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 verified methylenetetrahydrofolate reductase deficiency or glutamate formiminotransferase deficiency. 5 Other reviews are also available. 15,16,478–481
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 serine, glycine and histidine. In the 1930s, at about the same time that pteridine pigments of butterfly 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 and pteroylglutamate, respectively (Figure 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 γ-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 (THF) (Figure 155-2).
Structure of folic acid and its derivatives (Modified from Rowe. 1 Used with permission)
Structure of 5,6,7,8-tetrahydrofolic acid (THF). (Reproduced from Rowe. 1 Used with permission)
As summarized in Figure 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 γ-glutamyl peptide chains of varying length. 5-MethylTHF is the predominant form of folate in serum and in many tissues. 5-formylTHF, also known as "folinic acid," "leucovorin," or "citrovorum factor," is a reduced folate that has been used therapeutically because of its chemical stability.
Three distinct systems have been described for the transport of folates and folate antagonists (antifolates) across mammalian cell membranes. 20,21,482,483 One, the reduced folate carrier (RFC), encoded on chromosome 21q22.2-22.3 ([SLC19A1], NM_003056, MIM 600424), appears to be important for folate uptake by many types of cells, including hemopoietic cells, and has been studied extensively in cancer cells. It mediates a low affinity, high-capacity system for the uptake of reduced folates and methotrexate at high (μM) concentrations. 21–25 It shows considerable transcript heterogeneity. 25,26 Presence of functional RFC is necessary for fetal development in the mouse. Homozygous RFC knockout embryos die post implantation; survival can be prolonged by daily subcutaneous injection of the mother of folic acid, but embryos show absence of erythropoiesis and severe neural tube and limb bud defects. 484,485 RFC expression has been detected in enterocytes, 27 but it apparently is not responsible for intestinal folate uptake under physiological conditions.
The second system, a family of membrane-associated folate receptors (FR), is coded for by genes on chromosome 11q13.3-13.5. 21,28 These glycoproteins mediate a high affinity, low-capacity system and operate at low (nM) concentrations of exogenous folate. The FOLR1 (NM_000802, MIM 136430) and FOLR2 (NM_000803, MIM 136425) genes, encoding FR-α and FR-β respectively, have similar structures but differ in their 5′-untranslated regions and in their transcriptional regulatory elements. Both FR-α and FR-β are attached to the cell membrane by a glycosylphosphatidylinositol anchor. Folate binding protein (FBP), which carries a fraction of circulating folate, is derived from FR-α by cleavage from this anchor. 21,32,33 It has been argued that uptake of folate bound to FR-α and FR-β occurs by a process called “potocytosis” at non-clathrin-coated invaginations in the plasma membrane 29–31, but this is still debated. 21,32,33,486
Recently, a high-affinity proton-coupled folate transporter (PCFT) encoded on chromosome 17q11.2 ([SLC46A1] NP_542400, MIM611672) has been identified and cloned. Transport mediated by the PCFT is greatest at acid pH, and shows similar affinity for reduced and oxidized folates, 483,484,488 in agreement with the properties of the intestinal folate transporter in vivo. This transporter was initially identified as a heme carrier protein and designated HCP1 489 but was subsequently shown to display 100-fold greater affinity for reduced folates than heme. 483 Identification of mutations at the SLC46A1 locus in patients with hereditary folate malabsorption is strong evidence that the PCFT is responsible for folate uptake in the intestine and at the blood-brain barrier under physiologic conditions. 484,490
In addition to the above systems, there is evidence that passive diffusion may work together with folate receptors in transplacental folate transport. 34
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 fibroblasts to about 1 μM in human lymphocytes and certain tumor cells. 35 Although the K m for monoglutamate folates of many folate-dependent enzymes is greater than 1 μM, that for polyglutamate folates of appropriate chain length is generally much lower, allowing folate metabolism to progress at the concentration of folates present in cells. As well, folate polyglutamates of appropriate length can be channeled between the active sites of multifunctional folate-dependent enzymes, while monoglutamates must diffuse between active sites. 491 Folate polyglutamates also appear to play a role in accumulation of folate within cells and in partition of folates between cytoplasmic and mitochondrial compartments. Because folate polyglutamates do not readily traverse the cell membrane, they are retained in cells even though the intracellular folate concentration is greater than the extracellular concentration. Similarly, folates with polyglutamate chains longer than three do not traverse the mitochondrial membrane, and as a result separate pools of folate polyglutamates exist in the cytoplasm and mitochondria, with consequences for compartmentalization of cellular folates and control of folate metabolism. 492,493
Both a cytoplasmic and a mitochondrial folylpolyglutamate synthase exist. Both enzymes are encoded by the same gene on chromosome 9cen-q34 ([FPGS], NM_004957, MIM 136510) with alternative splicing resulting in the two folylpolyglutamate synthase proteins. 36–38 These enzymes form a peptide bond between the γ-carboxyl of the glutamate already present and the α-amino group of the glutamate to be added. Folylpolyglutamate synthase adds glutamate residues one at a time, requires ATP for its reaction, utilizes THF and other folates as well as antifolates as substrates with different affinities, and reacts poorly with folic acid and 5-methyl-THF. A mutant Chinese hamster ovary cell line deficient in folate polyglutamate synthesis is auxotrophic for glycine, adenosine, and thymidine, apparently because reactions generating these within the cell require folate polyglutamates. 40,41
Dietary folate exists predominantly in the polyglutamate form. These polyglutamates must be hydrolyzed in the intestine prior to absorption; folates traverse the intestinal epithelium as monoglutamates and are released into the circulation. 42,43 The γ-glutamyl chain is resistant to digestion by the common proteolytic enzymes and is hydrolyzed by specific 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. The genes for both the lysosomal ([GGH], NM_003878, MIM 601509) 44 and intestinal ([GCP2], AF007544, MIM 600934) 494–496 forms of conjugase have been identified. The latter is identical to the prostate-specific membrane antigen (PSMA). 46,496
Metabolic Pathways and Enzymes
The major metabolic pathways of the folates are shown in Figure 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-THF. This compound is used unchanged for the synthesis of thymidylate (Figure 155-3, reaction 4). 5,10-Methylene-THF is reduced to 5-methyl-THF for the biosynthesis of methionine (Figure 155-3, reaction 1), or is oxidized to 10-formyl-THF for use in purine synthesis 47 (Figure 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 (Figure 155-3, reaction 4), which also results in the oxidation of the folate moiety to dihydrofolate (DHF).
Scheme of folate-mediated 1-carbon transfer reactions: 1. Methionine synthase (methyl-THF:homocysteine methyltransferase); 2. Methylene-THF reductase (MTHFR); 3. Serine hydroxymethyltransferase; 4. Thymidylate synthase; 5. Dihydrofolate reductase; 6. Methylene-THF dehydrogenase (NAD and NADP-dependent forms have been described); 7. Methenyl-THF cyclohydrolase; 8. 10-Formyl-THF synthase; 9. 5-phosphoribosylglycineamide (GAR) transformylase; 10. 5-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylase; 11. Glutamate formiminotransferase; 12. Formimino-THF cyclodeaminase; 13. 5,10-Methenyl-THF synthetase; 14. 10-Formyl-THF dehydrogenase. AdoMet = adenosylmethionine; DHF = dihydrofolate; THF = tetrahydrofolate; methylCbl = methylcobalamin; GAR = 5-phosphoribosylglycinamide; FGAR = α-N-formyl-glycinamide ribonucleotide; AICAR = 5-phosphoribosyl-5-aminoimadazole-4-carbox- amide; C2, C8 = carbons number 2 and 8 or of purine ring.
Folic acid, a synthetic vitamin not found in nature, and dihydrofolate (DHF) are reduced by dihydrofolate reductase (Figure 155-3, reaction 5) to THF. The gene for dihydrofolate reductase is located on chromosome 5q11.1-q13.2 ([DHFR], NM_000791, MIM 126060). 49,50,497 Dihydrofolate reductase has long been known to be the primary site of action of the antifolate chemotherapeutic drug methotrexate. Unstable gene amplification resulting in resistance to methotrexate is associated with double-minute chromosomes, while in stably amplified cells that are resistant to methotrexate, the amplified genes are associated with elongated chromosomes. 48
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 with formation of 5,10-methylene-THF from THF (Figure 155-3, reaction 3). In mitochondria, glycine is also metabolized to 5,10-methylene-THF, plus carbon dioxide and ammonia, by the glycine cleavage system 16 (also see Chap. 90). There are two separate serine hydroxymethyltransferases, a cytoplasmic form ([SHMT1], NM_004169, MIM 182144) and a mitochondrial form ([SHMT2], NM_005412, MIM 138450). Both have been cloned: SHMT1 is on chromosome 17p11.2, and SHMT2 is on chromosome 12q13. 51–53 The cytosolic form has been crystallized, and its structure solved. 54 A mutant Chinese hamster ovary cell line deficient 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, cytoplasmic serine hydroxymethyltransferase may also play a role in gluconeogenesis. 16 Both forms of serine hydroxymethyltransferase are capable of catalyzing the hydrolysis of 5,10-methenyl-THF to 5-formyl-THF. 56
A secondary source of one-carbon units in some tissues is the catabolism of histidine. During this process, a formimino group is transferred from formiminoglutamate (FIGLU) to THF, followed by the release of ammonia with generation of 5,10-methenyl-THF. 139 The two enzyme activities, glutamate formiminotransferase (Figure 155-3, reaction 11) and formimino-THF cyclodeaminase (Figure 155-3, reaction 12), share a single polypeptide which channels folate polyglutamate molecules from one reaction to the next. This protein is encoded by the FTCD gene (AF169017, MIM 606806) on chromosome 21q22.3. 101,832 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 fibroblasts and blood cells.
Two enzyme systems carry out folate interconversions in mammals. 57 A trifunctional polypeptide, called C1-THF synthase, bears activities of NADP-dependent methylene-THF dehydrogenase, methenyl-THF cyclohydrolase, and 10-formyl-THF synthase (Figure 155-3, reactions 6, 7, and 8). 58,138 The trifunctional enzyme is found only in the cytosol, 59 and is encoded by a gene on chromosome 14q24 ([MTHFD1], NM_005956, MIM 172460). 60 A second, mitochondrial enzyme contains NAD-dependent methylene-THF dehydrogenase and methenyl-THF cyclohydrolase activities, without 10-formyl-THF synthase activity ([MTHFD2], NM_006636, MIM 604887). This bifunctional enzyme is not detected in normal adult tissue but is expressed in tissues that contain undifferentiated cells and in transformed mammalian cells. 57,59,61 It is encoded by a gene on chromosome 2, and appears to have arisen by duplication of the MTHFD1 gene followed by loss of the 10-formyl-THF synthase domain and a change in its cofactor dependence. 498 Another mitochondrial protein, encoded by a gene on chromosome 6q25.2 (MTHFD1L, AL117452, MIM 611427) has 10-formyl-THF synthase activity. This was originally described as a mitochondrial C1-THF synthase, 499 but has subsequently been shown to lack dehydrogenase and cyclohydrolase activities. 500 Thus the two mitochondrial enzymes between them contain the three activities present in the cytoplasmic trifunctional enzyme. The crystal structure of the dehydrogenase/cyclohydrolase domain of the human trifunctional enzyme has been determined in the presence of NADP and inhibitory folate analogues, allowing identification of NADP- and folate-binding sites. 62,63,501 The active sites for the dehydrogenase and cyclohydrolase activities of the trifunctional enzyme appear to overlap. 502,503.
The interconversion reactions link the major source of single-carbon units, methylene-THF, with the synthesis of purine (GAR and AICAR transformylase [Figure 155-3, reactions 9 and 10]). The cytoplasmic and mitochondrial systems also permit either the release of single carbons from folate as formate or, more probably, the scavenging of potentially toxic formate (Figure 155-3, reaction 8). Because folate polyglutamates do not cross the mitochondrial membrane, there are effectively two separate pools of folates within the cell, cytosolic and mitochondrial. It has been suggested that under normal conditions, cytoplasmic C1-THF synthase favors incorporation of formate into methylene-THF, while the mitochondrial system, when active, favors production of formate from methyleneTHF; this formate can cross the mitochondrial membrane to serve as substrate in the cytoplasm for synthesis of 10-formyl-THF required for purine synthesis. 492
10-Formyl-THF dehydrogenase ([Figure 155-3, reaction 14)]; [FTHFD], AF052732, MIM 600249) releases excess active single-carbon fragments from the folate pool and generates carbon dioxide. Its activity is restricted to the liver 64 and serves to maintain sufficient THF to permit acceptance of single carbons in folate-dependent reactions. A mitochondrial homolog of this enzyme, encoded by the ALDH1L2 gene on chromosome 12q24.11, has recently been identified. 504
5,10-MethenylTHF synthase is an ATP-dependent enzyme (Figure 155-3, reaction 13; [MTHFS], NM_006441, MIM 604197) 65–67 that catalyzes the interconversion of 5-formyl-THF (folinic acid) and 5,10-methenyl-THF. Activity of this enzyme is inhibited by 10-formyl-THF, 505 and it has been suggested that 5-formyl-THF, which is not a substrate for any known folate dependent reaction, serves as a storage form of folate that can re-enter the active folate pool when the 10-formyl-THF level falls. This enzyme is also important in supporting the clinical use of folinic acid for preventing methotrexate toxicity.
5,10-Methylene-THF reductase (MTHFR) (Figure 155-3, reaction 2; [MTHFR], AJ237672, MIM 236250) 68–70 converts 5,10-methylene-THF to 5-methyl-THF. 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 S-adenosylmethionine, which is bound by the C-terminal regulatory region. 72 The reaction catalyzed by MTHFR is bidirectional in vitro, but in vivo it is essentially unidirectional toward 5-methyl-THF. The human gene on 1p36.3 consists of 11 exons. 73,74
Methionine synthase, also known as 5-methyl-THF:L-homocysteine methyltransferase ([MTR], NM_000254, MIM 156570), is a cobalamin-dependent enzyme that catalyzes the transfer of a methyl group from methyl-THF (or S-adenosylmethionine) to homocysteine to form methionine (Figure 155-3, reaction 1). In the complete reaction, the methyl group from methyl-THF is transferred to enzyme-bound fully-reduced cob(I)alamin to form methylcobalamin. 506,507 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 is required to regenerate methylcobalamin by reductive methylation, using S-adenosylmethionine as methyl source. In bacteria this is accomplished by a two-component flavoprotein system 97,98 consisting of flavodoxin 99 and flavodoxin reductase. 100 In humans, this is accomplished by a single protein, methionine synthase reductase, a unique member of the ferredoxin-NADP+ reductase family of electron transferases that contains binding sites for FMN, FAD and NADPH. 83,508 The FMN domain of methionine synthase reductase has been shown to form a complex with the activation domain of methionine synthase. 509,510 Methionine synthase reductase 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
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 confirmed this assignment at 1q43.85–86,511 The predicted sequence of the human enzyme is 55 percent identical to the cobalamin-dependent methionine synthase from E. coli 86 (bacteria also have a noncobalamin-requiring methionine synthase). The bacterial enzyme has been extensively studied, and has been shown to be a modular protein, with an N-terminal homocysteine-binding domain, a 5-methyl-THF-binding domain, a cobalamin-binding domain, and a C-terminal activation domain that contains an S-adenosylmethionine-binding site. 506 The structure of each of these domains of bacterial methionine synthase has been determined by X-ray crystallography. 89,90,512,513 Cobalamin binds to methionine synthase in the “base-off histidine-on” conformation, with its dimethylbenzimidazole base displaced from the lower axial position of the central cobalt atom and replaced by a histidine residue of the protein. 80 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 methyl-THF monoglutamate and because the MTHFR reaction is essentially irreversible in the cell, folate entering cells must pass through the methionine synthase reaction in order to generate THF and, from THF, the other folate cofactors. 91–95 In cobalamin deficiency (acquired or inherited, see below), or when cobalamin is irreversibly oxidized by nitrous oxide, 514,515 methionine synthase activity decreases or is absent, methyl-THF and homocysteine accumulate, and methionine and, especially, S-adenosylmethionine are reduced. In addition to the folate being "trapped" as methyl-THF, most of it remains as the monoglutamate because methyl-THF is a poor substrate for folylpolyglutamate synthase. Folic acid or folinic acid can bypass this block until methyl-THF again accumulates as a result of MTHFR activity.
Disorders of Folate Nutrition, Transport, and Metabolism
Although a number of children born to mothers with a diet deficient in cobalamin have shown evidence of cobalamin deficiency (see "Cobalamin (Vitamin B12)" below), folate deficiency in the infant secondary to deficiency in the mother is unusual. 15 In nutritional folate deficiency 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 formiminoglutamate (FIGLU) levels. Psychologic and mental changes followed, but were rapidly reversed by folic acid supplementation. Red blood cell folate levels fall in folate deficiency significantly 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 infants 104 and, particularly, supplements given in the periconceptional period to women to reduce the frequency of neural tube defects. 105 The observation that periconceptional supplementation with folic acid can prevent occurrence as well as recurrence of neural tube defects has led to supplementation of grain foods in North America with folic acid since 1998. It has also been suggested that increased folate intake may serve to reduce serum concentration of homocysteine, a likely risk factor in cardiovascular disease (see Chap. 88). 106–108 However, several large studies have failed to demonstrate any reduction in occurrence of cardiovascular disease or death with reduction of serum homocysteine levels. 516,517
The processes and reactions affected by inherited disorders of folate transport and metabolism are shown in Figure 155-4. Those that are discussed in some detail below include hereditary folate malabsorption (reaction 5); cerebral folate deficiency; glutamate formiminotransferase deficiency (reaction 4); MTHFR deficiency (reaction 1); and functional methionine synthase deficiency (reactions 2 and 3).
Processes and reactions affected by inherited disorders of folate transport and metabolism; 1. Methylene-THF reductase (MTHFR) deficiency; 2. and 3. Functional methionine synthase deficiency (cblE, methionine synthase reductase deficiency; cblG, methionine synthase deficiency); see text; 4. Glutamate formiminotransferase deficiency; 5. Hereditary folate malabsorption (affecting folate transport in both the intestine and the choroid plexus; 6. Cerebral folate deficiency (affecting folate transport at the choroid plexus only. Suspect or unproven disorders: A, dihydrofolate reductase deficiency; B, methenyl-THF cyclohydrolase deficiency; C, cellular uptake defect of folate; and D, methyl-THF:homocysteine methyltransferase deficiency (original report from Japan9). Steps affected in 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 = adenosylmethionine; DHF = dihydrofolate; THF = tetrahydrofolate; methylCbl = methylcobalamin; GAR = 5-phosphoribosylglycinamide; FGAR = α-N-formyl-glycinamide ribonucleotide; AICAR = 5-phosphoribosyl-5-aminoimadazole-4-carbox- amide; C2, C8 = carbons number 2 and 8 or of purine ring.
Hereditary Folate Malabsorption (MIM 229050)
Clinical and Laboratory Findings
This disorder [Figure 155-4 (5)], which has also been called congenital malabsorption of folate because of its early clinical presentation, has been described in more than 20 patients, mostly females.109–123,518–522 The common clinical presentation in hereditary folate malabsorption is severe megaloblastic anemia in the first few months of life with low serum folates. Diarrhea, mouth ulcers, and failure to thrive are common, and most patients have shown progressive neurologic deterioration. Folinic acid-responsive peripheral neuropathy has been described. 115,123 Laboratory findings may include urinary excretion of FIGLU and orotic acid. 12,120 Uptake of folate in the intestine and its transport across the blood-brain barrier both appear to be affected in this disorder. All patients have been severely restricted in their ability to absorb oral folic acid and reduced folates. Parenteral therapy with large doses of folates has been effective in correcting blood folate levels and anemia, 111,112,120 but has been of limited effectiveness in correcting the levels of folate in the CSF. One of the patients 120 had additional findings, including a relative inability to retain plasma folate after parenteral folate administration, a finding 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 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 boys 121 had a partial deficiency in both humoral (surface Ig and response to pokeweed mitogen) and cellular (E-rosette forming and response to hemagglutinin and concanavalin A) immunity.
Mutations in the SLC46A1 gene on 17q11.2 that encodes the PCFT have been reported in 14 patients, including two sets of siblings, with hereditary folate malabsorption. 483,484,523–526 These mutations included a splice site mutation that resulted in loss of exon 3, a single-base deletion, a single-base duplication in exon 1, a nonsense mutation (affecting two adjacent bases and resulting in a stop codon in exon 1) and 6 missense mutations, all affecting highly conserved amino acid residues. Function of most of these mutations has been assessed in an in vitro system and they have been shown to result in decreased uptake of 5-methyl-THF at pH 5.5. 484,527 All patients were shown to have two mutated alleles, and the disorder was shown to segregate as an autosomal recessive trait in three families. 483,523,524 The reason for the apparent excess of affected females with hereditary folate malabsorption remains unknown.
It is essential to maintain folate levels in the serum, red blood cells, and CSF above levels associated with folate deficiency (4, 150, and 15 ng/ml, respectively). 15 Some patients may respond to large oral doses of folic acid, folinic acid, or methyl-THF. 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 Studies have suggested that folinic acid 118,119 or methyl-THF is more effective in increasing CSF folate levels. One patient treated for 15 years with intramuscular folinic acid has shown normal physical and mental development. 518
Cerebral Folate Deficiency (MIM 613068)
Patients with cerebral folate deficiency have markedly decreased CSF levels of 5-methyl-THF, the predominant form of folate in the brain, in the presence of normal or elevated serum folate levels. 130,528–534. This condition has also been referred to as idiopathic cerebral folate deficiency to differentiate it from the decrease in CSF folate that can occur during chronic use of antifolate or anticonvulsant drugs or in Rett syndrome, Aicardi-Goutières syndrome, 3-phosphoglycerate dehydrogenase deficiency, dihydropteridine reductase deficiency, aromatic amino acid decarboxylase deficiency or Kearns-Sayre syndrome. 529 Clinically, patients have been apparently normal at birth. Typical features have included marked unrest, irritability and sleep disturbances starting in the first year of life, followed by psychomotor retardation and regression, cerebellar ataxia, and dyskinesia. There has been deceleration in the rate of head growth starting at approximately six months of age in most patients. Visual disturbances typically begin to occur at three years of age, and sensorineural hearing loss at six years. MRI studies have shown cerebral atrophy in several cases. About one-third of patients with the disorder developed epilepsy 529 and many patients have been diagnosed with autism or autistic spectrum disorders. 529,532 One patient presented in adolescence with catatonic schizophrenia which did not respond to therapy with folinic acid. 535
Cerebral folate deficiency is caused by decreased function of folate receptor α (FR-α), which is required in addition to the proton-coupled folate transporter for transport of folate across the blood-brain barrier at the choroid plexus. In most cases the disorder appears to be of autoimmune origin, caused by the presence of blocking antibodies directed against FR-α. 536 However, mutations in the FOLR1 gene that encodes FR-α have been identified in some families. 537,538 Mutations identified included two nonsense mutations and an in-frame duplication of 6 amino acids. Expression studies showed an absence of mRNA for both of the nonsense mutations, while the duplication was shown to result in almost complete loss of FR-α function. 537
These disorders (Figure 155-4 (C)) appear in a group of reported patients with varied clinical findings, 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 therapy with high doses of folate. 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, affecting 34 individuals in four generations, and resulting in the death of 18. The proband showed a marked reduction in the uptake of methyl-THF 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-THF by bone marrow cells from the proband and his son. Of particular interest, however, was the finding 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 V max and total uptake of methyl-THF, whereas folic acid uptake was normal; the daughter showed only a possible elevation in the K m for methyl-THF, while the three clinically normal brothers resembled the proband kinetically. The status of both of these disorders of cellular uptake remains to be clarified.
Dihydrofolate Reductase Deficiency-Suspect Disorder
There are two published reports describing three cases of putative dihydrofolate reductase deficiency 132,133 [Figure 155-4 (A)]. Megaloblastic anemia developed in these patients soon after birth and showed a better clinical response to folinic acid (5-formyl-THF), a reduced folate, than to folic acid, an oxidized folate. In all three patients, dihydrofolate reductase activity was decreased in liver biopsies.
The original patient 132 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 μg/day of folic acid did not produce a clinical response; 5 mg/day of oral folic acid resulted in a sustained 3-year remission, with relapse when folate therapy was discontinued. 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 fibroblasts 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 deficiency. 133 Activity in a liver biopsy was not detectable in the routine assay in the first 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 deficiency (cblE complementation group, see below).
The second child was first 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 deficient in functional transcobalamin 135 (see cobalamin section below). There was absent unsaturated serum cobalamin-binding capacity, although immunoassay did show transcobalamin protein levels at 39 percent of the normal mean. There was no cobalamin-binding protein corresponding to transcobalamin 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 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 difficult 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 deficiency in an infant with severe megaloblastic anemia must be considered, all other known causes must be ruled out before this diagnosis can be confirmed.
Methenyltetrahydrofolate Cyclohydrolase Deficiency-Suspect Disorder
As previously discussed, methenyl-THF cyclohydrolase (Figure 155-3, reaction 7) is part of a trifunctional protein that contains the activities of methylene-THF dehydrogenase, methenyl-THF cyclohydrolase, and 10-formyl-THF synthase. 58 Methenyl-THF cyclohydrolase deficiency [Figure 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. 539 Clinically, the patients had mental retardation, microcephaly, ventricular dilatation, and abnormal electroencephalograms. A later report from the same laboratory 9 essentially retracted the diagnosis, and no additional cases have been reported.
Glutamate Formiminotransferase Deficiency (MIM 229100)
During catabolism of histidine, a formimino group is transferred from formiminoglutamate to tetrahydrofolate, followed by the release of ammonia and the formation of 5,10-methenyl-THF. The two enzyme activities involved in these steps, glutamate formiminotransferase (EC 220.127.116.11) (Figure 155-3, reaction 11) and formimino-THF cyclodeaminase (EC 18.104.22.168) (Figure 155-3, reaction 12), share a single polypeptide, which forms an octomeric enzyme 101,540 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 deficiency is associated with a disease state or whether the association of clinical findings with FIGLU excretion is a result of bias of ascertainment. 5,126,131 Individuals with glutamate formiminotransferase deficiency [Figure 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 direct analysis of the FTCD gene in patients does not support this hypothesis. 541
Diagnosis of this disease is hampered by the absence of enzyme activity in cultured human cells; 12 there is dispute as to whether the deficiency can be diagnosed using red blood cells. 5,141 In most cases in which the liver has been examined, enzyme activities were higher than would be expected for a complete block resulting in disease. 9 Erbe 5 has summarized most of the known patients with glutamate formiminotransferase deficiency. 140,142–155 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 findings, including hypersegmentation of neutrophils and macrocytosis. The reported biochemical findings 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-amino-5-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 fibromatous 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 deficiency.
Enzyme activity was measured in the livers of five 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
Mutations of the FTCD gene (MIM 606806, AF169017) on chromosome 21q22.3 540 have been identified in three patients with the mild phenotype of formiminotransferase deficiency. 541 Two siblings were heterozygous for two missense mutations (c.403C→T [p.R135C] and c.896G→C [p.R299P]) affecting the formiminotransferase domain of the bifunctional protein. The third patient was hemizygous for a c.990dupG mutation; the second FTCD allele in this patient was apparently completely deleted. Formimino transferase activity of a porcine protein engineered to express the missense mutations was decreased in both cases: R135C reduced activity to 61 percent of control activity, and R299P reduced activity to 57 percent of control. These activities were similar to those measured in liver from formiminotransferase deficient patients. The insertion mutation resulted in a truncated protein with formiminotransferase activity that lacked the cyclodeaminase domain. 541 The results of these studies show that formiminotransferase deficiency is inherited as an autosomal recessive trait.
The major difficulty 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 definitive diagnosis is difficult. In addition, FIGLU excretion may be caused by other defects in folate or cobalamin metabolism. Indeed, further studies of fibroblasts of one patient given a diagnosis of FTCD deficiency, who had megaloblastic anemia and folate-responsive homocystinuria, 141 showed low methionine biosynthesis, low methionine synthase activity, and low MeCbl synthesis; the patient was subsequently shown to have methionine synthase deficiency (cblG complementation group, see below). Thus, it is appropriate to study fibroblasts from all patients who show evidence of hypomethioninemia for evidence of a functional deficiency in methionine synthase.
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 patients 152,154 responded to methionine supplementation. Given that the correlation between clinical phenotype and FIGLU excretion remains uncertain, the basis for treating these patients is unclear.
Methylenetetrahydrofolate Reductase Deficiency (MIM 236250)
Methylene-THF reductase (MTHFR) (EC 22.214.171.124) is a cytoplasmic enzyme that catalyzes the NADPH-linked reduction of methylene-THF to methyl-THF (Figure 155-3, reaction 2). Methyl-THF serves as the methyl donor for the methylation of homocysteine in the reaction catalyzed by methionine synthase (5-methyl-THF:homocysteine methyltransferase (Figure 155-3, reaction 1). The combined action of MTHFR and methionine synthase supplies single-carbon units for methylation reactions that use S-adenosylmethionine. The reaction catalyzed by MTHFR reductase is essentially irreversible under physiological conditions, and enzyme activity is regulated by levels of S-adenosylmethionine, which is an inhibitor. 69,157,158
Clinical and Laboratory Findings
Since the first reports of MTHFR deficiency in 1972 160 [see Figure 155-4 (1)], more than 90 cases have been reported.161–163,166–169,172–184,189,190,192–194,196,197,199,200–202,542–556 The major biochemical findings have been moderate homocystinuria and hyperhomocysteinemia 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. 182,193,198. The most common clinical manifestation in MTHFR deficiency 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. Patients with this disease rarely have megaloblastic anemia. Homocystinuria was present in all patients, with a reported range of 15 to 667 μM/24 h and a mean of 130 μM/24 h. Homocystine, not normally detected in urine or free in plasma, was found in the plasma: mean value 57 μM (range: 12 to 233 μM). Values of total plasma or serum homocysteine (tHcy) ranging from 60 to 320 μM (controls: 4 to 14 μM) have been reported. 194,202–204,548,549,551–554 Plasma methionine levels were low in all patients, ranging from 0 to 18 μM, with a mean of 12 μM; normal is 23 to 35 μM, 5 although values vary among laboratories.
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 finding. 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 affected older brother was asymptomatic at age 37 years. 194
Although homocystinuria was consistently seen in all patients, and indeed is the clinical clue by which the diagnosis of MTHFR deficiency is made, the excretion of homocystine in urine is much less than that found in homocystinuria due to cystathionine β-synthase deficiency (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 MTHFR deficiency are always low-normal or low. This, again, distinguishes these patients from those with cystathionine β-synthase deficiency, who generally have hypermethioninemia. In contrast to patients who are functionally deficient in methionine biosynthesis because of abnormalities in methylcobalamin formation (complementation groups cblC, cblD, cblE, cblF, and cblG; see below), patients with MTHFR deficiency do not have megaloblastic anemia. In addition, in contrast to patients with the cblC, classic cblD, and cblF disorders, these patients have no methylmalonic aciduria or acidemia. Although serum folate levels were not always low, many patients with MTHFR deficiency 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 field have been measured in only a minority of patients, they have usually been low. 5,200
Studies on Cultured Cells
Deficiency of MTHFR has been confirmed by studies of liver, leukocytes, and cultured fibroblasts and lymphoblasts. The enzyme assay routinely used for these studies measures the activity in the nonphysiological direction, using radioactive methyl-THF as substrate and menadione as electron acceptor, but it can be assayed in the physiological direction as well. 68 Under the latter conditions, the concentration of S-adenosylmethionine required for inhibition is considerably smaller than that required for inhibition of the reverse reaction. 69 Activity is extremely sensitive to the stage of the culture cycle in fibroblasts, with the specific activity in control cells being highest in confluent cultures. 158 This variability is sufficiently great to allow for the misclassification of controls and heterozygotes if not taken into account. In general, there is rough correlation between residual enzyme activity and clinical severity. Levels and distribution of folate derivatives have been studied in cultured fibroblasts 164,171 and liver 177,186. A direct relationship was found in cultured fibroblasts between the proportion of cellular folate present as methyl-THF 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. 161,171 The synthesis of methionine from labeled formate in cultured cells 178,206 also correlates with clinical severity.
Control cultured fibroblasts can grow when homocysteine, together with folate and cobalamin, is substituted in the culture medium for methionine, an essential amino acid for these cells. In contrast, fibroblasts from patients with MTHFR deficiency do not grow under these conditions. 160,165 This inability to grow on homocysteine is shared by fibroblasts from patients who are functionally deficient in methionine synthase (cblC, cblD, cblE, cblF, and cblG; see cobalamin section below). 207
Genetic heterogeneity in the severe form of this disorder was suggested by the fact that fibroblast extracts from two of the original families showed differential heat inactivation at 55°C. 165 It was subsequently shown that thermolabile reductase occurs in patients with both early- and late-onset disease as well as in the general population. 208 In some patients, it was shown that thermolability was due to the presence of severe MTHFR mutations in combination with the common 677C→T polymorphism that is responsible for the majority of enzyme thermolability in the general population. 209,210
Neuropathologic changes in severe MTHFR deficiency include dilated cerebral ventricles, internal hydrocephalus, microgyria, and low brain weight. 5,163,166,168,184–186,189,192 Also seen in the brain are perivascular changes, demyelination, macrophage infiltration, gliosis, and astrocytosis. Other major pathologic findings are thromboses of arteries and cerebral veins; these appear to have been major factors in the death of these patients. Two reports 189,192 have described classical findings of subacute combined degeneration of the cord, similar to that observed in patients with untreated cobalamin deficiency, in patients dying with MTHFR deficiency. One patient with MTHFR deficiency had a fibrosarcoma. 192
In general, patients with disorders of cobalamin metabolism, who also have a block in methionine biosynthesis, have similar neurologic findings to patients with MTHFR deficiency, but the hematological abnormalities present in the inborn errors of cobalamin metabolism are absent in MTHFR deficient patients. It has been pointed out 192 that the neuropathologic vascular findings in MTHFR deficiency are similar to those seen in classical homocystinuria due to cystathionine β-synthase deficiency. Overall, the vascular findings may be attributed to elevated homocysteine levels, while the demyelination, astrogliosis, and lipid-filled macrophages, which are associated in many patients with a progressive course of seizures, microcephaly, and severe psychomotor retardation, may be consequences of methionine deficiency. It has been suggested that the combination of MTHFR deficiency and Factor V Leiden may contribute to the vascular pathology in some patients. 237,238
MTHFR is present in mammalian brain. 239,240 Several authors have suggested that only methyl-THF among the natural folates can cross the blood-brain barrier, 172,241 and thus MTHFR deficiency 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 suggested 77 that the neurologic dysfunction may occur as a result of impaired purine and pyrimidine synthesis in the brain, as opposed to low levels of S-adenosylmethionine. The relative importance of low folate levels, low methionine levels, and low levels of neurotransmitters in the pathology of MTHFR deficiency is uncertain. 242 Differences seen between functional methionine synthase deficiency 236,243 (cblC, cblD, cblE, cblF, and cblG) and MTHFR deficiency should be useful in sorting out the relative importance of low levels of reduced folates and low levels of methionine. These comparisons have the potential of being made more difficult by developmental and tissue differences in the distribution of these enzyme activities. 244,245
The most important finding in the clinical differential diagnosis is the absence of megaloblastic anemia in patients with MTHFR deficiency as compared to patients with functional methionine synthase deficiency (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 fibroblasts from some patients with MTHFR deficiency and that this could lead to the incorrect diagnosis of methionine synthase deficiency (cblE or cblG). 208
MTHFR deficiency has been very resistant to treatment but betaine has improved the overall prognosis. 5,15,126,131,195,557 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-THF to replace the missing product; (c) methionine to correct the cellular methionine deficiency; (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-specific enzyme that converts homocysteine to methionine; and (h) riboflavin, because of the flavin requirement of MTHFR. Criteria for the success of treatment 5 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 difficult to assess the efficiency of a single one.
Cooper 15 suggested a therapeutic regimen consisting of oral betaine, folinic acid, and methionine, with additional vitamin B6 and cobalamin. He recommended cobalamin because of the observations of subacute combined degeneration of the cord 189 in a child treated with methyl-THF alone. Interestingly, therapy with methionine alone or with methyl-THF is not particularly effective in most cases, even though S-adenosylmethionine deficiency 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 riboflavin. 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. 5,181,182,190,551 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. Two patients who were treated from the first month of life 190 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 Strauss et al described treatment of five Old Order Amish children homozygous for the same mutation with betaine; all patients showed improvement in neurological function, although the four older patients (age at diagnosis ranged from 7 months to 18 years) had irreversible brain damage. The youngest child, who was treated from the neonatal period, had normal brain growth and neurological function at the age of three years. 551
Thus, betaine appears to be the most promising agent for therapy of MTHFR deficiency, although, as mentioned above, some other therapies have been partially successful. There is not a great deal of data on the optimum dose of betaine in these patients. Ronge and Kjellman suggested a dose of 6 g/day (3g b.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 Sakura 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 μM, and suggested that this is the therapeutic threshold for serum betaine. Strauss et al treated their patients with 534 ± 222 mg/kg/d betaine resulting in decreased serum homocysteine and increased cerebral 5-methyl-THF, although neither measurement achieved control values. 551
Many authors 5,12,196,202,551 have stressed the importance of early diagnosis and therapy because of the poor prognosis in this disorder once there is evidence of neurologic involvement. Among Old Order Amish patients homozygous for the same mutation, efficacy of therapy was inversely related to the age at which therapy with betaine was started. 551 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.
Neurological deterioration and death of a child with MTHFR deficiency following exposure to nitrous oxide anesthesia has been reported. 558 Nitrous oxide is known to irreversibly inactivate methionine synthase by attacking enzyme-bound reduced cobalamin; the presence of severe MTHFR deficiency in this patient may have rendered him extremely sensitive to these toxic effects. Neurological deterioration responsive to therapy with folic acid and cobalamin has also been described in a patient homozygous for the c.677C→T polymorphism who was exposed to nitrous oxide anesthesia twice in an 8-week period. 563
Kang and his colleagues originally suggested that thermolability of reductase activity at 46°C for 5 min in lymphocyte extracts from adults might be associated with "intermediate homocystinemia" and an increased risk for vascular disease in adult life. 211–214 Cloning of the MTHFR gene 74 led to 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 prevalence of the TT genotype varies among populations, from less than 2% in Africans and African-Americans to 15-35% in Hispanics. 209,222,559,560 The association between homozygosity for the T allele and plasma homocysteine levels was found to be related to folate status, 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, including coronary artery disease, stroke and venous thrombosis, has been extensively studied. Meta-analyses indicate that the TT genotype is associated with a 20% increase in the risk of coronary artery disease, with variations among populations, perhaps as the result of differences in folate nutrition. 561–563 This effect has been attributed to elevation of homocysteine levels, but large-scale epidemiological studies have not shown any preventive effect of lowering plasma homocysteine. 516,564 The TT genotype has also been associated with increased risk of neural tube defects when present in either the mother or fetus. 565–567 Inconclusive results have been obtained for other birth defects, including congenital heart defects, cleft lip and/or palate, and Down syndrome. 560 The 677C→T polymorphism was found to be associated with a decreased risk of colorectal cancer, 232 although this protective effect may be lost if folate levels are low. 232,568,569 The T allele of the MTHFR 677C>T polymorphism may be associated with increased risk of other types of cancer, including gastric and breast cancer and leukemia. 560
Another polymorphism, 1298A→C, which converts glutamate to alanine at amino acid residue 429 (E429A), is also associated with decreased enzyme activity. 233–235 This variant has not been as well studied as the 677C>T polymorphism, and no clear effects on health have been demonstrated. 560 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
The crystal structure of the E. coli enzyme, which is considerably smaller than mammalian MTHFR 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.
MTHFR deficiency is inherited as an autosomal recessive trait. The MTHFR gene on chromosome 1p36.3 encodes a 2.2 kb product and consists of 11 exons. 73,74 Multiple transcripts have been identified, reflecting multiple translation start sites and alternative splicing. 570,571 To date, over 50 mutations have been identified in patients with severe MTHFR deficiency, affecting both the catalytic and regulatory domains of the enzyme (Figure 155-5). 73,203,210,247,547–549,551,553–556,572–576 Several nonsense and splice site mutations have been reported, as well as a deletion resulting in a frame shift, but the majority of mutations have been missense mutations. Nearly all of these have been reported in only one or two families. An exception is a c.1141C→T (R377C) mutation that has been identified in 5 affected Old Order Amish children; screening of 230 healthy members of the Amish community disclosed a 30 percent carrier frequency for this mutation. 551
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 enlarged in order to show all the amino acid changes. (Courtesy of D. Leclerc and R. Rozen, McGill University)
MTHFR deficiency has been diagnosed or excluded prenatally by enzyme assay or by measurement of the incorporation of labeled formate into methionine by cultured amniotic fluid cells. 182,188,190,205,248,249,577 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. In families in which the MTHFR mutations are not known, genotyping of common polymorphisms may allow identification of an affected fetus by linkage. 577 A newborn screening program has been set up among the Old Order Amish, where a specific MTHFR mutation (c.1141C→T) exists at a high frequency. 551
A knockout mouse model for MTHFR deficiency has been created. 578 Homozygous knockout mice had elevated homocysteine levels and decreased body weight and decreased cerebellar and cerebral size compared to wild type and heterozygous mutant mice, with enlarged lateral ventricles. There was depletion of cerebellar granule cells and disorganization of Purkinje cells. Survival of homozygous knockout mice was decreased during the first several weeks after birth, accompanied by motor and gait abnormalities or severe tremor. There was microvesicular steatosis of the liver, and deposition of lipid in the aorta, starting at 10 months of age in homozygotes and present at 14 months in both homozygotes and heterozygotes. 578,579 Treatment of dams with betaine throughout pregnancy and lactation resulted in decreased mortality and improved growth rate of homozygous mutant pups. 580
Administration of a folate-deficient diet to wild-type and heterozygous mutant dams during pregnancy resulted in elevated homocysteine, increased resorption rates and increased developmental delay compared to dams receiving a folate-replete diet. Folate deficiency was associated with increased kevels of neural tube defects and heart defects and of placental abnormalities. 581–583
Functional Methionine Synthase Deficiency
Three metabolic pathways intersect at methionine synthase: those of folate, cobalamin (Cbl), and sulfur-containing amino acids. Because deficiency in the activity of this enzyme results in diminished or absent methylcobalamin (MeCbl) synthesis, disorders affecting it have been considered and designated genetically as cobalamin metabolism defects (symbolized as cbl), along with others affecting synthesis of both MeCbl and adenosylcobalamin (AdoCbl) or of AdoCbl 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 cobalamin metabolism. Nevertheless, because methyl-THF participates in the methionine synthase reaction and because methionine synthase deficiency significantly 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). The others indirectly affecting methionine synthase activity (cblC, cblD, and cblF) will be considered in detail in the subsequent sections on cobalamin metabolism. 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 biochemically. Most patients so far reported with these disorders presented in the first few months of life with vomiting, poor feeding, and lethargy. Hypotonia, seizures, and developmental delay characterize their severe neurologic dysfunction.Table 155-1 summarizes some of the clinical findings in patients with these disorders. The prevalence of neurologic signs and symptoms is striking. Megaloblastic anemia and homocystinuria or hyperhomocysteinemia are generally present, and hypomethioninemia is often found. Serum cobalamin and folate concentrations are normal or elevated, and methylmalonic aciduria is absent, except in one patient, in whom it was a transient finding. 262
Table 155-1:Clinical and Laboratory Features of Patient with Hyperhomocysteinemia Due to Defects in MeCbl Synthesis* |Favorite Table|Download (.pdf) Table 155-1: Clinical and Laboratory Features of Patient with Hyperhomocysteinemia Due to Defects in MeCbl Synthesis*
|Finding ||Mutant Class |
| || cblE || cblG |
| ||N = 21 ||N = 38 |
|Megaloblastic anemia ||16 ||19 |
|Developmental Retardation ||15 ||27 |
|Cerebral Atrophy ||10 ||11 |
|Hypotonia ||5 ||25 |
|Feeding Difficulty ||3 ||11 |
|Lethargy ||2 ||5 |
|Seizures ||5 ||15 |
|Visual Abnormalities ||6 ||10 |
|Microcephaly ||6 ||7 |
Patients with the cblE disorder have usually presented in the first year. A majority of patients have megaloblastic anemia and neurologic abnormalities, 243,262,264,584–588 but some patients have presented with megaloblastic anemia alone. 589 Patients with the cblG disorder have generally presented within the first year of life with megaloblastic anemia and developmental retardation 141,243,252–254,259,261,590,591 but some have presented later in childhood with developmental regression. 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 Others have presented in adulthood with psychosis.
The constellation of homocystinuria and hypomethioninemia without methylmalonic aciduria suggested strongly that these patients had isolated deficiencies in the activity of methionine synthase, either primary or secondary to abnormal synthesis or utilization of MeCbl, its cofactor. Studies of fibroblasts derived from several of these patients supported this hypothesis and identification of the genes underlying the disorders has proven it. Incorporation of [14C]propionate into macromolecules (an indirect measure of AdoCbl-dependent methylmalonyl CoA mutase) was normal, while incorporation of [14C]methyl-THF (an indirect measure of methionine synthase function) 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-THF incorporation distinguished two complementation groups: 263 cblE (index patient reported in reference 264) and cblG (index patient reported in reference 252). Accumulation of cobalamin by fibroblasts was normal or increased in these groups, as was the fraction recovered as AdoCbl. In contrast, the fraction identified as MeCbl was much reduced. 243.
When methionine synthase activities were determined under standard conditions in extracts of fibroblasts from cblE patients, activities were normal or slightly reduced. 243 When the assay was performed in the presence of suboptimal concentrations of reducing agent, methionine synthase activities were less than those in controls. 265 These findings led to the hypothesis that the cblE group has defects in an enzyme required either to reduce cobalamin so that it can participate in the methionine synthase reaction or to maintain it in its active reduced form [cob(I)alamin] on methionine synthase. 265 For example, bacterial methionine synthase has accessory reductase proteins 266 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. 83 It has subsequently been shown that this protein binds to the activating domain of methionine synthase 510,592 and is capable of maintaining methionine synthase-bound cobalamin in its active reduced form. 508 Methionine synthase reductase may also act as a molecular chaperone, stabilizing newly synthesized methionine synthase and increasing the rate of binding of cobalamin to the apoenzyme. 593
In the cblG complementation group, methionine synthase activities are reduced even under optimal assay conditions, 243 although some heterogeneity has been noted. 255 This finding suggested that patients in this group have primary defects in methionine synthase itself. 263 In extracts of cell lines from most patients, about 75 percent of cellular cobalamin 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 cobalamin of any form migrated at this position. 256 This was highly suggestive of mutations in the cobalamin-binding domain of methionine synthase or absent methionine synthase protein in these patients, strengthening the possibility that the cblG group reflected primary deficiencies in the methionine synthase apoenzyme. With the identification and cloning of the gene encoding human methionine synthase, mutations were identified in cblG patients, confirming that mutations affecting this enzyme underlie the cblG disorder. 85,267
The association of isolated functional methionine synthase deficiency 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 cobalamin metabolism, rather than the AdoCbl-methylmalonyl CoA mutase one. Hall 268 and Shevell and Rosenblatt 269 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 deficiency, and the response of each to treatment may likewise be distinctive. The hematologic problems may reflect 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 cobalamin therapy. 268 Hall has suggested a wide range of possibilities for the disruption of function in the nervous system. They include toxicity of methyl-THF or homocysteine, the classic folate trap hypothesis or variants of it (discussed above), and reduced methylation of proteins and neurotransmitters due to deficiency 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 deficiencies 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.
Both of these diseases are inherited as autosomal recessive traits. 243,257,268 The MTRR gene (MIM 602568) encoding methionine synthase reductase is on chromosome 5p15.2-15.3. 83 Several disease-causing mutations have been identified in patients with the cblE disorder (Figure 155-6). 83,257,586,587,589 The most frequent of these is an intronic mutation (c.903+469T→C) that represents 25 percent of disease causing alleles that have been identified. This sequence change results in creation of a novel exonic splicing enhancer and inclusion of a pseudoexon of 140 bp in mRNA. 594 A c.1361C>T (p.S454L) mutation has been observed in patients of Iberian origin and may be associated with a milder cblE phenotype, characterized by megaloblastic anemia without neurological involvement. 589 In addition, a number of polymorphisms have been identified, 587,595,596 some of which are being investigated for influences on the frequency of birth defects, in the same way as polymorphisms in the MTHFR gene. The activity of methionine synthase reductase containing one disease-causing mutation, c.166G→A (V156M), has been shown to be modulated by the forms of two of these MTRR polymorphisms, c.66A→G (p.I22M) and c.524C→T (p.S175L). 597
Mutations in the MTRR gene and corresponding changes in the methionine synthase reductase amino acid sequence. The locations of the FMN-binding, FAD-binding and NADPH-binding domains in the protein are shown.
The human methionine synthase gene ([MTR] U75743, MIM 157570) on chromosome 1p43 consists of 33 exons spanning at least 60 Kb of genomic DNA. 85,86,511,598 Twenty-two disease-causing mutations have been identified in patients with the cblG disorder (Figure 155-7) 85,260,267,556,598 affecting all domains of the modular methionine synthase enzyme. Notably, all of the cblG variants so far examined appear to be effectively null for the methionine synthase protein rather than cobalamin-binding mutants. 260 The most frequently observed MTR mutation was c.3518C→T (p.P1173L), which appears to disrupt the S-adenosylmethionine binding site in the activation domain of methionine synthase; 267 this accounts for 42 percent of all identified pathogenic alleles. 598 Despite the frequency of this allele, no homozygotes have been identified, suggesting that homozygotes may not be viable.
Mutations in the MTR gene and corresponding changes in the sequence of the methionine synthase protein. Locations of the homocysteine-binding domain, the methyl-THF-binding domain, the cobalamin-binding domain and the activation domain (which contains the adenosylmethionine-binding site) are shown. (Modified from Watkins et al, 2002 598)
Knockout mouse models of both the cblE and the cblG disorders have been created. Complete inactivation of either the MTRR or the MTR gene is embryonic lethal. 599,600. In transgenic mice homozygous for a hypomorphic MTRR allele, methionine synthase activity was decreased to 40-60% of its level in wild type mice. There were significantly elevated homocysteine levels with decreased levels of methionine, and significantly decreased growth rate for males although not for females. 600 Mothers homozygous for the gene trapped allele had more resorptions, smaller placentas and embryos, and a higher frequency of embryos with ventricular septal cardiac defects than wild type mothers; the homozygous genotype in embryos was associated with reduced embryo length, decreased placental and embryo weight and higher frequency of ventricular septal defects. 601
Diagnosis, Treatment, and Prognosis
The clinical hallmarks of these disorders are developmental delay and megaloblastic anemia, with homocystinuria and without methylmalonic aciduria. Although most patients have been diagnosed early in life, one (a cblG) did not come to medical attention until age 21. 250 Differentiation from other possible diagnoses such as transcobalamin deficiency (see below), other folate transport or metabolism defects, or cystathionine β-synthase deficiency (Chap. 88) can be accomplished by studies of cultured cells, particularly by incorporation of 14C from [14C]methyl-THF and complementation analysis. These two assays are especially important because deficiency of MTHFR has a similar clinical presentation and may result in decreased cellular MeCbl accumulation and even decreased methionine synthase activity. 208 Prenatal diagnosis has been performed for both the cblE and cblG disorders using amniotic fluid cells; 271,586,602 in some cases measurement of homocysteine levels in amniotic fluid by mass spectrometry has also been used. With the cloning of the genes for both methionine synthase reductase and methionine synthase, molecular diagnosis is possible in families where the mutations are known.
Because the patients reported to date have responded to hydroxocobalamin (OHCbl) therapy with normalization of their biochemical parameters and at least partial resolution of their clinical symptoms 236,243,255,268 and because it seems likely, as in cblC patients (below), that delays in treatment may result in incompletely reversible developmental delays or neurologic deficits, 243,250,268 institution of OHCbl administration should occur as soon as the diagnosis is made or suspected. Dosages of 1 mg OHCbl 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, a 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 cobalamin 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 OHCbl alone, may be justified to avoid the vascular injury and thromboembolism associated with hyperhomocysteinemia (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 hyperhomocysteinemia.
Because patients with these disorders have been described relatively recently, the long-term prognosis in these conditions remains unknown. The index cblE patient was thriving at 18 years of age, although he was mildly developmentally delayed, 236 while his prenatally diagnosed and treated brother (14 years old) appeared normal, except for a slight speech impediment. 269 In contrast, the index cblG patient, although clinically well, remains significantly retarded with major visual defects. 252 It seems likely that patients in the cblE and cblG groups will show a range of clinical outcomes, 243,268,586,588,589 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 deficiency. 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 MTHFR deficiency 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 deficiency. Homocystinuria has been described in MTHFR deficiency 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 deficiency, except in the original Japanese patients, and only rarely in MTHFR deficiency.
Table 155-2:Inherited Defects of Folate Metabolism |Favorite Table|Download (.pdf) Table 155-2: Inherited Defects of Folate Metabolism
| ||Hereditary Folate Malabsorption ||MTHFR Deficiency ||Glutamate Formimino-transferase Deficiency ||Methionine Synthase Reductase Deficiency (cblE) ||Methionine Synthase Deficiency (cblG) |
|Clinical findings |
|Prevalence ||20 cases ||>90 cases ||<20 cases ||21 cases ||38 cases |
|Megaloblastic anemia ||A ||N ||N* ||A ||A |
|Developmental delay ||A ||A ||N* ||A ||A |
|Seizures ||A ||A ||N* ||A ||A |
|Speech abnormalities ||N ||N ||A* ||N ||N |
|Gait abnormalities ||N ||A ||N* ||N ||A* |
|Peripheral neuropathy ||N* ||A ||N* ||N ||A* |
|Apnea ||N ||A ||N* ||N* ||N |
|Biochemical findings |
|Homocystinuria/hyperhomocysteinemia ||N ||A ||N ||A ||A |
|Hypomethioninemia ||N ||A ||N ||A ||A |
|Formiminoglutamic aciduria ||A* ||N ||A ||N ||N* |
|Folate absorption ||A ||N ||N ||N ||N |
|Serum cobalamin ||N ||N ||N* ||N ||N |
|Serum folate ||A ||A ||N* ||N ||N |
|Red blood cell folate ||A ||A* ||N* ||N ||N |
|Defects detectable in cultured whole cells/fibroblasts |
|MethylTHF fixation ||N ||N ||N ||A ||A |
|MethylTHF content ||N ||A ||N ||N ||N |
|MeCbl content ||N ||N* ||N ||A ||A |
|Extracts – specific activity ||Activity undetectable in cultured cells ?Abnormal in liver and erythrocytes |
|Methionine synthase ||N ||A ||N ||N** ||A |
|Glutamate formiminotransferase |
|MTHFR ||N || ||N ||N ||N |
|Treatment ||Folic acid, Reduced folate ||Betaine, folate, methionine ||?folate ||OH-Cbl, betaine, reduced folate |
Defects detectable in cultured cells include a decreased incorporation of label from methyl-THF into protein in cblE and cblG disease, or from formate into methionine in MTHFR deficiency, cblE and cblG disease, and a decreased content of methyl-THF in fibroblasts from patients with MTHFR deficiency. Cells from patients with cblE and cblG show decreased levels of MeCbl, as may cells from some patients with MTHFR deficiency. 208 In cell extracts of cultured fibroblasts, activity of MTHFR is decreased in MTHFR deficiency. In extracts of cblE and cblG cell lines, abnormalities in methionine synthase activity can be detected. Abnormalities of glutamate formiminotransferase have not been detected in any cultured cell system.