28 Disorders of Cobalamin and Folate Transport and Metabolism

David S. Rosenblatt, Brian Fowler

28.1 Disorders of Absorption and Transport of Cobalamin – 343 28.1.1 Hereditary Deficiency – 343 28.1.2 Defective Transport of Cobalamin by Enterocytes (Imerslund-Gräsbeck Syndrome) – 343 28.1.3 Haptocorrin (R Binder) Deficiency – 344 28.1.4 Deficiency – 344

28.2 Disorders of Intracellular Utilization of Cobalamin – 345 28.2.1 Combined Deficiencies of Adenosyl cobalamin and Methylcobalamin – 345 28.2.2 Adenosylcobalamin Deficiency – 347 28.2.3 Methylcobalamin Deficiency – 348

28.3 Disorders of Absorption and Metabolism of Folate – 351 28.3.1 Hereditary Folate Malabsorption – 351 28.3.2 Glutamate-Formiminotransferase Deficiency – 351 28.3.3 Methylenetetrahydrofolate Reductase Deficiency – 352

References – 353 342 Chapter 28 · Disorders of Cobalamin and Folate Transport and Metabolism

Cobalamin Transport and Metabolism

Cobalamin (cbl or ) is a cobalt-containing multiple intracellular conversions. As methylcobalamin, water-soluble vitamin that is synthesized by lower it is a cofactor of the cytoplasmic enzyme methionine organisms but not by higher plants and animals. In the synthase. As adenosylcobalamin, it is a cofactor of human diet, its only source is animal products in which the mitochondrial enzyme methylmalonyl-coenzyme it has accumulated by microbial synthesis. Cbl is needed A mutase, which is involved in the catabolism of valine, for only two reactions in man, but its metabolism in- threonine and odd-chain fatty acids into succinyl-CoA, volves complex absorption and transport systems and an intermediate of the Krebs cycle.

V

. Fig. 28.1. Cobalamin (Cbl) endocytosis and intracellular me- TC, transcobalamin (previously TCII); V1, variant 1; V2, variant 2; tabolism. The cytoplasmic, lysosomal, and mitochondrial com- 1+,2+,3+ refer to the oxidation state of the central cobalt of Cbl. partments are indicated. AdoCbl, adenosylcobalamin; CoA, co- Letters A-H refer to the sites of blocks. Enzyme defects are indicat- enzyme A; MeCbl, methylcobalamin; OHCbl, hydroxycobalamin; ed by solid bars 343 28 28.1 · Disorders of Absorption and Transport of Cobalamin

affinity for Cbl, receptor or increased susceptibility to pro- For patients with inherited disorders affecting cobala- teolysis [7–9]. min (Cbl) absorption, the main clinical finding is mega- loblastic anemia. Except for transcobalamin (TC) defi- Genetics ciency, the serum Cbl level will usually be low. Patients At least 45 patients of both sexes have been reported, and with disorders of intracellular Cbl metabolism show inheritance is autosomal recessive. A cDNA has been cha- elevations of homocysteine or methylmalonic acid, racterized, and the is localized on 11q13 either alone or in combination. The serum Cbl level is [10]. A recently described variant of the gastric IF (GIF) not usually low. For those disorders that affect methyl- gene, 68AoG, is probably not a disease causing mutation cobalamin (MeCbl) formation, the major manifestations but could serve as a marker for inheritance of the disorder include megaloblastic anemia secondary to folate [11]. A 4-bp deletion (c183_186delGAAT) in the coding deficiency and neurological abnormalities presumably region of the GIF gene was identified as the cause of intrinsic secondary to methionine deficiency or homocysteine factor deficiency in an 11 year-old girl with severe anemia elevation. The main findings in those disorders that and Cbl deficiency [12]. affect adenosylcobalamin (AdoCbl) formation, are sec- ondary to elevated methylmalonic acid and resultant Diagnostic Tests acidosis. The hematological abnormalities in the defects of Cbl ab- sorption and transport should be detected by measurement of red blood cell indices, complete blood count and bone Inherited disorders of cobalamin (Cbl) metabolism are di- marrow examination. Low serum Cbl levels are present. vided into those involving absorption and transport and A deoxyuridine suppression test on marrow cells is useful those involving intracellular utilization [1–5]. but is not easily available in most clinical laboratories. In hereditary IF deficiency, in contrast to acquired forms of pernicious anemia, there is normal gastric acidity and 28.1 Disorders of Absorption normal gastric cytology. Cbl absorption, as measured by and Transport of Cobalamin the Schilling test, is abnormal but is normalized when the labeled Cbl is mixed with a source of normal IF, such as Absorption of dietary Cbl involves first binding to a glyco- gastric juice from an unaffected individual. (R binder, haptocorrin) in the saliva. In the intes- tine, haptocorrin is digested by proteases, allowing the Cbl Treatment and Prognosis to bind to intrinsic factor (IF), which is produced in the IF deficiency can be treated initially with hydroxycoba- stomach by parietal cells. Using a specific receptor, the IFCbl lamin (OHCbl, 1 mg/day intramuscularly) to replete body complex enters the enterocyte. Following release from this stores until biochemical and hematological values nor- complex Cbl binds to transcobalamin (TC), the physiologi- malize. The subsequent dose of OHCbl required to main- cally important circulating Cbl-binding protein, forming tain normal values may be as low as 0.25 mg every 3 months. TC-Cbl, which is then slowly released into the portal vein. If treatment is delayed, some neurological abnormalities Inherited defects of several of these steps are known. may persist in spite of complete reversal of the hematologi- cal and biochemical findings.

28.1.1 Hereditary Intrinsic Factor Deficiency 28.1.2 Defective Transport of Cobalamin by Enterocytes Clinical Presentation (Imerslund-Gräsbeck Syndrome) Presentation is usually from one to 5 years of age but in cases of partial deficiency, can be delayed until adolescence Clinical Presentation or adulthood. Patients present with megaloblastic anemia Defective transport of Cbl by enterocytes, also known as as the main finding, together with failure to thrive, often Imerslund-Gräsbeck syndrome or megaloblastic anemia 1 with vomiting, alternating diarrhea and constipation, ano- (MGA1), is characterized by prominent megaloblastic rexia and irritability [6–8]. Hepatosplenomegaly, stomatitis anemia manifesting once fetal hepatic Cbl stores have been or atrophic glossitis, developmental delay, and myelopathy depleted. The disease usually appears between the ages of or peripheral neuropathy may also be found. 1 year and 5 years, but onset may be even later [13–19]. Most patients have proteinuria and, in a few cases, this is Metabolic Derangement of the tubular type, with all species of represented IF is either absent or immunologically detectable but non- rather than albumin alone. The literature on the renal functional. There have been reports of IF with reduced pathology has been reviewed [20]. Although patients who 344 Chapter 28 · Disorders of Cobalamin and Folate Transport and Metabolism

excreted protein during childhood continued to excrete 28.1.3 Haptocorrin (R Binder) Deficiency protein in adulthood, the renal lesions were not progressive [14]. Neurological abnormalities, such as spasticity, trun- Clinical Presentation cal ataxia and cerebral atrophy, may be present as a con- Very few cases have been described and it is not clear sequence of the Cbl deficiency. whether this entity has a distinct phenotype. Hematological findings are absent and neurological findings such as sub- Metabolic Derangement acute combined degeneration of the spinal cord in one man This disorder is caused by defects of the IF-Cbl receptor, in the fifth decade of life [29] and optic atrophy, ataxia, which has been recently shown to comprise two compo- long-tract signs and dementia in another, may be coinci- nents. was first purified as the IF-Cbl receptor from dental. the proximal renal tubule [21–23]. Fyfe et al. demonstrated that a second component, amnionless, co-localizes with Metabolic Derangement V cubilin in the endocytic apparatus of polarized epithelial The role of haptocorrin is uncertain but it could be involved cells, forming a tightly bound complex that is essential for in the scavenging of toxic Cbl analogs or in protecting endocytic function [24]. Thus defective function of either methylcobalamin from photolysis [30]. Deficiency of hapto- protein may cause this disorder. corrin has been described in isolation and in association with deficiency of other specific granule proteins such as Genetics lactoferrin [31]. About 250 cases have been reported and inheritance is au- tosomal recessive [19], with environmental factors affecting Genetics expression [22, 25]. Most patients are found in Finland, The haptocorrin gene has been cloned and mapped to Norway, Saudi Arabia, Turkey, and among Sephardic Jews. chromosome 11q11-q12 [32, 33]. No mutations have been The cubilin gene (CUBN) has been mapped to 10p12.1. A described in any patient with haptocorrin deficiency. P1297L mutation was found in 31 of 34 disease chromo- Heterozygosity for haptocorrin deficiency appears to be somes from 16 of 17 Finnish families segregating megalo- associated with low serum cobalamin [34]. blastic anemia [26]. Linkage studies in families from Nor- way, without mutations of the CUBN gene, led to the Diagnostic Tests discovery of the amnionless gene (AMN). A study of 42 Serum Cbl levels are low, because most circulating Cbl is MGA1 sibships confirmed CUBN mutations in Finnish bound to haptocorrin. TC-Cbl levels are normal, and there and AMN mutations in Norwegian patients, and either are no hematologic findings of Cbl deficiency. A deficiency among patients from other countries. Evidence was also or absence of haptocorrin is found in plasma, saliva and provided for a possible additional MGA1 causing gene leukocytes. locus [27, 28]. Treatment and Prognosis Diagnostic Tests It is uncertain whether treatment is warranted due to the In contrast to patients with IF deficiency, the Schilling test lack of a clearly defined phenotype. is not corrected by providing a source of human IF with the labeled Cbl [1]. The diagnosis is aided by finding low serum Cbl levels, megaloblastic anemia and proteinuria. Most of 28.1.4 Transcobalamin Deficiency the reports in the literature do not comment on the levels of homocysteine and methylmalonic acid. Gastric morpho- Clinical Presentation logy and pancreatic function are normal, there are no IF In TC deficiency, symptoms usually develop much earlier autoantibodies and IF levels are normal. than in other disorders of Cbl absorption, mainly within the first few months of life. Even though the only TC in Treatment and Prognosis cord blood is of fetal origin, patients are not sick at birth. Treatment with systemic OHCbl corrects the anemia and Presenting findings include pallor, failure to thrive, weak- the neurologic findings, but not the proteinuria. As with ness and diarrhea. Although the anemia is usually megalo- hereditary IF deficiency, once Cbl stores are replete, low blastic, patients with pancytopenia or isolated erythroid doses of systemic OHCbl may be sufficient to maintain hypoplasia have been described. Leukemia may be mis- normal hematological and biochemical values. takenly diagnosed because of the presence of immature white cell precursors in an otherwise hypocellular marrow. Neurologic disease is not an initial finding but may develop with delayed treatment, with administration of folate in the absence of Cbl, or with inadequate Cbl treat- ment [35]. Neurological features include developmental 345 28 28.2 · Disorders of Intracellular Utilization of Cobalamin

delay, neuropathy, myelopathy and encephalopathy and 28.2 Disorders of Intracellular rarely retinal de generation [36]. Defective granulocyte Utilization of Cobalamin function with both defective humoral and cellular im- munity may occur. A number of disorders of intracellular metabolism of Cbl have been classified as cbl mutants (A-H), based on the Metabolic Derangement biochemical phenotype and on genetic complementation The majority of patients have no immunologically detect- analysis. able TC, although others have some detectable TC that is able to bind Cbl but lacks normal function [1, 37, 38]. 28.2.1 Combined Deficiencies of Adenosyl- Genetics cobalamin and Methylcobalamin Inheritance is autosomal recessive; there have been at least 36 cases, including both twins and siblings [1, 35]. The TC Three distinct disorders are associated with functional gene has been mapped to chromosome 22q11.2-qter. Di- defects in both methylmalonyl-coenzyme A (CoA) mutase sease causing deletions, nonsense mutations, activation of and methionine synthase. They are characterized by both an intra exonic cryptic splice site, as well as a number of methylmalonic aciduria and homocystinuria. polymorphic variants have been described [39–41]. Cobalamin F Diagnostic Tests Clinical Presentation. Of the eight known patients with cblF Serum Cbl levels are not usually low, because the majority disease, seven presented in the first year of life. In this di- of serum Cbl is bound to haptocorrin and not to TC. sease, a complete blood count and bone marrow examina- However Cbl bound to TC, as reflected by the unsaturated tion may reveal megaloblastic anemia, neutropenia and vitamin-B12-binding capacity, is low but this test must thrombocytopenia. Other clinical findings can include be performed before Cbl treatment is started. Since TC failure to thrive, recurrent infections, developmental delay, is involved in the transcytosis of Cbl through the entero- lethargy, hypotonia, aspiration pneumonia, hepatomegaly cyte, the Schilling test may be abnormal in TC-deficient and encephalopathy, pancytopenia, and heart anomalies patients. In those patients in whom the Schilling test is (personal communication). The original infant girl had normal, immunoreactive TC is found. Reports of levels glossitis and stomatitis in the first week of life [45, 46]. She of Cbl related metabolites are scarce and inconsistent. had severe feeding difficulties requiring tube feeding. Tooth For example, normal plasma total homocysteine and abnormalities and dextrocardia were present. One infant moderately increased urine methylmalonic acid was died suddenly at home in the first year of life. One boy reported in three patients and methylmalonic aciduria developed juvenile rheumatoid arthritis at the age of 4 years and homocystinuria, without specified levels in one patient and a pigmented skin abnormality at 10 years. [36, 42]. Study of TC synthesis in cultured fibroblasts or amnio- Metabolic Derangement. The defect in cblF appears to be cytes allows both pre- and post-natal diagnosis in patients a failure of Cbl transport across the lysosomal membrane who do not synthesize TC [43]. DNA testing is possible following degradation of TC in the lysosome. As a result, for both diagnosis and heterozygote detection, in families Cbl cannot be converted to either adenosylcobalamin in which the molecular defect has been identified. Recently (AdoCbl) or methylcobalamin (MeCbl). The inability of developed assays, utilizing antibodies generated against cblF patients to absorb oral Cbl suggests that IFCbl also has recombinant human TC, allow reliable measurement of to pass through a lysosomal stage in the enterocyte before serum TC even in patients who have been treated with Cbl is released into the portal circulation. Cbl [44]. Genetics. As both male and female patients of unaffected Treatment and Prognosis parents have been reported, inheritance is presumed to be Adequate treatment requires administration of oral or autosomal recessive. The gene responsible for cblF has not systemic OHCbl or cyanocobalamin (CNCbl) of 0.5–1 mg, been identified. initially daily then twice weekly, to maintain serum Cbl levels in the range of 1000–10,000 pg/ml. Intravenous Cbl Diagnostic Tests. The serum Cbl level may be low, and is not recommended, because of the rapid loss of vitamin in the Schilling test has been abnormal in all patients tested. the urine. Folic acid or folinic acid can reverse the megalo- Usually, increased plasma total homocysteine, low to blastic anemia and has been used in doses up to 15 mg normal plasma methionine, homocystinuria and methyl- orally four times daily. However folates must never be given malonic aciduria are found, although urine and plasma as the only therapy in TC deficiency, because of the danger elevations of homocysteine were not reported in the original of neurological deterioration. patient. Precise diagnosis of the inborn errors of Cbl me- 346 Chapter 28 · Disorders of Cobalamin and Folate Transport and Metabolism

tabolism requires tests in cultured fibroblasts. The incor- Metabolic Derangement. The exact defect in the cblC poration of [14C] propionate into macromolecules is a good disorder remains undefined but clearly involves an early screen for the integrity of the methylmalonyl-CoA mutase step in intracellular Cbl processing, such as the reduction reaction, and the incorporation of [14C] methyltetrahydro- of the oxidation state of the central cobalt of Cbl from 3+ to folate or the conversion of labeled formate to methionine 2+ following efflux of Cbl from the lysosome. Decreased reliably measures the function of methionine synthase. The activities of microsomal Cbl3+ reductase, CNCbl-ligand total incorporation of [57Co] CNCbl by fibroblasts and its transferase and a mitochondrial, reduced-nicotinamide- conversion to both MeCbl and AdoCbl, can differentiate adenine-dinucleotide-linked aquacobalamin reductase a number of the disorders. In fibroblasts from cblF pa- have been described in fibroblast extracts but findings were tients, total incorporation of labeled CNCbl is elevated, but not consistent [54–56]. Regardless of the exact mechanism CNCbl is not converted to either AdoCbl or MeCbl. The if the reduction of Cbl does not occur, neither AdoCbl nor entire label is found as free CNCbl in lysosomes. There is MeCbl can be formed, and Cbl does not bind to the two V decreased incorporation of both labeled propionate and intracellular enzymes and leaves the cell. labeled methyltetrahydrofolate. Genetics. The gene responsible for cblC has been localized Treatment and Prognosis. Treatment with parenteral to chromosome 1 and recently identified [56a]. A common OHCbl (first daily and then biweekly, or even less frequently) mutation, 271 dup A, accounts for 40% of all disease at a dose of 1 mg/day seems to be effective in correcting alleles. Inheritance is autosomal recessive. Prenatal diag- the metabolic and clinical findings. Despite the fact that nosis can be performed by measuring the incorporation of two Schilling tests showed an inability to absorb Cbl with or labeled propionate or labeled methyltetrahydrofolate and without IF, the original patient responded to oral Cbl before the synthesis of MeCbl and AdoCbl in cultured chorionic being switched to parenteral Cbl. villus cells (but not native chorionic villus) and amniocytes and by measuring methylmalonic acid and total homo- Cobalamin C cysteine levels in amniotic fluid. These methods cannot Clinical Presentation. This is the most frequent inborn detect heterozygotes [56b]. error of Cbl metabolism, and several hundred patients are known [2, 47–50, 50a] (plus personal experience). Many Diagnostic Tests. Increased plasma total homocysteine, were acutely ill in the first month of life, and most were low to normal plasma methionine, homocystinuria and diagnosed within the first year. The early-onset group shows methylmalonic aciduria are the biochemical hallmarks of feeding difficulties and lethargy, followed by progressive this disease. In general, methylmalonic acid levels seen neurological deterioration, including hypotonia, hyper- are lower than those found in patients with methylmalonyl- tonia or both, abnormal movements or seizures, and coma. CoA mutase deficiency, but higher than those seen in the Severe pancytopenia or a non-regenerative anemia, which Cbl transport defects. A complete blood count and bone is not always associated with macrocytosis and hyper- marrow examination allow detection of the hematologic segmented neutrophils, but which is megaloblastic on abnormalities. bone-marrow examination, may be present. Patients may Fibroblast studies show decreased incorporation of develop multisystem pathology, such as renal failure, he- label from propionate, methyltetrahydrofolate (or formate) patic dysfunction, cardiomyopathy, interstitial pneumonia and CNCbl, and there is decreased synthesis of both A doCbl or the hemolytic uremic syndrome characterized by wide- and MeCbl. Cells fail to complement those of other cblC spread microangiopathy. Additional features include an patients. unusual retinopathy consisting of perimacular hypo- pigmentation surrounded by a hyperpigmented ring and Treatment and Prognosis. Treatment with 1 mg/day OHCbl a more peripheral salt-and-pepper retinopathy sometimes (parenteral) decreases the elevated metabolite levels, but accompanied by nystagmus, microcephaly and hydro- these are not usually completely normalized. In one com- cephalus. A small number of cblC patients were not diag- prehensive study, oral OHCbl was found to be insufficient, nosed until after the first year of life and some as late as and both folinic acid and carnitine were ineffective. Daily the end of the fourth decade of life [51–53, 53a]. The earlier- oral betaine (250 mg/kg/day) with twice weekly systemic diagnosed patients in this group had findings overlapping OHCbl (1 mg/day) resulted in normalization of methionine those found in the younger-onset group. Major clinical and homocysteine levels and decreased methylmalonic findings in this late-onset cblC group included confusion, aciduria [57]. Even though oral administration of OHCbl disorientation and gait abnormalities and incontinence. appears not to be effective, this route was reported to be Macrocytic anemia was seen in only about a third of the successful in one patient [58]. oldest patients. Therefore, it is important to search for the Of a group of 44 patients with onset in the first year of cblC disorder by determination of metabolite levels in the life, 13 died, and only one patient was neurologically intact, presence of neurological findings alone. with other survivors described as having severe or mo derate 347 28 28.2 · Disorders of Intracellular Utilization of Cobalamin

impairment. Survival with mild to moderate disability was in bone-marrow abnormalities and produce anemia, leuko- found in the patients who had a later onset [50]. penia and thrombocytopenia. Hyperammonemia, hyper- glycinemia and ketonuria may be found. Cobalamin D Clinical Presentation. Until recently just two males from Metabolic Derangement one sibship were known to belong to the cblD complemen- The defect in cblA had been thought to lie in the reduction tation group [59–61]. The elder sibling was diagnosed with of the central cobalt of Cbl from the 2+ to the 1+oxidation behavioral problems and mild mental retardation at the age state in mitochondria. The MMAA gene was proven to of 14 years. He had ataxia and nystagmus. Suormala et al. correspond to the cblA complementation group. Based on recently described three patients indicating heterogeneity the domain characteristics of the protein sequence deduced of the cblD defect [62]. One patient with isolated methyl- from this gene it was proposed that the cblA protein is a malonic aciduria presented prematurely with respiratory transporter or an accessory protein involved in the trans- distress, cranial hemorrhage, necrotizing enterocolitis and location of Cbl into mitochondria [63]. convulsions but without anemia. Two unrelated patients A patient with all the clinical and biochemical features presented with isolated homocystinuria, megaloblastic of cblA has been described, but cells from this patient com- anemia and neurological changes but without metabolic plement those from other cblA patients. This implies that decompensation. more than one step may be involved in the intramitochon- drial reduction of Cbl or that intragenic complementation Metabolic Derangement. The cblD defect can cause defi- may occur among cblA lines [64]. cient synthesis of both AdoCbl and MeCbl together or The defect in cblB is deficiency of adenosyltransferase, either in isolation. This points to a multifunctional protein, the final intramitochondrial catalyst in the synthesis of or at least three different gene products involved in Cbl AdoCbl [65]. metabolism between the reduction of Cbl-3+ and specific cobalamin coenzyme synthesis. Genetics Male and female patients with cblA and cblB have been Genetics. All five subjects belonging to the cblD comple- described, and parents of cblB patients have decreased mentation group are male so that sex linkage cannot be adenosyltransferase activity, indicating autosomal-reces- excluded. sive inheritance. The MMAA gene has been localized to chromosome 4q31.1-2 [63]. It encodes a predicted poly- Diagnostic Tests. Methylmalonic aciduria with or without peptide of 418 amino acid residues and its deduced se- increased plasma total homocysteine and homocystinuria, quence represents a domain structure belonging to the or isolated methylmalonic aciduria may be found. Although AAA ATPase superfamily. The precise role for the gene the original patient showed no megaloblastic anemia, the product has not been determined. Many mutations in the deoxyuridine-suppression test was abnormal. In fibroblast MMAA gene have now been described among cblA patients studies findings can be similar to those of the cblC, cblA or [66, 67]. cblE/G defects although differences in the severity and The gene for adenosyltransferase has also been cloned, responsiveness to addition of OHCbl to the culture medium is localized to chromosome 12q24, and encodes a predicted may be seen. This heterogeneity emphasis the necessity of protein of 250 amino acids. Examination of cblB patient complementation analysis to make a specific diagnosis in cell lines revealed several disease causing mutant alleles, the cbl defects. confirming that the MMAB gene, corresponds to the cblB complementation group [68, 68a].

28.2.2 Adenosylcobalamin Deficiency Diagnostic Tests Total serum Cbl is usually normal, and there is markedly Clinical Presentation increased methylmalonic aciduria (0.8–1.7 mmol/day; Adenosylcobalamin (AdoCbl) deficiency comprises cblA normal <0.04 mmol/day) but no increase of plasma total and cblB, two disorders characterized by methylmalonic homocysteine or homocystinuria. A decrease in the level of aciduria (MMA) which is often Cbl-responsive [2]. The methylmalonic-acid excretion in response to Cbl therapy phenotype resembles methylmalonyl-CoA mutase defi- is useful in distinguishing these disorders from methyl- ciency (7 Chap. 19). Most patients have an acidotic crisis in malonyl-CoA-mutase deficiency. The exact differentiation the first year of life, many in the neonatal period. Symptoms of cblA and cblB from mutase deficiency depends on fibro- are related to methylmalonic-acid accumulation and in- blast studies. In both cblA and cblB levels of methylmalonyl- clude vomiting, dehydration, tachypnea, lethargy, failure to CoA mutase are normal in the presence of added AdoCbl. thrive, developmental retardation, hypotonia and encepha- The incorporation of labeled propionate is decreased in lopathy. The toxic levels of methylmalonic acid may result both cblA and cblB and is usually responsive to the addition 348 Chapter 28 · Disorders of Cobalamin and Folate Transport and Metabolism

of OHCbl to the culture medium. Uptake of labeled CNCbl cloning of the cDNA for the gene on chromosome 1q43 [78, is normal but there is decreased synthesis of AdoCbl. Adeno- 79]. Patients with the cblG variant of methionine-synthase syltransferase activity is clearly deficient in cblB, but normal deficiency have null mutations [80]. Where both mutations in cblA fibroblast extracts. Complementation analysis al- are known in a patient, molecular analysis can be used for lows confirmation of the mutant class. carrier detection in the family and for prenatal diagnosis.

Treatment and Prognosis Diagnostic Tests Most of these patients respond to protein restriction and Homocystinuria and hyperhomocysteinemia are almost to OHCbl treatment, either 10 mg orally daily or 1 mg always found in the absence of methylmalonic acidemia. intra muscularly, once or twice weekly. For details of the However, one cblE patient had transient unexplained planning of a protein-restricted diet, 7 Chap. 17. Some pa- methylmalonic aciduria. Hypomethioninemia and cysta- tients appear to become resistant to Cbl treatment. Therapy thioninemia may be present, and there may be increased V with AdoCbl has been attempted in cblB with and without serine in the urine. A complete blood count and bone success, and it may be that AdoCbl does not reach the target marrow examination will detect the hematological manifes- enzyme intact. There have been reports of prenatal therapy tations. Fibroblast extracts from cblE patients have normal with Cbl in AdoCbl deficiency. Most (90%) cblA patients activity of methionine synthase in the standard assay, but improve on Cbl therapy, with 70% doing well long term. It deficient activity can be found when the assay is performed must be noted that only 40% of cblB patients respond to under limiting reducing conditions [70, 76]. Cell extracts Cbl, and their long-term survival is poorer [69]. from cblG patients have decreased methionine synthase activity in the presence of excess reducing agent. Incorpo- ration of labelled methyltetrahydrofolate or formation of 28.2.3 Methylcobalamin Deficiency methionine from labeled formate is decreased in cultured fibroblasts from both cblE and cblG patients. Uptake of Clinical Presentation CNCbl is normal but synthesis of MeCbl is decreased in Methylcobalamin (MeCbl) deficiency comprises cblE and both disorders. In some cblG patients (cblG variants) no Cbl cblG. The most common clinical findings are megaloblastic forms are bound to methionine synthase following incuba- anemia and neurological disease [70, 72–74]. The latter tion in labeled CNCbl. Complementation analysis distin- include poor feeding, vomiting, failure to thrive, cerebral guishes cblE from cblG patients. atrophy, developmental delay, nystagmus, hypotonia or hypertonia, ataxia, seizures and blindness. Cerebral atrophy Treatment and Prognosis may be seen on imaging studies of the central nervous Both of these disorders are treated with OHCbl or MeCbl, system, and at least one cblE patient showed a spinal-cord 1 mg intramuscularly, first daily and then once or twice cystic lesion on autopsy. Most patients are symptomatic in weekly. Although the metabolic abnormalities are nearly the first year of life, but one cblG patient was not diagnosed always corrected, it is difficult to reverse the neurologic until age 21 years and carried a misdiagnosis of multiple findings once they have developed. Treatment with betaine sclerosis [75]. Another cblG patient, who was diagnosed (250 mg/kg/day) has been used, and one cblG patient was during his fourth decade of life, had mainly psychiatric treated with L-methionine (40 mg/kg/day) and had neuro- symptoms. Two patients with minimal findings and with- logical improvement. Despite therapy, many patients with out clear neurological features have also been reported cblG and cblE show a poor outcome. In one family with cblE, [76]. there was successful prenatal diagnosis using cultured amniocytes, and the mother was treated with OHCbl twice Metabolic Derangement per week beginning during the second trimester, and the The defect in cblE is deficiency of the enzyme, methionine baby was treated with OHCbl from birth. This boy has synthase reductase, which is required for the activation by developed normally to age 14 years, in contrast to his older reductive methylation of the methionine synthase apo- brother, who was not treated until after his metabolic enzyme. The cblG defect is caused by deficient activity of the decompensation in infancy and who is now 18 years old methionine synthase apoenzyme itself. and has significant developmental delay. Some patients may benefit from high dose folic or folinic acid treatment. Genetics There are at least 27 cblE and 27 cblG patients known. A cDNA for methionine-synthase reductase has been cloned, and mutations have been detected in cblE patients [77]. The methionine-synthase-reductase gene has been localized to chromosome 5p15.2–15.3. Mutations in the methionine- synthase gene have been found in cblG patients following 350 Chapter 28 · Disorders of Cobalamin and Folate Transport and Metabolism

Folate Metabolism Folic acid (pteroylglutamic acid) is plentiful in foods carbon unit is essential for the endogenous formation of such as liver, leafy vegetables, legumes and some fruit. methionine, thymidylate (dTMP) and formylglycine- Its metabolism involves reduction to dihydro- (DHF) amide ribotide (FGAR) and formylaminoimidazole- and tetrahydrofolate (THF), followed by addition of a carboxamide ribotide (FAICAR), two intermediates of single-carbon unit, provided by histidine or serine; this purine synthesis. These reactions also allow regenera- carbon unit can be in various redox states (methyl, tion of DHF and THF. methylene, methenyl or formyl). Transfer of this single-

V

. Fig. 28.2. Folic acid metabolism. 1, methionine synthase; dihydrofolate, dTMP, deoxythymidine monophosphate; dUMP, 2, methylenetetrahydrofolate reductase; 3, methenyltetrahydro- deoxyuridine monophosphate; FAICAR, formylaminoimidazole folate cyclohydrolase; 4, dihydrofolate reductase; 5, glutamate carbox amide ribotide; FGAR, formylglycinamide ribotide; FIGLU, formiminotransferase; 6, formiminotetrahydrofolate cyclode- formi minoglutamate; GAR, glycinamide ribotide; THF, tetrahydro- aminase; AICAR, aminoimidazole carboxamide ribotide; DHF, folate. Enzyme defects are indicated by solid bars 351 28 28.3 · Disorders of Absorption and Metabolism of Folate

mediate levels of folate absorption, making autosomal- Three confirmed inborn errors of folate absorption recessive inheritance likely. A cDNA for a putative intestinal and metabolism have been described. folate transporter has been cloned, and it is identical to that Hereditary folate malabsorption presents with for the reduced folate carrier [83]. To date, no report of severe megaloblastic anemia, due to the importance mutations in these patients has appeared. The defect in of dTMP and purine synthesis in hematopoiesis, and hereditary folate malabsorption is not expressed in amnio- is usually associated with progressive nerurological cytes or chorionic villus cells. deterioration. Glutamate-formiminotransferase deficiency has Diagnostic Tests been reported in association with various degrees of Measurement of serum, red blood cell and CSF folate levels psychomotor retardation and megaloblastic anemia. and a complete blood count and bone marrow analysis Severe methylenetetrahydrofolate reductase (MTHFR) should be performed. The most important diagnostic fea- deficiency presents mainly with developmental delay, tures are the severe megaloblastic anemia in the first few often accompanied by seizures, microcephaly and months of life, together with low serum folate levels. Mea- findings related to cerebrovascular events. Patients surements of related metabolite levels have been sporadi- typically show hyperhomocysteinemia without mega- cally reported and inconsistently found abnormalities loblastic anemia. include increased excretion of formiminoglutamate, orotic aciduria, increased plasma sarcosine and cystathionine and low plasma methionine. Folate levels in CSF remain low even when blood levels are high enough to correct the 28.3 Disorders of Absorption megaloblastic anemia [84]. As mentioned, a number of and Metabolism of Folate patients have been reported with only neurological mani- festations and low levels of CSF folate. Folate absorption 28.3.1 Hereditary Folate Malabsorption may be directly investigated by measuring serum folate levels following an oral dose of between 5 and 100 mg of Clinical Presentation folic acid. This rare condition presents in the first months of life with severe megaloblastic anemia, diarrhea, stomatitis, failure to Treatment and Prognosis thrive and usually progressive neurological deterioration High-dose oral folic acid (up to 60 mg daily) or lower with seizures and sometimes with intracranial calcifications parenteral doses in the physiological range correct the [81]. Peripheral neuropathy has been seen, as have partial hematologic findings but are less effective in correcting defects in humoral and cellular immunity. the neurological findings and in raising the level of folate in the CSF. Both methyl-THF or folinic acid may be more Metabolic Derangement effective in raising CSF levels and have been given in com- All patients have severely decreased absorption of oral folic bination with high-dose oral folic acid. The clinical response acid or reduced folates, such as formyltetrahydrofolic acid to folates has varied among patients and, in some cases, (formyl-THF, folinic acid) or methyltetrahydrofolic acid. seizures were worse after folate therapy was started. It is These patients provide the best evidence for the existence of important to maintain blood and CSF folate in the normal a single transport system for folate at both the intestine and range. If oral therapy does not raise CSF folate levels, the choroid plexus. Transport of folates across other cell parenteral therapy should be used. Intrathecal folate therapy membranes is not affected in this disorder. The hematologi- may be considered if CSF levels of folate cannot be raised cal and gastrointestinal manifestations of this disease, but by other treatments although the required dose of folate is not the neurological manifestations, can be reversed by phar- unknown. A recent report stresses that in some cases high macologic, but relatively low levels of folate. Folate meta- oral doses of folinic acid (up to 400 mg orally daily) may bolism in cultured fibroblasts is normal. Recently a novel eliminate the need for parenteral therapy [81]. The cerebral disorder was described with psychomotor retardation, spas- folate deficiency syndrome responds exclusively to folinic tic paraplegia, cerebellar ataxia and dyskinesia, associated acid (10–20 mg/day) and not to folic acid [82a]. with normal blood folate levels and low folate levels only in the cerebrospinal fluid (CSF) [82]. This cerebral folate defi- ciency syndrome has been recently found to be caused by an 28.3.2 Glutamate-Formiminotransferase immune process against the cerebral folate carrier [82a]. Deficiency

Genetics Clinical Presentation Several female patients are known, consanguinity has been Over a dozen patients have been described, but the clinical noted in four families, and one patient’s father had inter- significance of this disorder is still unclear [4, 85, 86]. A 352 Chapter 28 · Disorders of Cobalamin and Folate Transport and Metabolism

mild and severe form has been postulated, although it is hydantoin-5-propionate, a stable oxidation product of the difficult to determine the importance of this distinction formiminoglutamate precursor, 4-imidazolone-5-propio- given the small number of patients. In the severe form of nate and 4-amino-5-imidazolecarboxamide, an interme- formiminotransferase deficiency there is both mental and diate of purine synthesis. physical retardation, abnormal electroencephalograms and dilatation of cerebral ventricles with cortical atrophy. Treatment and Prognosis Several of the patients had a folate-responsive megaloblastic It is not clear whether reducing formiminoglutamate ex- anemia with macrocytosis and hypersegmentation of neu- cretion is of any clinical value. Although two patients in one trophils. Patients ranged in age from 3 months to 42 years. family responded to folate therapy by reducing excretion Two had mental retardation, two had seizures and three had of formiminoglutamate, six others did not. One of two delayed speech as their presenting findings, and two were patients responded to methionine supplementation. Pyrid- studied because they were the siblings of known patients. In oxine and folic acid have been used to correct the megalo- V the mild form there is no mental retardation, but there is a blastic anemia in one infant. greater excretion of formiminoglutamate. Although mental retardation was described in most of the original patients from Japan, of the remaining eight patients, only three 28.3.3 Methylenetetrahydrofolate showed mental retardation. Reductase Deficiency

Metabolic Derangement This section is restricted to the severe form of this defi- Histidine catabolism is associated with a formimino-group ciency. The role of polymorphisms in methylenetetra- transfer to THF, with the subsequent release of ammonia hydrofolate reductase (MTHFR) with respect to the risk and the formation of 5,10-methenyl-THF. A single octa- for common disease, such as neural tube defects or car- meric enzyme catalyzes two different activities: glutamate diovascular disease, is beyond the scope of this chapter formiminotransferase and formiminotetrahydrofolate (7 [88] for a review]. cyclod eaminase. These activities are found only in the liver and kidney, and defects in either of these activities will Clinical Presentation result in formiminoglutamate excretion. It has been sug- Approximately 100 patients with severe MTHFR deficiency gested (without any direct enzyme measurements) that have been described [2, 48, 85, 89–91], or are known to the the severe form of this disease is due to a block in the cyclo- authors. Most commonly, they were diagnosed in infancy, deaminase activity, whereas the mild form is due to a block and more than half presented in the first year of life. The in the formiminotransferase activity. most common early manifestation was progressive ence- phalopathy with apnea, seizures and microcephaly. How- Genetics ever, patients became symptomatic at any time from in- Glutamate formiminotransferase deficiency has been found fancy to adulthood and, in the older patients, ataxic gait, in both male and female children of unaffected parents. psychiatric disorders (schizophrenia) and symptoms relate d Consanguinity has not been reported; it has been presumed to cerebrovascular events have been reported. An infant that the disease is inherited in an autosomal-recessive had extreme progressive brain atrophy, and the magnetic manner. Because of the lack of expression of the enzyme in resonance image showed demyelination [92]. A 10-year-old cultured cells, prenatal diagnosis has not been possible, but boy had findings compatible with those of Angelman it may be possible to measure formiminoglutamate levels in syndrome [93]. At least one adult with severe enzyme defi- amniotic fluid. This has not been reported. The human ciency was completely asymptomatic. Autopsy findings gene has been cloned and localized to chromosome 21q22.3. have included dilated cerebral vessels, microgyria, hydro- Hilton et al. found mutant alleles in three patients and cephalus, perivascular changes, demyelination, gliosis, concluded that they represent the molecular basis for this astrocytosis and macrophage infiltration. In some patients, disease, although expressed residual activity was 60% [87]. thrombosis of both cerebral arteries and veins was the major cause of death. There have been reports of patients Diagnostic Tests with findings similar to those seen in subacute degenera- Elevated formiminoglutamate excretion and elevated levels tion of the spinal cord due to Cbl deficiency. Of note is the of formiminoglutamate in the blood, only following a his- fact that MTHFR deficiency is not associated with mega- tidine load in the severe form, help to establish the diag- loblastic anemia. nosis. A complete blood count and bone marrow examina- tion may detect megaloblastic anemia. Normal to high Metabolic Derangement serum folate levels are found, particularly in the mild form. Methy-THF is the methyl donor for the conversion of Hyperhistidinemia and histidinuria have been reported. homocysteine to methionine and, in MTHFR deficiency, Two other metabolites that may be found in the urine are the result is an elevation of total plasma homocysteine levels 353 28 References

and decreased levels of methionine. The block in the con- Treatment and Prognosis version of methylene-THF to methyl-THF does not result It is important to diagnose MTHFR deficiency early be- in the trapping of folates as methyl-THF and does not in- cause, in the infantile forms, the only patients that have terfere with the availability of reduced folates for purine and done well have been those who have been treated from pyrimidine synthesis. This explains why patients do not have birth. Early treatment with betaine following prenatal diag- megaloblastic anemia. It is not clear whether the neuro- nosis has resulted in the best outcome [103–105]. Suggested pathology in this disease results from the elevated homo- doses have been in the range of 2–3 g/day (divided twice cysteine levels, from decreased methionine and resulting daily) in young infants and 6–9 g/day in children and interference with methylation reactions or from some other adults. Betaine is a substrate for betaine methyltransferase, metabolic effect. It has been reported that individuals with an enzyme that converts homocysteine to methionine, but a severe deficiency in MTHFR may be at increased risk is mainly active in the liver. Therefore, betaine may be ex- following exposure to nitrous oxide anesthesia [94]. pected to have the doubly beneficial effect of lowering homocysteine levels and raising methionine levels. Because Genetics betaine methyltransferase is not present in the brain, the MTHFR deficiency is inherited as an autosomal-recessive central nervous system effects must be mediated through disorder. There have been multiple affected children of the effects of the circulating levels of metabolites. The dose both sexes with either unaffected parents or affected families of betaine should be modified according to plasma levels with consanguinity. Prenatal diagnosis has been reported of homocysteine and methionine. Other therapeutic agents using amniocytes, and the enzyme is present in chorionic that have been used in MTHFR deficiency include folic acid villi. A cDNA has been isolated, and the gene coding for or reduced folates, methionine, pyridoxine, cobalamin, and MTHFR has been localized to chromosome 1p36.3. Over carnitine. Most of the treatment protocols omitting betaine 50 mutations causing severe deficiency have been described, have not been effective. Dramatic improvement was report- in addition to polymorphisms that result in intermediate ed in a patient with severe enzyme deficiency following enzyme activity and that may contribute to disease in the early introduction of methionine supplements [106]. general population [95–101].

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