting in deficiency of other folate coenzyme forms.

Mthfr Deficiency

The most common inborn error of folate metabolism is methylene-THF reductase (MTHFR) deficiency (OMIM 236250) due to mutations of the MTHFR gene on chromosome 1p36.3. Over 90 patients with this disorder have been identified (see also Chapter 138). Decreased MTHFR activity results in deficiency of methyl-THF, the source of the methyl group used by methionine synthase. Thus MTHFR deficiency is characterized by hyperhomocysteinemia and homocystinuria with hypomethioninemia. Clinical presentation varies, but patients usually show symptoms during infancy or early childhood that include feeding difficulties, lethargy, hypotonia, developmental delay, and seizures.39 Cerebral atrophy and demyelination are often present. Patients with later onset may have mental retardation, ataxia, or psychiatric problems. Unlike patients with inborn errors of cobalamin metabolism, in which methionine synthase activity is impaired, patients with MTHFR deficiency do not have megaloblastic anemia. Cultured fibroblasts have decreased incorporation of label from [14C]formate into cellular macromolecules, but incorporation of label from [14C]methyl-THF is normal. Diagnosis depends on enzyme assay of cell extracts demonstrating reduced MTHFR-specific activity.

MTHFR deficiency is inherited as an autosomal recessive trait. Over 40 mutations of the MTHFR gene have been reported.40,41Almost all of these mutations have been restricted to one or two families—an exception is a c.1129CÆT mutation that is present at a high frequency among the Old Order Amish.42

Patients with MTHFR deficiency have been treated with a variety of agents, including methyl-THF and other folates, methionine, pyridoxine, cobalamin, carnitine, betaine, and riboflavin, individually and in various combinations. Of these, betaine appears to be the most effective. Treatment is most successful when the disease is diagnosed at an early stage, before irreversible neurological damage can occur.

Polymorphisms of the methylene-THF reductase (MTHFR) gene have been identified that result in decreased enzyme activity less severe than that seen in patients with severe MTHFR deficiency. The most well studied of these is a c.677CÆT mutation that results in reduction of enzyme activity to 50% of control levels. Homozygosity for the T allele at this polymorphism is associated with increased serum homocysteine levels in individuals with low folate intake.

Glutamate Formiminotransferase Deficiency

A small number of patients have been identified who have deficiency of the bifunctional enzyme that catalyzes the transfer of the formimino group from formiminoglutamate (FIGlU), generated during histidine catabolism, to tetrahydrofolate (THF) to form formimino-THF, which is then converted to 10-formyl-THF. Patients with glutamate formiminotransferase deficiency (OMIM 229100) are characterized by elevated FIGlu levels either constitutively or in response to a histidine load. Because the affected enzyme is expressed only in liver and kidney, using enzyme assay to confirm the diagnosis is rarely possible. Two classes of patients have been reported: one was characterized by mental retardation, physical retardation, and cortical atrophy, while the second showed no mental retardation but massive FIGlu excretion. However, it has been suggested that the severe manifestations in the first group of patients were the result of ascertainment bias.43

The gene encoding the bifunctional enzyme, FTCD, has been identified on chromosome 21q22.3, and causative mutations have been identified in three patients.44 It remains unclear whether there is any clinical phenotype beyond FIGlu excretion in this disorder.

Hereditary Folate Malabsorption

A specific defect in intestinal folate uptake (OMIM 229050) has been described in approximately 20 patients. Transport across the blood-brain barrier is also affected in these individuals. Presentation typically occurs in the first months of life, with diarrhea, failure to thrive, developmental delay, megaloblastic anemia, and neurological deterioration.47 Serum and CSF folate levels are reduced. Patients with this disorder have mutations in the SLC46A1 gene on chromosome 17, which encodes a proton-coupled folate transporter.48,49 Optimal treatment involves maintaining adequate levels of folate in the CNS to avoid neurological impairment; the appropriate level must be determined for each patient individually and may involve administration of very large doses of folate.