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Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. C H A P T E R 94 Disorders of Propionate and Methylmalonate Metabolism Wayne A. Fenton _ Roy A. Gravel 1. Propionyl coenzyme A (CoA) Ð formed in the catabolism of several essential amino acids (isoleucine, valine, methionine, threonine), odd-chain fatty acids, and cholesterol Ð is metabolized primarily by enzymatic conversion to methylmalonyl CoA, which is subsequently isomerized to succinyl CoA. This sequence depends on the activity of several enzymes (see Fig. 94-2): propionyl CoA carboxylase, methylmalonyl CoA racemase, and methylmalonyl CoA mutase. Propionyl CoA carboxylase requires biotin as a cofactor, whereas methylmalonyl CoA mutase requires adenosylcobalamin (AdoCbl), a cobalamin (Cbl; vitamin B12) coenzyme. 2. Propionyl CoA carboxylase and methylmalonyl CoA mutase are oligomeric enzymes. Propionyl CoA carboxylase is composed of nonidentical subunits (a and b); biotin binds to the a subunit. The holocarboxylase contains six a and six b subunits (a6b6). The a subunit is encoded by a gene on chromosome 13 (NM_000282) in humans, the b subunit by a gene on chromosome 3 (NM_000532). Methylmalonyl CoA mutase is a dimer of identical subunits (a2), encoded by a gene on chromosome 6 (NM_000255). 3. Inherited de®ciency of propionyl CoA carboxylase activity in humans results from genetically distinct defects at four loci. Isolated de®ciency is caused by mutations at the a and b loci coding for the carboxylase subunits. De®ciency of multiple biotin-dependent carboxylases occurs in two forms: one resulting from de®ciency of holocarboxylase synthase (the enzyme that attaches biotin to apocarboxylase subunits), the other from de®ciency of biotinidase (the enzyme that cleaves biotin from the lysine residue in the carboxylase to which the biotin is attached). Multiple carboxylase de®ciency is discussed in detail in Chapter 156. 4. Isolated de®ciency of propionyl CoA carboxylase, a major cause of the ketotic hyperglycinemia syndrome, results in the accumulation of propionate in blood and of 3hydroxypropionate, methylcitrate, tiglylglycine, and unusual ketone bodies in urine. Two complementation groups, pccA (OMIM 232000) and pccBC (OMIM 232050) have been de®ned among propionyl CoA carboxylase±de®cient patients. These groups correspond to mutations affecting A list of standard abbreviations is located immediately preceding the index in each volume. Nonstandard abbreviations used in this chapter include: AdoCbl adenosylcobalamin; cbl cobalamin metabolism locus (cblA, cblB, etc.); Cbl cobalamin; CN-Cbl cyanocobalamin; CoA coenzyme A; CPS I carbamylphosphate synthetase I; DMB dimethyl benzimidazoyl; H4folate tetrahydrofolate; IF intrinsic factor; MeCbl methylcobalamin; MeH4folate N5-methyltetrahydrofolate; mut methylmalonyl CoA mutase locus; OH-Cbl hydroxocobalamin; OLCFA odd-numbered long-chain fatty acids; pcc propionyl CoA carboxylase locus; TC (I, II, or III) transcobalamin (I, II, or III). 5. 6. 7. 8. _ David S. Rosenblatt genes coding for the a subunit and the b subunit, respectively, of the carboxylase apoprotein. Clinically, the disorder is characterized by severe metabolic ketoacidosis, which often appears in the neonatal period and requires vigorous alkali therapy and protein restriction. Oral antibiotic therapy to reduce gut propionate production also may prove useful. Multiple carboxylase de®ciency (OMIM 253270) leads to impaired activity of four biotin-dependent enzymes: acetyl CoA carboxylase, propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase, and pyruvate carboxylase. The clinical hallmarks of this disorder include ketoacidosis, a diffuse erythematous rash, alopecia, seizures, hypotonia, and developmental retardation (see Chapter 156). Inherited de®ciency of methylmalonyl CoA mutase activity in humans is caused by mutations at many different loci. Isolated de®ciency results from mutations at the apomutase locus (OMIM 251000) and at two loci coding for gene products required, speci®cally for the biosynthesis of AdoCbl. Combined de®ciency of mutase and of the other major Cbl-dependent enzyme in mammalian cells, methionine synthase (formally, N5-methyltetrahydrofolate:homocysteine methyltransferase), results from inherited defects in Cbl transport and from three distinct defects in the intracellular pathway of Cbl coenzyme synthesis affecting the synthesis of both AdoCbl and methylcobalamin (MeCbl), the coenzyme required by methionine synthase. These several defects in intracellular Cbl metabolism are discussed in detail in Chapter 155. Neonatal or infantile metabolic ketoacidosis is the clinical hallmark of isolated methylmalonyl CoA mutase de®ciency. Cells from some apomutase-de®cient children have no functional mutase (designated mut 0); cells from others contain a structurally altered mutase with reduced af®nity for AdoCbl and with reduced stability (mut 2). Such children exhibit methylmalonic acidemia and methylmalonic aciduria that do not respond to Cbl supplementation but can sometimes be treated with dietary protein restriction. Carnitine, as well as oral antibiotic therapy to reduce gut ¯ora, may be effective as well. Two abnormalities in AdoCbl synthesis only, designated cblA (OMIM 251100) and cblB (OMIM 251110), lead to impaired methylmalonyl CoA mutase activity and are characterized by a clinical and chemical picture virtually identical to that seen in apomutase-de®cient children. In most cblA patients and some cblB patients, pharmacologic supplements of CN-Cbl or hydroxocobalamin (OH-Cbl) produce distinct reductions in methylmalonate accumulation and offer a valuable therapeutic adjunct to dietary protein limitation. 2165 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2166 PART 9 / ORGANIC ACIDS 9. Three other distinct defects Ð cblC (OMIM 277400), cblD (OMIM 277410), and cblF (OMIM 277380) Ð lead to impaired synthesis of both AdoCbl and MeCbl and, accordingly, to de®cient activity of both methylmalonyl CoA mutase and methionine synthase. Such children have methylmalonic aciduria and homocystinuria. Most children with a cblC mutation appear to be more severely affected clinically than the two known sibs in the cblD group, although a number of cblC patients have had an onset of disease in adult life. Major clinical problems in cblC patients include failure to thrive, developmental retardation, and such hematologic abnormalities as megaloblastic anemia and macrocytosis. The precise defect in the cblC and cblD patients is not yet known, but it involves an early step in the intracellular metabolism of cobalamins. The defect in cblF cells involves impaired ef¯ux of free Cbl from lysosomes. Therapy includes protein restriction, pharmacologic doses of OH-Cbl, and betaine supplementation. 10. The discriminating biochemical features of the known forms of inherited methylmalonic acidemia are shown in Table 94-4. 11. All of the disorders of propionate and methylmalonate metabolism for which there are adequate data are inherited as autosomal-recessive traits. Heterozygotes for the following defects can be detected biochemically: pccA, mut 0, mut2, and cblB. Genetic complementation analyses with somatic cell heterokaryons have been particularly useful in demonstrating genetic heterogeneity and in con®rming the existence of autosomal-recessive inheritance among the propionic acidemias and the methylmalonic acidemias. Precise molecular defects have been described for pccA, pccBC, mut 0, and mut2 patients. 12. Prenatal detection of fetuses with propionyl CoA carboxylase de®ciency, methylmalonyl CoA apomutase de®ciency, and defective synthesis of AdoCbl or of both coenzymes is best done using assays in cultured amniotic cells and gas chromatographic/mass spectroscopic determinations on amniotic ¯uid or maternal urine. Methylmalonic acid and its immediate precursor, propionic acid, are detectable in only trace amounts in normal human blood, urine, and cerebrospinal ¯uid. The minuscule quantities of these compounds in extracellular ¯uids have obscured the key role that these acids play in human metabolism. Biochemists investigating animal nutrition have been interested in propionate metabolism for many years because ruminants derive most of their energy requirements from the oxidation of propionate and acetate produced by bacterial fermentation in their rumens.1 Although propionate and methylmalonate are of little quantitative importance in humans as direct sources of energy, these acids, found intracellularly largely as their coenzyme A (CoA) esters, are vital intermediates in the catabolism of fat and protein. Several independent, and seemingly unrelated, lines of evidence drew the attention of the physician and the clinical investigator to the study of propionate and methylmalonate metabolism. In 1959 and 1960, several groups reported that adenosylcobalamin (AdoCbl), one of the coenzyme forms of cobalamin (Cbl) (vitamin B12), is an essential cofactor in the enzymatic conversion of L-methylmalonyl CoA to succinyl CoA.2± 4 Shortly thereafter, patients with acquired Cbl de®ciency were shown to excrete large amounts of methylmalonic acid in their urine.5,6 The methylmalonic aciduria was rapidly reversed by administration of physiologic doses of Cbl and was attributed to an acquired block in methylmalonate catabolism caused by inadequate amounts of the needed Cbl coenzyme. In 1961, Childs and associates7 described a young boy with recurrent attacks of severe ketoacidosis who had elevated concentrations of glycine and several other amino acids in his blood and urine. A series of detailed metabolic studies demonstrated that the attacks were precipitated by protein feeding and more speci®cally by ingestion of the branched chain amino acids, methionine and threonine. Because elevation in plasma glycine level was the most striking biochemical abnormality, the disorder was called ketotic hyperglycinemia. Later evidence has established that this disorder is caused by an inherited defect in the catabolism of propionate, not by a primary abnormality in glycine utilization or biosynthesis.8,9 Since 1967, a number of critically ill children have been described who draw these seemingly disparate observations together and focus attention on the enzymes and coenzymes that participate in the pathway responsible for the formation of propionate and its conversion to succinate. Oberholzer,10 Stokke,11 and their colleagues described infants with profound metabolic acidosis and hyperglycinemia (or hyperglycinuria) who excreted huge amounts of methylmalonic acid in their urine, but who were not Cbl de®cient. Subsequently, Rosenberg and his colleagues8 reported that urine from the index patient with ketotic hyperglycinemia and from his affected sister contained no methylmalonic acid. This observation indicated that primary methylmalonic acidemia and ketotic hyperglycinemia were different disorders with identical clinical manifestations.3 The latter group and Lindblad et al.12,13 also described children with ketoacidosis and methylmalonic acidemia who were not Cbl de®cient, but who responded to administration of pharmacologic doses of cyanocobalamin (CN-Cbl) or its coenzyme with a marked decrease in concentration of urinary methylmalonic acid. The index patient8 was subsequently shown to suffer from a primary defect in AdoCbl synthesis,14,15 not from a defect of the apoenzyme that catalyzes the conversion of methylmalonyl CoA to succinyl CoA. These observations and others, which will be discussed in detail subsequently, emphasize that numerous inherited abnormalities in the metabolic pathway for propionate and methylmalonate occur and that these defects lead to profound illness and, in many cases, death due to a disturbed acid-base balance or developmental failure. The study of these disorders has led to important insights in our understanding of the role of this pathway in humans and has illustrated, once again, that a group of clinically identical disorders can be produced by several different mutations affecting the synthesis of related apoenzymes and coenzymes. Several reviews of this subject matter have been published.16±21 BIOCHEMICAL PATHWAYS Propionate Metabolism Formation of Propionate and Methylmalonate. Most of the propionic acid used by ruminant animals is formed by bacterial fermentation in the rumen.1 By contrast, nonruminant mammals derive most of their propionate from the catabolism of lipid and protein. As noted in Fig. 94-1, catabolism of the branched chain amino acid isoleucine leads to the formation of propionyl CoA, as does the degradation of methionine and threonine.22 Studies with [13C]valine in a patient with methylmalonic acidemia23 and with the valine catabolite [13C]isobutyrate in rats24 indicate that valine is also a propionate precursor and is not catabolized directly to methylmalonyl CoA, as had been suggested. Catabolism of these amino acids accounts for much of the propionate formed in humans; data presented by Thompson et al.25 indicate that their contribution is on the order of 50 percent in patients with inborn errors of propionate or methylmalonate metabolism. Other sources of propionate include b-oxidation of fatty acids with an odd number of carbon atoms, which ultimately leads to the formation of 1 mole of propionyl CoA per mole of fatty acid.25± 27 Degradation of the side chain of cholesterol also leads to the synthesis of propionyl CoA, but this pathway appears to be of little quantitative signi®cance.28 Finally, there have been suggestions Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM cycle and is ultimately glycogenic because of its conversion to pyruvate by way of oxaloacetate. The sum of these reactions may be written as follows: Propionate ATP ! pyruvate 4H ADP Pi In bacteria, propionate is formed from pyruvate by reversal of the reaction sequence just described,26 but in mammalian systems the equilibrium of the system is far in the direction of propionate catabolism, rather than biosynthesis. Apoenzymes that gut bacteria may contribute a substantial amount of propionate, at least under some circumstances. These ®ndings are based on the ability of oral antibiotics to decrease urine29 and plasma25 propionate concentrations in some propionic and methylmalonic acidemia patients. From stable isotope studies in patients with methylmalonic aciduria and propionic aciduria, Leonard estimated that about 50 percent of propionate production is due to amino acid catabolism, 25 percent is due to the odd chain fatty acid catabolism, and 25 percent is due to bacterial activity in the gut.30 Methylmalonyl CoA is synthesized essentially only from propionyl CoA (Fig. 94-1). Propionate has long been known to be glycogenic in animals,31 but the pathway by which propionate is converted to carbohydrate became clear only when Lardy and Adler demonstrated that liver mitochondria contain enzymes that synthesize succinate from propionate.31 The discovery in 1955 that methylmalonate is an intermediate in the formation of succinate from propionate (Fig. 94-1) provided an important further step in the characterization of this pathway.32,33 Kaziro and Ochoa ®rst de®ned the individual steps of propionate catabolism in animal tissues and characterized the enzymes involved.26 Propionyl CoA, formed either by the degradative reactions discussed above or by the enzymatic esteri®cation of propionate itself,34 may be considered the precursor of this reaction sequence (Fig. 94-2). Three enzymatic reactions are responsible for the conversion of propionyl CoA to succinyl CoA. The ®rst involves the carboxylation of propionyl CoA to methylmalonyl CoA,34,35 a reaction catalyzed by propionyl CoA carboxylase (EC 6.4.1.3). Although two stereoisomers of methylmalonyl CoA exist, only the D form is produced in the carboxylation reaction.36,37 This isomer is not a substrate for the subsequent mutase reaction and must be racemized to the L con®guration by another enzyme, methylmalonyl CoA racemase (EC 5.1.99.1).38 The third reaction, catalyzed by methylmalonyl CoA mutase (EC 5.4.99.2), isomerizes L-methylmalonyl CoA to succinyl CoA.39 The latter compound enters the tricarboxylic acid Propionyl CoA Carboxylase. This enzyme, ®rst crystallized from pig heart,40 has been puri®ed to homogeneity from bovine kidney41 and human liver.42,43 The enzyme is composed of nonidentical subunits (a and b): the required cofactor, biotin, is bound exclusively to the larger (a) subunit. The molecular masses of the human enzyme are 750 to 800 kDa for the native form, 72 kDa for the a subunit, and 56 kDa for the b subunit. In the enzyme from Mycobacterium smegmatis, each mole contains 6 moles of biotin.44 This suggests that the native enzyme is a hexamer of protomers, each protomer containing a single a and a single b subunit. The size of the human enzyme implies a similar structure, namely an (ab)6 quaternary structure. The carboxylation of propionyl CoA occurs in a two-step reaction.26 In the ®rst step, which requires adenosine triphosphate (ATP) and Mg2 and is stimulated by K, bicarbonate is attached to the ureido (N 1) nitrogen of biotin in the apoenzyme±biotin complex (Fig. 94-3), forming a carboxybiotin±apoenzyme intermediate. This complex, in turn, reacts with propionyl CoA to transfer the carboxyl group from biotin to the second carbon of propionyl CoA, forming D-methylmalonyl CoA. As with other biotin-catalyzed carbon dioxide ®xation reactions, the biotin molecule is directly responsible for the transfer of the carboxyl group.45 The complementary DNAs (cDNAs) for the a and b subunits have been cloned from human46±48 and rat liver 49±51 libraries. A 2.9-kb messenger RNA (mRNA) codes for the a chain and a major 2.0-kb mRNA codes for the b chain.46,50 Based on the sequence of the full-length cDNAs, the human a chain contains 703 amino acids52 and the b chain 539 amino acids.53,54 As is the case for most nuclear-encoded mitochondrial proteins, the a and b subunits are synthesized on free cytoplasmic polyribosomes as larger precursors, bearing cleavable N-terminal leader peptides.55 The Nterminus of the mature human a subunit has been determined and implicates a leader peptide of 26 amino acids.48 In studies on the rat enzyme, the positively charged leader peptides of both subunits were shown to direct mitochondrial uptake and processing in vitro.51,56 Assembly of the mature a and b subunits into the oligomeric holoenzyme is expected to occur after mitochondrial import and removal of the leader peptides have occurred. The polypeptide sequences of the a and b subunits have revealed a number of highly conserved regions by comparison with related enzymes from bacteria to mammals. Most striking is the near universal tetrapeptide, Ala-Met-Lys-Met, that de®nes the biotin-binding site (Lys residue) of the a subunit.47,51 In addition, the a subunit contains a recognizable biotin carboxylase domain Fig. 94-2 Enzymatic details of the major catabolic pathway for propionyl CoA and methylmalonyl CoA. Succinyl CoA has several metabolic fates, including oxidation through the tricarboxylic acid cycle and condensation with glycine to form d-aminolevulinic acid. Two coenzymes act in the reaction sequence: biotin, in the carboxylation of propionyl CoA, and adenosylcobalamin (AdoCbl), in the isomerization of methylmalonyl CoA to succinyl CoA. Fig. 94-1 Precursors of and the major catabolic pathway for propionate and methylmalonate. The free acids are derived from their CoA esters by hydrolysis. A number of clinical disorders arise from errors at various steps in these pathways. Broken arrows indicate the presence of several reactions. 2167 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2168 PART 9 / ORGANIC ACIDS Fig. 94-3 A model for the mammalian propionyl CoA carboxylase protomer containing two nonidentical subunits (a and b), a biotin carrier site on the a subunit, and multiple substrate and effector sites. and, within it, an ATP binding site57; the b subunit contains sequences related to carboxybiotin and propionyl CoA binding domains (Fig. 94-3) . Expression of the C-terminal 67 amino acids of the human a subunit in Escherichia coli results in its biotinylation by the bacterial biotin ligase, BirA, or by coexpressed human holocarboxylase synthase.58 The human PCCA gene, encoding the a subunit, has been mapped to chromosome 13q32.46,59 The PCCA gene is at least 100 kbp long, but its intron±exon organization remains to be elucidated. The PCCB gene, encoding the b subunit, is on chromosome 3q13.3-q2246,49 and has been positioned within a YAC contig containing the blepharophimosis-ptosis-epicanthus inversus syndrome locus.60 The PCCB gene contains 15 exons, ranging in size from 57 to 183 bp.61 Methylmalonyl CoA Racemase. This enzyme owes its discovery to the observation that methylmalonyl CoA synthesized chemically is a substrate for the mutase reaction (Fig. 94-2), whereas methylmalonyl CoA formed enzymatically from the carboxylation of propionyl CoA will not react with the mutase unless it is ®rst heated. Ultimately, the demonstration that heating converts D-methylmalonyl CoA to DL-methylmalonyl CoA led to the conclusion that only the L form of the ester will react with the mutase enzyme. This interpretation was con®rmed by separating mutase activity from racemase activity using Sephadex chromatography.26,38,62 The racemase has been puri®ed extensively from sheep liver.38 It has no known cofactor requirements and catalyzes the conversion of D- to L-methylmalonyl CoA by inducing a shift in the a-hydrogen atom.38,62 Methylmalonyl CoA Mutase. In 1955, Flavin et al.32 and Katz and Chaikoff33 observed independently that the isomerization of methylmalonyl CoA to succinyl CoA was catalyzed by an enzyme found in sheep kidney and rat liver. The chemical analogy between this isomerization reaction and the isomerization of glutamate to b-methylaspartate in bacteria,63 along with the demonstration by Barker and his colleagues64,65 that a coenzyme form of Cbl was needed for the latter reaction, led to the ®nding in several laboratories that a Cbl coenzyme is also required for the isomerization of methylmalonyl CoA.2 ±4 The enzyme, originally called methylmalonyl CoA isomerase but now designated methylmalonyl CoA mutase, was ®rst crystallized from sheep kidney and bacteria. More recently, it has been puri®ed to homogeneity from human placenta66 and human liver.67 From both human sources, the enzyme appears to be a dimer (145 to 150 kDa) of identical subunits (72 to 77 kDa). The holoenzyme contains 1 mole AdoCbl per mole of subunit,67 the Cbl cofactor being very tightly bound to the apoenzyme (Km of the puri®ed, expressed human enzyme for AdoCbl is 5 10 8 M).68 Under certain conditions, the human enzyme displays complex kinetics with regard to the binding of methylmalonyl CoA and AdoCbl, leading to the thesis that the active sites of the dimeric enzyme are not equivalent.69 In this regard, it is signi®cant that hydroxocobalamin (OH-Cbl) appears to act as both a competitive and an irreversible inhibitor of human mutase.69 As indicated in Figure 94-2, the isomerization reaction could occur by exchange of a hydrogen for either the free carboxyl group or the CoA-carboxyl moiety of methylmalonyl CoA. Studies using isotopically labeled substrate demonstrated convincingly that it is the CoA-carboxyl group that is transferred 70 through an intramolecular isomerization.71,72 The role of the Cbl coenzyme in this reaction has been established by electron paramagnetic resonance and ultraviolet (UV)-visible spectroscopy. As suggested by Halpern,73 AdoCbl serves as the source of a pair of free radicals, generated initially by homolysis of the cobalt±carbon bond. Although no evidence for such radicals has been obtained with holoenzyme alone, in the presence of substrate, a tightly exchange-coupled free radical pair can be observed.74,75 One electron is clearly on cob(II)alamin, as further identi®ed by its UVvisible spectrum.76 The nature of the other radical species is less clear, but isotope effect data suggest that it is substrate derived, rather than a radical form of the 50 -deoxyadenosyl group.76 For mutase from Propionibacterium shermanii, about 20 to 25 percent of the cofactor is present in a radical form under steady-state conditions, and stopped-¯ow kinetic observations suggest that homolysis is much faster than the overall reaction.76 This implies that a later, slow step (such as product release) must be rate controlling, a conclusion supported by tritium isotope effect studies77 and rationalized by structural investigations of the enzyme. The mutase subunit is synthesized as a larger cytoplasmic precursor, bearing a 3- to 4-kDa cleavable leader peptide.78 In a cell-free system, the precursor is imported by mitochondria via an energy-dependent mechanism and cleaved to its mature form by a divalent cation-dependent protease (the mitochondrial processing peptidase). In intact cells, the precursor is rapidly (halflife 6±9 min) converted to its mature form.78 Both cDNA and genomic DNA encoding human mutase have been cloned, as have the corresponding murine sequences.79±82 The cDNA sequence predicts a leader peptide 32 amino acids long that is strongly positively charged (4 Arg, 2 Lys, 1 Glu).80,82 Analysis of Southern blots of DNA from human/hamster somatic cell hybrids and in situ hybridization, using the mutase cDNAs as probes, mapped the human gene for mutase and the MUT locus to region 6p12-p21.283 and uncovered one highly informative restriction fragment length polymorphism.79 Mutase from P. shermanii has been puri®ed, cloned, and overexpressed in E. coli. It is an ab heterodimer with a single active subunit (a) that binds one molecule of AdoCbl.84,85 The a subunit is about 60 percent identical (75 percent similar) to the human enzyme, with many of the nonconservative substitutions occurring in the N-terminal region.86 Several forms of P. shermanii mutase have been crystallized and their radiographic structures solved (PDB codes 1REQ±5REQ).87±89 Based on these, mutase consists of two major domains, with an N-terminal region involved in subunit±subunit interaction and an interdomain linker (see Fig. 94-10). The ®rst domain is a (ba)8 TIM barrel, with the substrate binding site threaded through its center. The second domain is the so-called Cbl-binding domain, by virtue of its close sequence and structural homology with the Cbl-binding domain of methionine synthase.90 It is a (ba)5 barrel that has a groove for binding the 5,6-dimethylbenzimidazolyl (DMB) side chain of Cbl, the stabilized histidine side chain that displaces the DMB from the lower axial position of the Cbl, and interaction sites for the lower face of the corrin ring. The interface between these domains Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM Fig. 94-4 Minor pathways of propionate catabolism. Note that both pathways can ultimately generate acetyl CoA. The signi®cance of these minor pathways is discussed in the text. accommodates the upper face of the corrin and the 50 deoxyadenosyl group. Comparison of substrate-free and substrate-bound structures indicates both how substrate and product come and go from the active site and how the enzyme protects the radicals at its active site.88 In the substrate-free (open) conformation, four of the ba pairs in the ®rst domain have moved as a rigid body, relative to the other four and the second domain, to split open the TIM barrel and provide access to the active site and the binding sites for the pantotheine chain of the substrate. In the bound (closed) con®guration, however, the domain has come back together to close the active site around the methylmalonyl group, the rest of the substrate blocks access through the middle of the barrel, the active site cavity is reduced in volume, and the binding site for the 50 -deoxyadenosyl group is destroyed. Thus, the substrate-triggered conformational change appears to simultaneously drive homolysis of the cobalt±carbon bond while sequestering the radicals formed at the active site, permitting a free-radical rearrangement protected from the surrounding aqueous environment. Because a corresponding opening of the TIM barrel must occur before product can be released, it has been suggested that this is likely the slow step in the overall forward reaction.88 Alternative Pathways of Propionate Metabolism Although the catabolism of propionate to succinate through methylmalonate is the major pathway for propionate utilization in mammalian systems, alternative pathways exist. Propionyl CoA can replace acetyl CoA as a ``primer'' for long-chain fatty acid synthesis91 and lead to the formation of odd-chain fatty acids, notably heptanoate, nonanoate, and undecanoate. There are also alternative catabolic mechanisms, one of which is described in Fig. 94-4.26 The ®rst step in this sequence involves the formation of an a,b-unsaturated fatty acid, acrylyl CoA, which is subsequently hydrated, leading to the formation of either lactyl CoA or b-OHpropionyl CoA. The former compound is hydrolyzed to lactate, thus providing a second means by which propionate may be converted to pyruvate. Catabolism of b-OH-propionyl CoA leads ultimately to the synthesis of acetyl CoA or b-alanine, compounds discussed in Chapter 91. In addition, propionyl CoA may condense with oxaloacetate to form methylcitrate in a reaction analogous to the biosynthesis of citric acid from acetyl CoA and oxaloacetate.92 These alternative pathways are of little quantitative importance in normal subjects but become much more prominent in patients with blocks in the major pathway of propionate metabolism.93±95 Coenzymes Biotin. Biotin is widely distributed in plants and animal tissues and is readily synthesized by a variety of microorganisms. It was ®rst isolated from egg yolk in 1936 by the Dutch biochemist Kogl,96 and its structure was de®ned soon thereafter by du Vigneaud and colleagues.97 Our understanding of this watersoluble cofactor is inextricably linked with the evolution of our knowledge concerning avidin, an egg white protein that binds biotin very tightly. Comprehensive reviews on biotin98,99 and on biotin-dependent enzymes100,101 exist. The reader is referred to Chapter 156 for further discussion of biotin. Structure and Function. Biotin is the essential cofactor of the four bicarbonate-utilizing carboxylases in mammals. Structurally, it is composed of fused imidazole and thiophene rings to which is attached an n-valeric acid side chain; it has a molecular mass of 244 daltons. It is covalently attached to carboxylases through a linkage between the carboxyl group and the e-amino group of a lysine residue in a well-de®ned biotin-binding domain, which is highly conserved among carboxylases from bacteria to mammals (Fig. 94-3).102 Its role is to act as the carboxyl carrier in the carboxylation of substrate molecules (Fig. 94-5). The four biotin-dependent carboxylases are enzymes of intermediary metabolism.103 One of these, acetyl CoA carboxylase, occurs in two forms104,105 and is a cytosolic enzyme. It catalyzes the key step in long-chain fatty acid biosynthesis, the formation of malonyl CoA from acetyl CoA. The three other biotin-dependent carboxylases are found in the mitochondrial matrix, where they catalyze critical steps in amino acid and organic acid metabolism. Pyruvate carboxylase is a key enzyme of gluconeogenesis; b-methylcrotonyl CoA carboxylase and propionyl CoA carboxylase are involved in the catabolism of amino acids, and the latter also performs the ®nal step in the oxidation of fatty acids of odd-numbered chain lengths and cholesterol. All four carboxylases are biotinylated by the same enzymatic reaction (reviewed in Chapter 156). This is con®rmed genetically by the inherited metabolic disorder biotin-responsive multiple carboxylase de®ciency, in which the activity of all four biotindependent carboxylases is impaired.106 The responsible enzyme, 2169 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2170 PART 9 / ORGANIC ACIDS Fig. 94-5 Schematic representation of biotin uptake and metabolism by tissue cells. Neither the mechanism by which biotin is transported across the plasma membrane nor that by which it enters the mitochondrion is well understood. Abbreviations: bio 5 biotin; ACC 5 acetyl CoA carboxylase; PC 5 pyruvate carboxylase; MCC 5 b-methylcrotonyl CoA carboxylase; PCC 5 propionyl CoA carboxylase. holocarboxylase synthase, has been puri®ed from bovine liver and is found in both the mitochondria and cytosol.107,108 Although mitochondrial apocarboxylases are expected to be biotinylated subsequent to their import, it has been shown experimentally that biotinylation of the a subunit of propionyl CoA carboxylase can be accomplished in either compartment.109 Holocarboxylase synthase shows a striking ability to cross species barriers and is fully interchangeable with the orthologous E. coli biotin ligase BirA, with which it shares sequence similarity across its biotin binding region.110,111 Either enzyme will biotinylate the E. coli biotin carboxyl carrier protein, a component of the bacterial acetyl CoA carboxylase, or the 67±amino acid, biotin-carrier component of the human propionyl CoA carboxylase a subunit.58,111,112 Biotin remains bound to the carboxylases until they are degraded. On proteolysis of the enzymes, biotin is released from small biotinylated peptides or from biocytin (biotinyl lysine) by the enzyme biotinidase. The latter enzyme, widely distributed in tissues and most abundant in serum, has been puri®ed to homogeneity.113,114 In addition to its biocytin hydrolase activity, the enzyme has been shown to have a biotinyl-transferase activity in which biotin is transferred from biocytin to lysine-containing acceptors, including poly-lysine and histones, the latter suggesting a novel physiologic role for biotin.115,116 (discussed in detail in Chapter 156). Absorption and Distribution. It seems likely that free biotin is formed in the intestinal lumen, either by enzymatic hydrolysis of ingested, protein-bound biotin or by release from intestinal microorganisms. Biotinidase may play a role in releasing biotin from foods.117 Saturable, carrier-mediated, sodium-dependent systems for biotin transport and absorption have been demonstrated in human118 and rat119 intestine and in human intestinal brush-border membranes.120 In addition, a second, sodiumindependent system has been described in basolateral membrane vesicles from human intestine,121 thus accounting for both intestinal absorption and release into the portal circulation. Although nonmediated transport can occur, particularly at low pH, it seems likely that the mediated system predominates at physiologic biotin concentrations and pH. Biotin in blood appears to be largely free.122 Although experiments in cultured cell systems have been rather uninformative concerning the mechanism of cellular transport, basolateral membrane vesicles prepared from rat123 or human124 liver show a sodium-dependent, carriermediated transport, as do brush-border membrane vesicles from human placenta,125 suggesting that biotin transport is generally a mediated process. Biotin De®ciency. Spontaneous biotin de®ciency has almost never been reported in humans, probably because the daily requirement is very small (estimated at l20 mg/day) and because intestinal microorganisms synthesize suf®cient amounts of the cofactor even in the absence of nutritional sources. Biotin de®ciency has been reported, however, in patients with the short bowel syndrome being fed exclusively by parenteral alimentation.126 Experimental biotin de®ciency has been produced in animals and humans by ingestion of large amounts of egg white, which contains the potent biotin binder avidin.127 Under these conditions, cutaneous pallor, dermatitis, depression, lassitude, muscle pains, hyperesthesia, and ®nally anemia and electrocardiographic changes developed in four experimental human subjects. All these symptoms and signs were reversed rapidly by administration of 150 to 300 mg biotin daily for several days. In animals, experimental biotin de®ciency has been shown to produce decreased activity of biotin-dependent carboxylases in tissues.98± 100 Cobalamin (Vitamin B12). The structure and function of this compound have intrigued students of human biology since 1926, when Minot and Murphy demonstrated that oral administration of crude liver extract was effective in the treatment of pernicious anemia.128 In 1948, this anti-pernicious anemia factor was isolated from liver and kidney129,130 and was named vitamin B12. Administration of as little as 1 mg of the vitamin daily was shown to prevent relapse of pernicious anemia. Although the vitamin is widely distributed in animal tissues, there is strong evidence that it is synthesized only in microorganisms found in soil, water, or the rumen and intestine of animals. For further information on Cbl, the reader is referred to Chapter 155. For comprehensive reviews of Cbl chemistry and metabolism, the reader is referred elsewhere.131 Structural Features. The isolation of vitamin B12 culminated in the elucidation of its three-dimensional structure by Hodgkin and co-workers using x-ray crystallographic techniques.132 Vitamin B12, or, as it is now of®cially designated, cobalamin, is composed of a central cobalt atom (Co) surrounded by a planar corrin ring and a complex side chain extending down from the corrin plane consisting of a 5,6-dimethylbenzimidazolyl phosphoribosyl moiety (Fig. 94-6). The benzimidazole is linked to the Co atom through one of its nitrogens, whereas the phosphate is bonded to the D ring of the corrin. The molecule is completed by coordinate linkage of one of several different radicals to the Co nucleus from above the corrin plane. Thus, CN-Cbl or, more strictly, a-(5,6dimethylbenzimidazolyl)-cobamide cyanide, is formed by the Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM Fig. 94-7 Reactions catalyzed by cobalamin coenzymes in mammalian tissues. Note the speci®city of adenosylcobalamin for the isomerization of methylmalonyl CoA and of methylcobalamin for the methylation of homocysteine. Me-H4folate 5 N 5-methyltetrahydrofolate; H4folate 5 tetrahydrofolate. Fig. 94-6 Structure of adenosylcobalamin (AdoCbl). R 5 CH2CONH2; R0 5 CH2CH2CONH2. Other radicals that may be coordinately linked to the cobalt atom include CH3 (methylcobalamin), OH2 (hydroxocobalamin), and CN2 (cyanocobalamin). (Reprinted with permission from Babior BM: Cobamides as cofactors: Adenosylcobamide dependent reactions, in: Cobalamin Biochemistry and Pathophysiology. New York, Wiley, 1975, p 141.) attachment of a cyanide radical to the Co atom. Although this compound is the most common commercial form of the vitamin, it is an artifact of isolation and does not occur naturally in microorganisms, plants, or animal tissues. Many other cobalamins can be formed by replacement of the cyanide radical, but only four have been isolated from mammalian tissue: OH-Cbl, glutathionylcobalamin (GSCbl), methylcobalamin (MeCbl), and AdoCbl. The latter two compounds are unique for two reasons: They are the only two compounds in nature known to have a direct carbon± cobalt bond, and they are the only two forms of Cbl known to act as speci®c coenzymes in mammalian systems. The structure and nomenclature of the cobalamins are further complicated by oxidation and reduction of the Co atom. In OHCbl, the Co atom is trivalent [cob(III)alamin], and this compound has been called vitamin B12a. When the Co is reduced to a divalent state [cob(II)alamin], the molecule is called vitamin B12r and, in the monovalent state [cob(I)alamin], it is called vitamin B12s. These oxidation-reduction states are important because there appear to be speci®c reductase enzymes that sequentially convert cob(III)alamin to cob(I)alamin, with cob(II)alamin acting as an intermediate.133 The Co atom must be reduced to its monovalent state prior to formation of MeCbl or AdoCbl. Cobalamin Coenzymes. In 1958, Barker and his colleagues demonstrated that the glutamate mutase reaction in Clostridium tetanomorphum required vitamin B1263 and, more speci®cally, that the active coenzyme form of the vitamin was AdoCbl.64,65 One year later, Smith and Monty reported that the analogous isomerization of methylmalonyl CoA to succinyl CoA was defective in the liver of Cbl-de®cient rats.2 They suggested that Cbl is a cofactor for the latter isomerization system, a thesis borne out by Gurnani et al.3 and Stern and Friedmann,4 who showed in vitro that the activity of methylmalonyl CoA mutase in liver from Cbl-de®cient animals could be restored to normal by the addition of AdoCbl, but not by CN-Cbl or other vitamin B12 analogues (Fig. 94-7). For several years, because AdoCbl was the only known coenzyme form of vitamin B12, it was designated coenzyme B12. In 1966, Weissbach and his colleagues134 demonstrated that MeCbl is a cofactor in the complex reaction by which homocysteine is methylated to methionine (Fig. 94-7). This reaction requires S-adenosylmethionine and N5-methyltetrahydrofolate (Me-H4folate), as well as the methionine synthase apoenzyme and MeCbl. The mechanism of homocysteine methylation probably involves the following sequence: Me-H4folate is converted to tetrahydrofolate (H4 folate) by transferring its methyl group to a reduced Cbl prosthetic group on the methionine synthase holoenzyme; in turn, the methyl group is transferred from MeCbl to homocysteine, leading to the formation of methionine135,136 (see Chapter 155). The conversion of methylmalonyl CoA to succinyl CoA and the methylation of homocysteine to methionine are the only Cbldependent reactions that have been demonstrated conclusively in mammalian systems. Poston reported that AdoCbl acts as a cofactor in the enzymatic reaction by which a-leucine is isomerized to b-leucine,137 but this has not been con®rmed in other laboratories. In microorganisms, several other enzymes require AdoCbl138,139: glutamate mutase, diol dehydrase, glycerol dehydrase, ethanolamine ammonia-lyase, and ribonucleotide reductase. In addition, MeCbl is involved in the formation of methane and acetic acid and the fermentation of lysine in bacteria. Cbl Absorption and Distribution. The Cbl vitamins have a unique and highly specialized mechanism of intestinal absorption that has been reviewed in detail.140±142 The ability to transport physiologic quantities of the vitamin depends on the combined action of gastric, ileal, and pancreatic components. The gastric substance, called intrinsic factor (IF) by Castle,138 who ®rst demonstrated its existence, is a glycoprotein, synthesized by gastric parietal cells, that binds cobalamins in the intestinal lumen after they have been released from dietary protein by acid and peptic hydrolysis. Subsequently, the IF±Cbl complex interacts through its protein moiety with a speci®c ileal receptor protein, called cubilin.143 The vitamin is transcytosed across the ileal epithelial cells and appears in the portal blood bound to transcobalamin II (TC II), the transport protein for newly absorbed vitamin.140,144,145 When 2171 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2172 PART 9 / ORGANIC ACIDS labeled Cbl is administered intravenously or orally, most of it is immediately bound to TC II and disappears from the plasma in a few hours.146,147 Two other Cbl-binding proteins, transcobalamin I (TC I) and transcobalamin III (TC III), are also found in serum. TC I and TC III are glycoproteins of the R-binder, or haptocorrin, family, which carry the majority of Cbl found in plasma, but their physiologic role is unclear. For example, only a small fraction of newly absorbed Cbl binds to TC I, and this component turns over very slowly. MeCbl is the major circulating Cbl species, accounting for 60 to 80 percent of total plasma Cbl; OH-Cbl and AdoCbl make up the remainder.148 Because over 90 percent of total plasma Cbl is bound to TC I, it is clear that most of the circulating MeCbl travels with this R-binder. This Cbl distribution pattern is puzzling, particularly in the face of evidence indicating that AdoCbl accounts for approximately 70 percent of total hepatic Cbl, whereas MeCbl constitutes a mere 1 to 3 percent.148 A preponderance of AdoCbl is also present in such other tissues as erythrocytes, kidney, and brain. The physiologic signi®cance of these widely different fractional amounts of Cbl compounds in extracellular and intracellular compartments remains obscure. Transcobalamin II facilitates Cbl uptake by mammalian tissues. Finkler and Hall149 showed that CN-Cbl bound to TC II was accumulated by HeLa cells much more rapidly than free CNCbl or CN-Cbl bound to TC I, IF, or other binding proteins. Such TC II±mediated uptake has been subsequently con®rmed in a variety of cell types, both in vivo and in culture.141 These ®ndings, coupled with the observations in vivo that TC II disappeared from plasma as TC II±Cbl was absorbed150 and appeared in lysosomal fractions of hepatic151 and kidney152 cells, led to the proposal that the circulating TC II±Cbl complex is recognized by a speci®c, widely distributed plasma membrane receptor, a hypothesis supported by considerable experimental evidence. YoungdahlTurner and associates153 showed that the complex binds to a speci®c, high-af®nity (Ka 10 10 M) cell surface receptor on cultured skin ®broblasts, that the TC II±Cbl complex is internalized intact via adsorptive endocytosis,154 and that the degradation of TC II and release of Cbl from the complex occur as a result of lysosomal protease activity.153,154 Cbl then exits from the lysosome and is either converted to MeCbl and bound to the methionine synthase in the cytosol or enters the mitochondrion, where, after reduction and adenosylation, it is bound to methylmalonyl CoA mutase (Fig. 94-8).155,156 The intricate process just described is surely the most widely distributed physiologic means by which mammalian cells obtain Cbl, but it is not the only one. Hepatocytes, for instance, contain a surface receptor for asialoglycoproteins, and this receptor interacts with TC I±Cbl (and perhaps TC III±Cbl) complexes, thereby providing a second potential means by which this particular tissue obtains Cbl.157 There is also evidence that at least some tissues are capable of taking up free (unbound) Cbl if the concentration of unbound vitamin is increased to suf®ciently high concentrations. In cultured ®broblasts, this uptake process for free Cbl is saturable, Ca2 independent, and sensitive to inhibitors of protein synthesis and sulfhydryl reagents.158 Its functional role under most circumstances is probably negligible, however. Coenzyme Biosynthesis and Compartmentation. Because methylmalonyl CoA mutase, the mammalian enzyme dependent on AdoCbl, is a mitochondrial protein,159 whereas the MeCbldependent methionine synthase is cytoplasmic,160 it becomes important to relate the cellular biology of the vitamin to its cellular and molecular chemistry. The chemical pathway of AdoCbl synthesis was de®ned initially in bacteria.133,161 Three enzymes are required for coenzyme synthesis: two reductases and an adenosyltransferase. The reductases are ¯avoproteins that require NAD as a cofactor. The ®rst (EC 1.6.99.8) is responsible for converting cob(III)alamin (i.e., OH-Cbl) to cob(II)alamin, and the second (EC 1.6.99.9) for catalyzing the further reduction to cob(I)alamin. The latter compound and ATP are substrates for an adenosyltransferase (EC 2.5.1.17), which completes the synthesis of AdoCbl. Neither of the reductases has been puri®ed extensively, but the adenosyltransferase has. It has an optimal pH of 8, requires Mn2, and has a Km of 1 10 5 M for cob(I)alamin and 1.6 10 5 M for ATP.161 The biosynthetic steps leading to MeCbl formation are somewhat analogous, likely involving reduction followed by methylation directly by the methionine synthase apoenzyme.162,163 Evidence has accumulated that indicates that mammalian cell metabolism of Cbl may proceed by a very similar set of reactions (Fig. 94-8). In 1964, Pawalkiewicz et al.164 showed that human liver and kidney homogenates could convert CN-Cbl to AdoCbl. Several years later, AdoCbl synthesis from OH-Cbl was observed in HeLa cell extracts incubated with ATP and a reducing system that presumably bypassed the enzymatic reduction of OH-Cbl [cob(III)alamin] to cob(I)alamin.165 Subsequently, Mahoney and Rosenberg166 demonstrated the synthesis of both AdoCbl and MeCbl by intact human ®broblasts growing in a tissue culture medium containing OH-[57Co]Cbl. This system was subsequently characterized in cell extracts.167,168 As with the HeLa cell system, chemical reductants were employed to bypass both Cbl Fig. 94-8 General pathway of the cellular uptake and subcellular compartmentation of Cbl, and of the intracellular distribution and enzymatic synthesis of Cbl coenzymes. TC II 5 transcobalamin II; OH-Cbl 5 hydroxocobalamin; GSCbl 5 glutathionylcobalamin; MeCbl 5 methylcobalamin; MeFH4 5 methyltetrahydrofolate; FH4 = tetrahydrofolate; AdoCbl 5 adenosylcobalamin; CblIII, CblII, CblI cobalamins with cobalt valence of 31, 21, and 11, respectively. Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM reductases.168 Such extracts synthesized AdoCbl, thereby demonstrating that the adenosyltransferase found in bacteria also exists in normal human cells. These experiments also revealed that the adenosyltransferase was mitochondrial in location, implying that both the synthesis and cofactor activity of AdoCbl take place in this organelle. In contrast, MeCbl synthesis takes place in the cytosol in conjunction with the methionine synthase reaction (Fig. 94-8). Metabolic Abnormalities in Cbl De®ciency. The biochemical abnormalities in plasma and urine of patients with Cbl de®ciency re¯ect the dysfunction of the enzymes dependent on Cbl coenzymes. The ®rst relevant observation in this context was the demonstration by Cox and White5 and by Barness and his colleagues6 that methylmalonic acid excretion in the urine was distinctly increased in Cbl-de®cient patients with classic pernicious anemia. The methylmalonic aciduria in these patients was reversed rapidly by administration of physiologic doses of Cbl, indicating that repletion of Cbl restored the methylmalonyl CoA mutase reaction to normal. Later, Cox et al. reported that patients with Cbl de®ciency also have distinctly increased amounts of propionic acid in the urine, this abnormality again being reversed by treatment.169 Interestingly, they also found excessive amounts of acetic acid in the urine of Cbl-de®cient subjects. The mechanism of this abnormality is not clear, because acetate does not participate in the major pathway of propionate catabolism. The ®nding could re¯ect increased utilization of the alternative pathways of propionate metabolism in the face of a block in the major pathway, because each of the alternative routes leads eventually to the formation of acetyl CoA (Fig. 94-4). Excessive excretion of homocystine also has been documented in Cblde®cient patients,170,171 as has combined methylmalonic aciduria and homocystinuria.172 The latter report is particularly interesting because it documents congenital but not hereditary Cbl de®ciency, in this instance due to acquired Cbl de®ciency in the offspring of a strictly vegetarian mother who also was de®cient in the vitamin. A number of such cases have now been reported.21 DISEASE STATES The Propionic Acidemias In 1961, Childs et al.7 described a male infant with episodic metabolic ketoacidosis, protein intolerance, and remarkably elevated plasma glycine concentration. Several hundred children with similar clinical and biochemical ®ndings have since been described. Many of these children were subsequently found to have methylmalonic acidemia173; a few had b-ketothiolase de®ciency (see Chapter 93). However, the patient described by Childs et al., and many reported subsequently, had propionic acidemia due to a primary and speci®c de®ciency of propionyl CoA carboxylase activity (Fig. 94-2). This conclusion was derived independently from the description of a patient with massive propionate accumulation in blood,174 from another with impaired propionate oxidation in leukocytes9 and defective carboxylase activity in ®broblast extracts,175 and from a third with both propionic acidemia and defective carboxylase activity.176 We now recognize that propionyl CoA carboxylase de®ciency also occurs in children with inherited abnormalities in biotin metabolism, leading to the de®ciency of multiple biotin-dependent carboxylases (see Chapter 156). Hence, we must now use the term propionic acidemias to refer to this heterogeneous group of related inborn errors. As will be discussed subsequently, a similar heterogeneity exists among the methylmalonic acidemias. Propionyl CoA Carboxylase De®ciency. Clinical Manifestations. As mentioned above, this disorder was originally referred to as ketotic hyperglycinemia. E.G., the patient described by Childs and Nyhan and their colleagues,7,177,178 presented with dehydration, lethargy, and coma on the ®rst day of life. He was found to be severely ketoacidotic and responded slowly to massive alkali replacement. The clinical course was characterized by recurrent attacks of ketoacidosis, precipitated by infections or protein ingestion, and by developmental retardation, electroencephalographic (EEG) abnormalities, and osteoporosis. The patient had episodic neutropenia and thrombocytopenia prior to death at age 7. A sister (A.G.) also became ketotic and acidotic during the ®rst 4 days of life, but the course of her condition has been modi®ed dramatically because of the extensive experience gained in studying her brother. Although she has had mild attacks of ketoacidosis during intercurrent infections, maintenance on a lowprotein diet has resulted in little need for hospital care and normal somatic and mental development up to 15 years of age.179 In 1968, Hommes and his colleagues174 described a male infant with hyperventilation, are¯exia, and grunting at 60 h of age. There was a profound metabolic acidosis (arterial pH 6.98), and despite administration of massive amounts of sodium bicarbonate and tris(hydroxymethyl)-aminomethane, the infant died on the ®fth day of life. Leukocytes and platelets were normal. Postmortem examination showed only a fatty liver and degeneration of Purkinje cells and the granular layer of the cerebellum. Subsequent descriptions of patients with propionic acidemia have con®rmed that most patients present in the newborn period with severe metabolic acidosis manifested by refusal to feed, vomiting, lethargy, and hypotonia; dehydration, seizures, and hepatomegaly occur less often.180,181 Other patients have presented later, either with acute encephalopathy or episodic ketoacidosis or with developmental retardation apparently uncomplicated by attacks of ketosis or acidosis.182,183 A 5-year-old boy presented with a fatal necrosis of the basal ganglia without either metabolic acidosis or hyperammonemia.184 Propionic acidemia has been identi®ed in a 29-year-old man who presented initially with adult-onset chorea and dementia.185 Still other children, with almost complete de®ciency of propionyl CoA carboxylase activity as measured in extracts of cultured ®broblasts, have had no clinical abnormalities whatever and have been identi®ed only during family studies.186,187 No satisfactory explanation for this striking lack of clinical enzymatic correlation exists at present. Based on a survey of 65 patients with propionic acidemia, Wolf et al.181 reported that the clinical course of symptomatic patients is characterized by repeated relapses, usually precipitated by excessive protein intake, constipation, or intercurrent infection. Treatment of these children has been quite dif®cult, and neurologic sequelae have been common. Among the neurologic complications often observed, developmental delay, focal and general seizures, cerebral atrophy, and EEG abnormalities have been the most prominent. Surtees et al.183 also have reported a high prevalence of neurologic sequelae, including dystonia, severe chorea, and pyramidal signs, particularly in patients who survive longer. The cranial computer tomographic and magnetic resonance imaging ®ndings in propionic acidemia were reviewed by Bergman et al., who showed spectroscopic abnormalities, speci®cally an increase in glutamine/glutamate, even when the patients appeared to be stable.188 Walter et al. described 11 newborn patients with elevated blood ammonia levels and neurologic symptoms; only 4 had clinically important acidosis.189 Leukopenia and thrombocytopenia, perhaps due to marrow suppression by one or more of the toxic metabolites produced, is also not uncommon. Parathyroid hormone resistance and B-cell lymphopenia was described in a 7-week-old patient.190 Biochemical Abnormalities. Childs and Nyhan7,177,178,191 studied their index patient extensively. Because of the hyperglycinemia, they focused their attention on the pathways of glycine formation and utilization but found no consistent abnormalities. Normal hemoglobin concentration in the peripheral blood indicated that the pathway from glycine to d-aminolevulinic acid was not blocked. Slices of the patient's liver incorporated [14C]glycine into protein and carbon dioxide as well as slices of rat liver did. Salicylate and benzoate were normally conjugated with glycine, 2173 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2174 PART 9 / ORGANIC ACIDS and the glutathione concentration of whole blood was normal. Although the rate of conversion of tritiated glycine to serine in vivo was slower than in controls, this difference may have re¯ected the enlarged glycine pool rather than a speci®c block in the conversion of glycine to serine.191 Moreover, several observations suggested an abnormality in the catabolism of the branched chain amino acids, methionine, and threonine. Plasma concentrations of valine, isoleucine, and leucine were elevated intermittently; administration of leucine, valine, isoleucine, threonine, and methionine each precipitated attacks of ketoacidosis, but no other amino acids were toxic. Menkes192 reported that the urine contained large amounts of butanone (a four-carbon ketone that is a by-product of isoleucine catabolism) and the longer chain ketones, pentanone and hexanone. These long-chain ketones were not detected in the urine of patients with ketosis due to diabetes, starvation, or ketogenic diets. Because isoleucine, valine, threonine, and methionine are all precursors of propionate, a defect in propionate metabolism seemed likely, but patient E.G. died before any other studies of propionate catabolism could be performed. Subsequently, Hsia et al.9 demonstrated a striking defect in propionate catabolism in A.G., the affected sister of E.G. When leukocytes isolated from her peripheral blood were incubated with [3-14C]propionate, negligible quantities of 14CO2 were evolved as compared with values in controls, but her cells oxidized methylmalonate and succinate normally. Identical ®ndings were obtained using ®broblasts grown in tissue culture. These data showed that the primary metabolic defect in E.G. and A.G. was in the conversion of propionyl CoA to D-methylmalonyl CoA, a reaction catalyzed by propionyl CoA carboxylase. This conclusion was con®rmed subsequently by direct assay of carboxylase activity in ®broblast extracts.175 In their child with lethal neonatal acidosis, Hommes et al.174 found that the serum propionic acid concentration was 400 mg/dl (5.4 mM), a value more than 100 times that reported in normal infants. The liver contained fatty acids with 15 and 17 carbon atoms in addition to the even-chain fatty acids found in control livers. From these data, Hommes et al. also postulated a defect in propionyl CoA carboxylation in their patient. Subsequent investigations have con®rmed and extended these early ®ndings. Analysis of body ¯uids in several additional patients92±94,176 showed that propionate accumulation in blood and urine occurs regularly, its magnitude being related to the severity of the clinical course and the time at which sampling is performed. Ando and colleagues92 have stressed that other propionate derivatives also accumulate in urine. These include methylcitrate, which is probably formed from the intramitochondrial condensation of propionyl CoA with oxaloacetate92; propionylglycine, which results from the conjugation of propionate with glycine93; b-hydroxypropionate, an intermediate in one of the alternative pathways of propionate catabolism94 (Fig. 94-4); and tiglic acid,193 an isoleucine catabolite several steps proximal to the block. Although the exact amounts of these compounds in urine have not always been determined, they appear to account for a small fraction of the propionate pool that accumulates in vivo in this disease. Their presence may be important in mitigating the toxic effects of propionate excess. Wendel et al. showed elevated levels of odd-numbered fatty acids (OLCFA) in the erythrocyte lipids of ®ve patients with propionic acidemia and suggested that OLCFA levels re¯ect the continuous burden of propionyl CoA toxicity within cells and could serve as a means of evaluating the quality of long-term metabolic control.194 Other compounds, not directly concerned with the propionate pathway, also have been found in signi®cantly increased amounts. In addition to hyperglycinemia and hyperglycinuria, which were discussed earlier, marked hyperammonemia has been documented in several patients,181,195 and a distinct correlation between plasma propionate and blood ammonia has been noted in two patients.196 The Enzymatic Defect. The molecular pathology of propionic acidemia is both complex and interesting. Cell extracts from a number of affected patients share a common ®nding, namely, reduction in propionyl CoA carboxylase activity to 1 to 5 percent of that in controls.197±199 Because the enzyme is composed of two independently encoded enzyme subunits, the causative mutations will necessarily occur in one of two genes. This was ®rst illustrated in complementation experiments in which ®broblast heterokaryons formed between pairs of affected cell lines were assayed for recovery of functional propionyl CoA carboxylase by ®xation of 14 C-propionate.200±202 Two major complementation groups, pccA and pccBC, were identi®ed, the latter group showing intragroup complementation (subgroups pccB and pccC) compatible with the occurrence of interallelic complementation (see Fig. 94-11). It was shown subsequently that patients in the pccA group have a primary defect in the PCCA gene encoding the a subunit of propionyl CoA carboxylase, whereas patients in the pccBC group and subgroups have defects of the PCCB gene encoding the b subunit.203,204 There are several unusual features of mutant ®broblast lines from patients belonging to the different complementation groups. First, many individuals in the pccA group lack detectable a subunit protein; when this is the case, they invariably lack detectable b subunits as well.203,205 This has been explained by the inherent instability and consequent degradation of the b subunit in the absence of a subunit with which to assemble to form the native enzyme. Among a-minus/b-minus cell lines, some lack a subunit mRNA but contain b subunit mRNA, con®rming the assignment of the pccA complementation group to mutations of the PCCA gene.204 Second, a number of pccBC or subgroup ®broblasts lack b subunits but have a subunits. These have unstable b subunit protein due to mutation, although b subunit protein is present in at least some cases.205,206 Importantly, the small amount of residual propionyl CoA carboxylase activity observed in extracts of most mutant ®broblasts appears to be present even in those with absent a or b subunits. This suggests that the ``background'' activity is due to the minimal activity of other carboxylases acting on propionyl CoA as substrate, not to propionyl CoA carboxylase itself.207 A third unusual feature of propionic acidemia is that many heterozygotes of the pccBC group or subgroups have propionyl CoA carboxylase activity indistinguishable from that in controls, whereas obligate heterozygotes of the pccA group have the expected 50 percent of control activity.199 This has proved to be due to relative differences in the synthesis of the enzyme subunits. It has been shown that b subunits are synthesized in fourto ®vefold excess over a subunits, so that heterozygotes for nulltype b subunit mutations still have more than enough wild-type b subunits to interact with the limiting amount of a subunits to form normal amounts of carboxylase enzyme.205 Conversely, any reduction in the amount of a subunit, as in pccA heterozygotes, is directly re¯ected in a proportionate reduction in carboxylase activity.199 All three of these features of mutant cell lines can be used diagnostically to identify the affected gene, but caution is warranted because patients with point mutations in either gene may have both subunits present, despite having a defective holoenzyme. Pathologic Physiology. A defect in the carboxylation of propionate provides a satisfactory explanation for many of the ®ndings reported in this disorder. This defect would be expected to lead to an elevated concentration of propionate in the blood and an inability of leukocytes to catabolize propionate to carbon dioxide. Because isoleucine, valine, threonine, and methionine are precursors of propionate, such a block also should lead to the observed protein and speci®c amino acid intolerance. The appearance of long, odd-chain fatty acids in the liver suggests that when propionyl CoA carboxylation is blocked, odd-chain fatty acid biosynthesis may be augmented because propionyl CoA is the ``primer'' for such compounds. Finally, the presence of such compounds as butanone, methylcitrate, b-hydroxypropionate, propionylglycine, and tiglic acid very likely results from reversal of reactions proximal to the primary carboxylase block or from increased utilization of alternative pathways. Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM It is not at all clear from the foregoing, however, why some patients have a severe and often life-threatening course, and others are only mildly affected clinically. Major differences in dietary protein uptake and quality, in the contributions of gut bacteria to total propionate load, or in the activity of alternative mechanisms for propionate disposal are possible explanations for the wide clinical spectrum, but the prominent intrafamilial differences in severity are not easily explained this way.186 Furthermore, several other features of the disease are not adequately explained by the block in propionate catabolism. The ketosis produced in E.G. by leucine is not understood, because this amino acid is not catabolized to propionate. However, it is ketogenic in normal subjects, suggesting that its effect in E.G. was nonspeci®c. The cause for the hyperglycinemia seen in many, but not all, of these patients also has not been adequately de®ned. Because the infant described by Hommes et al.174 with massive propionic acidemia never demonstrated signs of hyperglycinemia, the latter cannot be ascribed simply to the acidosis or ketosis. Numerous theses have been put forth in explanation. For example, one or more products of isoleucine catabolism may interfere with glycine cleavage or glycine±serine interconversion.191,208,209 Ando et al.92 speculated that methylcitrate cleavage in the cytosol may yield propionate and glyoxylate, the latter being used as a substrate for glycine overproduction. Impaired glycine conjugation systems have been suggested, but no data in support of this notion have been forthcoming. Because plasma glycine concentration may increase in sick children with negative nitrogen balance of many causes,210 the hyperglycinemia may be nonspeci®c. The hyperammonemia often observed in this disorder has been the subject of considerable investigation. It appears likely that this secondary but clinically important ®nding results from inhibition of the ®rst enzyme of the urea cycle, mitochondrial carbamyl phosphate synthetase (CPS I), by the organic acids and CoA esters that accumulate intramitochondrially behind the block in propionyl CoA carboxylation. This conclusion rests on data from studies with experimental animals and animal tissues. For example, propionate inhibits ureagenesis in rat liver slices when ammonia, but not citrulline or aspartate, is the nitrogen-donating substrate.211 Administration to rats of suf®ciently large amounts of propionate or methylmalonate to produce hyperammonemia is associated with a marked decrease in hepatic concentration of N-acetyl glutamate,212 the required allosteric effector of CPS I, probably by competitively inhibiting N-acetyl glutamate synthetase.213 That such CPS I inhibition occurs in vivo as well as in vitro is supported by case reports that describe selective impairment of CPS I activity in the livers of patients with propionic acidemia214 or methylmalonic acidemia.215 Genetics. As a prelude to mutation identi®cation in propionic acidemia, it is ®rst necessary to identify the affected gene. This is most readily done by conducting complementation tests and determining whether the affected patient belongs in the pccA or pccBC group or a subgroup. Determination of the subgroup (e.g., pccB or pccC vs. pccBC) is not speci®cally required, but does provide insight into the functional impact of mutations affecting the b subunit (see Fig. 94-11). Alternatively, as described above, demonstration of some of the peculiarities of propionyl CoA carboxylase activity or of mRNA or enzyme subunit expression can also reveal the affected gene. Thus, the demonstration of normal enzyme activity in parents of an affected child would be compatible with mutations in the PCCB gene, although obtaining the converse, 50 percent activity, does not necessarily implicate the PCCA gene. Absence of both a and b subunits by Western blotting does identify the PCCA gene as responsible for the disease. Other more straightforward ®ndings, such as absence or abnormality of one of the two mRNA species or polypeptide, also will identify the affected gene. Performing the required experiments is worthwhile because there is a great diversity of mutations in the PCCA gene, and although the PCCB gene shows bias toward a small number of mutations that account for about 30 to 60 percent of alleles in different populations, there remains a large diversity of mutations that account for the rest. Mutations in the PCCA Gene. Nineteen disease-causing mutations have been identi®ed in the PCCA gene, eight of which fail to produce a complete a subunit. There are four splicing mutations, two nonsense mutations and two small deletions causing frameshifts. All four splicing mutations cause exon skipping. Three of them Ð 1771IVS-2del9, 1824IVS+3del4, and 1824IVS 3insCTÐ affect the same exon.216 The nonsense mutations, R288X and S537X, and the small deletions, 700del5 and 1115del4, are expected to produce truncated proteins. However, ®broblasts from a patient homozygous for 1115del4 failed to show mRNA by Northern blot, although it could be detected by reverse transcriptase polymerase chain reaction (RT-PCR). A similar ®nding was made for a patient heteroallelic for R288X and 700del5, indicating that both mutant mRNA species are unstable.217 These results suggest a propensity for mutations disrupting normal mRNA translation to produce mRNA-minus outcomes, as has been noted for other genes. An unusual ®nding in patient cells with mRNA destabilizing mutations at both alleles is the detection of an RT-PCR product showing an 84 bp insertion at nucleotide 1209, containing two inframe stop codons.217 The insertion is an anomalous exon derived by aberrant splicing within the adjacent intron. The cryptic transcript is part of the background ``noise'' of abnormal mRNAs occurring at very low level in normal cells. Cell lines in which the 84 bp insertion is detected as a predominant (but low-level) species share at both alleles severely deleterious mutations, which are consistent with rendering the normally structured mRNA species unstable and rapidly degraded. In a study of 12 mutant cell lines, 4 showed the characteristic transcript. The mutations included a splicing mutation (1671IVS 5G ! C), a nonsense mutation (R288X, three occurrences), and two small deletions (700del5, 1115del4) causing frameshifts.217 Screening for the 84 bp insertion by RT-PCR may provide a diagnostic bene®t, given the high proportion of such cell lines. The remaining mutations cause amino acid substitutions, which are expected to produce inactive or unstable a subunits, although most have yet to be evaluated in expression experiments. Two subunits with point mutations, G643R and Cdel687, have been expressed in E. coli and shown to abolish biotin binding.57 Another mutation, M348K, has been shown to reduce the intramitochondrial stability of the mutant a chain without appreciably affecting its import.218 This a chain failed to be biotinylated, as also was found for two other nearby mutations, D343G and G354V, and one near the N-terminus, A50P,57 indicating that all four mutations likely result in unstable a chains in vivo, accounting for the lack of biotinylation, because the biotin binding domain is far removed toward the C-terminus of the protein. Mutations in the PCCB Gene. Twenty-eight disease-causing mutations have been identi®ed in the PCCB gene. There are 16 missense mutations, three nonsense, three insertions, and one complex insertion/deletion (ins/del). Five other mutations cause disease through disruption of normal splicing. The most frequent mutation in white individuals is ins/del,219±221 present in 32 percent of alleles of mixed white heritage. A similar value also was obtained in a study involving 29 patients of Spanish or Latin American heritage.61 Two other mutations have been found in signi®cant frequency in the same populations. These are 1170insT and E168K, with combined frequencies of 14 percent and 17 percent, respectively, in Spanish and Latin American patients.61,222 The remaining mutations occur singly or have been found in only two or three patients among whites. Six mutation have been identi®ed among Japanese patients. Two of them, R410W and T428I, appear to be prevalent, occurring in 25 and 31 percent of alleles, respectively.221,223 There are also two splicing mutations (IVS43del4 and IVS123del8), both of 2175 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2176 PART 9 / ORGANIC ACIDS which result in exon skipping; one nonsense mutation (R499X), and one other point mutation (R165W).54,223,224 Of these, R410W and R165W also have been found in whites.61,225 The ins/del mutation is a complex mutation resulting from a deletion of 14 nucleotides and their replacement by 12 nucleotides unrelated to those that were lost. The outcome is the loss of an Msp I site, which has been used diagnostically, and generation of a frameshift that results in the production of an unstable, truncated protein.220 The origin of the ins/del is not obvious, but it is attractive to speculate that it is derived from duplication of nine nucleotides just upstream of the site, with random ®lling of three more nucleotides.219 The R410W mutation occurs in the same location and also results in loss of the Msp I site. This leaves ambiguous the results of diagnosis by Msp I digestion of PCR products. Dot blotting can be used to distinguish these possibilities,219 or PCR products that show at least one allele not cleaved by Msp I can be sequenced. Two amino acid substitutions, L519P and R512C, are unusual in that they are associated with apparently absolute de®ciency of b subunits.61 Both are present in patients with null-type second alleles (1170insT and ins/del, respectively) and both patients' cells are negative for the b subunit by Western blot. Other point mutations, including R44P, G131R, R165W, E168K, R410W, and A497V are associated with unstable b subunits, which appear reduced in quantity and may show smaller-sized fragments by Western blot.61,223 The abundance of b subunit mutations that are CRM negative (in the presence of detectable a subunit) highlights the affected gene (PCCB) and focuses the analysis for mutations. The identi®cation of mutations in the PCCB gene has made it possible to assess the basis of interallelic complemention that de®nes the pccB and pccC subgroups. Each complementing cell line would have one, if homozygous, or two candidate mutations that could be responsible for the interallelic complementation observed between mutant subgroups. The identity of the complementing allele in several cell lines was determined by microinjecting b subunit cDNA plasmids containing the candidate mutations into ®broblasts of each subgroup and assaying for recovery of 14C-propionate metabolism by autoradiography.225,226 The results paralleled the original complementation results, and the distribution of complementing alleles shows a pattern suggestive of functional domains within the b subunit. For example, two mutations from the pccB subgroup in the N-terminal half of the protein, dupKICK140 and P228L, are both complementing alleles. These mutations reside in the putative carboxybiotin binding domain by homology with related sequences in the 12S subunit of P. shermanii transcarboxylase. Similarly, two complementing alleles from the pccC subgroup, Idel408 and R410W, are close together in the C-terminal half of the protein. They are located in a region of high homology with the propionyl CoA binding site of the P. shermanii 12S subunit. These ®ndings suggest the importance of b±b interactions in the enzyme and indicate the bene®t of determining subgroup complementation within the PCCB gene as a way of assessing functional characteristics of the enzyme. A particular dif®culty for investigating the impact of mutations is the requirement for a two-subunit expression system and successful biotinylation to obtain functional propionyl CoA carboxylase. So far, this has been achieved in only one instance. Kelson et al.227 coexpressed cDNAs encoding the a and b subunits in E. coli, along with the chaperonin proteins GroES and GroEL to facilitate folding and assembly, and obtained fully assembled, biotinylated propionyl CoA carboxylase. Using this system, they evaluated the Japanese T428I mutation in the b subunit and showed that it resulted in complete obliteration of enzyme activity. Diagnosis and Mutation Analysis. A defect in propionate carboxylation must be considered in any child in whom ketosis or acidosis develops in the neonatal period. Other inborn errors of metabolism must be ruled out, as must the more common causes of acidosis in the newborn period. Determinations of propionic acid and its metabolites in blood or urine and studies of propionyl CoA carboxylase activity in leukocyte or ®broblast extracts are required for de®nitive diagnosis. The enzymatic test is, in fact, the only absolutely speci®c one, because propionate accumulation can occur in patients with defects of methylmalonate metabolism as well as in those with propionyl CoA carboxylase de®ciency. Such assays on cord blood leukocytes should allow immediate diagnosis in a high-risk newborn. Prenatal diagnosis has been accomplished reliably by measuring carboxylase activity in cultured amniotic ¯uid cells228 or chorionic villous biopsies,229 by measuring [14C]propionate ®xation in amniotic ¯uid cells,230 or by measuring methylcitrate in amniotic ¯uid.231 The identi®cation of the speci®c gene defect, in PCCA or PCCB, responsible for the enzyme de®ciency is important for monitoring future pregnancies and to contribute to genotype± phenotype correlations. The gene assignment can be achieved most unequivocally through complementation analysis. However, this is an esoteric procedure that is not readily available in most laboratories.200 ±202 Other methods can be used that take advantage of some of the peculiarities of each complementation group, although the techniques do not apply universally. For example, estimation of PCC activity in parents' ®broblasts will indicate PCCB gene mutations if the activity is within the normal range, although this does not apply in all cases of b subunit defects.199 Western blot, or 3H-biotin labeling in the case of the a subunit, will indicate defects of the PCCA gene, if both the a and b subunits are absent, or implicate the PCCB gene if only the b subunit is absent, reduced in quantity, or partially degraded.205 Finally, Northern blot or semiquantitative RT-PCR will identify the defective gene if the level or mobility of one or the other mRNA species is adversely affected.204 After identi®cation of the defective gene, cell lines can be analyzed for mutation by conventional techniques. In the PCCB gene, it is worthwhile to ®rst screen for common mutations. These include ins/del, which appears widespread, and 1170insT and E168K, which are frequent in Spanish and Latin American populations.61,219± 221 In Japanese patients, the common mutations include R410W and T428I.221,223 When screening for the ins/del by loss of the Msp I site, it should be recalled that R410W is also associated with loss of the same site. Although ins/del has not been observed in Japanese patients, R410W and R165W do occur in both Japanese and white individuals. It would be important to con®rm the mutation by sequencing if it is identi®ed through loss of the Msp I site. The PCCA gene does not have predominant mutations. Because a number of patients have had null mutations on both alleles, however, it is worthwhile to test for the characteristic 84-bp insertion detectable by RT-PCR in patients with absent or unstable mRNA.217 For determining the identity of unknown mutations, genomic DNA can be analyzed in the case of the PCCB gene. With the structure of the PCCB gene completed, it is possible to amplify each of the 15 exons and ¯anking intronic sequences for analysis of PCR products.61 Alternatively, RT-PCR and sequencing have been used to identify mutations at the mRNA level222,223 and to examine the effect of splicing mutations.54,224 In the case of the PCCA gene, where complete genomic structure is still lacking, analysis of overlapping cDNA segments generated by RT-PCR has been the method of choice.57,217,218 Treatment. A low-protein diet (0.5±1.5 g/kg per day) or one selectively reduced in the content of propionate precursors appears to be the best treatment for the disorder at this time. Such diets will minimize the number of attacks of ketoacidosis but will not necessarily prevent them or allow normal development in all patients. Because fasting has been shown to increase the excretion of propionate metabolites in patients, frequent feeding has been recommended.27 Attacks of ketoacidosis should be treated vigorously by withdrawing all dietary protein and administering sodium bicarbonate parenterally; glucose is also required to avoid catabolism. Acute attacks, particularly those accompanied by Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM hyperammonemia, have been treated with peritoneal dialysis.232 Total parenteral nutrition also has been used to treat critically ill patients.233 Because propionyl CoA carboxylase requires biotin as a coenzyme and because some patients' cells show a biotindependent increase in enzyme activity,234 it is possible that certain patients could improve when given supplementary biotin, but no clear example of a biotin-responsive patient with isolated propionyl CoA carboxylase de®ciency has yet been documented. On the other hand, dramatic biotin responsiveness has been described in several children in whom propionyl CoA carboxylase de®ciency is part of the constellation now called multiple carboxylase de®ciency (see Chapter 156). Two additional therapeutic adjuncts deserve mention. Roe and Bohan reported marked, transient clinical improvement in a child with propionic acidemia given a single oral dose (100 mg/kg) of L-carnitine.173 Because this child's urinary hippurate concentration increased markedly after carnitine, because free plasma carnitine was reduced in three other patients with propionic acidemia,235 and because urinary propionylcarnitine was present in large amounts in such patients,235 these workers proposed that patients with propionic acidemia have a relative carnitine de®ciency. Subsequently, Wolff et al.236 observed that L-carnitine supplements signi®cantly reduced the ketogenic response to fasting in patients with propionic acidemia. Thus, it appears that long-term L-carnitine supplementation in these patients warrants serious consideration. To date, no results of long-term treatment with carnitine have been published. The recognition that gut bacteria may contribute signi®cantly to propionate production in at least some individuals25 has led to the suggestion that speci®c antimicrobial therapy may be of clinical bene®t to some children with propionic acidemia by reducing the total amount of propionate in their serum and tissues.237 Metronidazole (10 mg/kg) has been reported to reduce fecal propionate substantially, concomitant with a reduction in the anaerobic bacterial count.237 Plasma propionate also decreased by 50 to 60 percent in two patients, whereas urinary excretion of propionate metabolites was reduced by an average of 34 percent in four.237 No comment was made on the effect of this treatment on the clinical status of these patients. Others have reported using a similar treatment, but also without any ®rm evidence of clinical ef®cacy183 (see the later subsection on Diagnosis and Treatment under the section on The Methylmalonic Acidemias). Further study is clearly required to establish whether such therapy improves the management of acute episodes of metabolic decompensation or provides any long-term bene®t with respect to growth, mental development, or neurologic outcome for this condition. In a retrospective study of 17 patients (12 early-onset, 5 lateonset) from a single hospital over 20 years, 7 patients died (5 early-onset, 2 late-onset).238 The neurologic outcome of the surviving patients was felt to be satisfactory, and was even better for early-onset patients. The investigators felt that other treatments such as liver transplantation or somatic gene therapy could improve the quality of life of propionic acidemia patients in the future. Not unexpectedly, successful application of therapy, particularly in mildly affected patients, is leading to the survival of women with propionic acidemia into their child-bearing years. One report documents a relatively uneventful pregnancy and delivery in a woman mildly affected with propionic acidemia treated with protein restriction and carnitine supplementation.239 Other such pregnancies are likely to occur; each will need to be handled individually according to the mother's speci®c clinical and biochemical status. Multiple Carboxylase De®ciency. In 1971, Gompertz et al.240 reported a male infant (J.R.) thought to have speci®c de®ciency of the mitochondrial, biotin-dependent enzyme, b-methylcrotonyl CoA carboxylase (Fig. 94-5). This infant developed a diffuse, erythematous skin rash at 5 weeks of age and was admitted to the hospital at 5 months of age because of a worsening rash, recurrent vomiting, irritability, and a mild metabolic acidosis. His urine, which smelled like ``tomcats' urine,'' was analyzed for organic acids and was found to contain large excesses of b-methylcrotonylglycine, tiglylglycine, and b-hydroxyisovaleric acid. When he was given 10 mg biotin (about 100 times the estimated human requirement) by mouth daily for several days, the rash, vomiting, irritability, and abnormal urine metabolites all disappeared dramatically. Several years later, it became clear that J.R. had multiple Ð not speci®c Ð carboxylase de®ciency. His reanalyzed urine contained metabolites characteristic of propionyl CoA carboxylase de®ciency, as well as b-methylcrotonyl CoA carboxylase de®ciency241; his cultured ®broblast extracts were de®cient in pyruvate carboxylase,202 as well as in propionyl CoA and b-methylcrotonyl CoA carboxylase242,243; and supplementation of the ®broblast growth medium with biotin led to complete correction of the de®ciency of all three biotin-dependent enzymes.202,242,243 Subsequently, more than 50 children with multiple carboxylase de®ciency have been described.106 These children are now known to suffer from defects in one of two steps in biotin metabolism: the transfer of biotin to apocarboxylases, catalyzed by holocarboxylase synthase244± 247; or the hydrolysis of biocytin, the biotin-containing product of degraded holocarboxylases, to release biotin, catalyzed by biotinidase.248± 250 As a group, children with holocarboxylase synthase de®ciency tend to present in the ®rst days or weeks of life with feeding dif®culties, hypotonia, lethargy, and seizures; some have a diffuse skin rash or alopecia.106 The ®rst described patient with multiple carboxylase de®ciency, J.R., proved to have a defect in holocarboxylase synthase. Early studies showed that his enzyme had a reduced af®nity for biotin when assayed in vitro.251,252 This was con®rmed by the identi®cation of a point mutation in the biotinbinding region of the enzyme and the production of a biotinresponsive mutant enzyme when it was expressed in E. coli.253,254 Several additional mutations have been identi®ed in patients, many of them clustering within the biotin-binding region of the enzyme.253±257 A second, larger group of children is de®cient in biotinidase activity.248±250 These children usually present later in life (mean age of onset 3 months) with a variety of neurologic problems (seizures, hypotonia, developmental delay, hearing loss, optic atrophy). Although a large number of mutations have been identi®ed in the biotinidase gene, several are highly prevalent and account for the majority of alleles in patients.258±260 Both groups respond dramatically to biotin supplements (10 mg daily) with prompt and sustained clinical improvement. Thus, multiple carboxylase de®ciency differs markedly from isolated propionyl CoA carboxylase de®ciency in response to biotin and, hence, in long-term prognosis. It should be emphasized, however, that the clinical presentations may be very similar. For this reason, urinary metabolite identi®cation is of important therapeutic signi®cance. For more information on multiple carboxylase de®ciency, the reader is referred to Chapter 156. The Methylmalonic Acidemias In 1967, Oberholzer,10 Stokke,11 and their colleagues described critically ill infants with profound metabolic ketoacidosis and developmental retardation who accumulated huge amounts of methylmalonate in their blood and urine. These children had none of the hematologic or neurologic stigmata of Cbl de®ciency, failed to respond to Cbl supplements, and excreted much larger amounts of methylmalonate than those observed in patients with pernicious anemia.6,7 They were presumed to have a congenital defect of methylmalonyl CoA racemase or of the methylmalonyl CoA mutase apoenzyme (Fig. 94-2). Shortly thereafter, Rosenberg,261 Lindblad,12,13 and their co-workers reported children with similar clinical presentations whose methylmalonic aciduria responded dramatically to pharmacologic but not physiologic amounts of CN-Cbl or AdoCbl. Such children were found subsequently to 2177 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2178 PART 9 / ORGANIC ACIDS have a primary defect of AdoCbl synthesis that resulted in impaired mutase activity.14,15 The array of different biochemical and clinical disturbances of methylmalonate metabolism was broadened still further in 1969 and 1970, when Mudd,262 Goodman,263 and their associates described children with methylmalonic aciduria whose clinical and chemical ®ndings differed from those described above. Ketoacidosis was not present, and the increased methylmalonate excretion was accompanied by homocystinuria, cystathioninuria, and hypomethioninemia. This biochemical constellation was interpreted as evidence for defective synthesis of both Cbl coenzymes, with secondary impairment of AdoCbl-dependent methylmalonyl CoA mutase and MeCbl-dependent methionine synthase (Fig. 94-7). These early descriptions, coupled with a body of data to be discussed below, have demonstrated that there are many different biochemical bases for inherited forms of methylmalonic acidemia: two distinct defects of the mutase apoenzyme, one producing complete mutase de®ciency (mut 0), the other partial de®ciency (mut ); two distinct defects of AdoCbl synthesis, one probably due to de®ciency of a mitochondrial Cbl reductase (cblA), the other to de®ciency of mitochondrial cob(I)alamin adenosyltransferase (cblB); and three distinct defects of both AdoCbl and MeCbl synthesis due to abnormal cytosolic or lysosomal metabolism of cobalamins (cblC, cblD, and cblF). Patients with lesions producing methylmalonic acidemia only (mut 0, mut , cblA, cblB) share many clinical features and will be discussed as a group; discussion of the other group of patients whose lesions produce methylmalonic acidemia and homocystinuria (cblC, cblD, and cblF) will follow. Methylmalonyl CoA Mutase De®ciency. Clinical and Laboratory Presentation. More than 100 children with isolated mutase de®ciency have been documented. Although, as mentioned above, there are four known etiologies for such de®ciency, the clinical ®ndings in affected patients from the four groups are remarkable more for their similarities than for their differences. Matsui et al. surveyed 264 the natural history in 45 such patients: 15 were mut 0; 5 were mut , 14 were cblA, and 11 were cblB. There were approximately equal numbers of males and females in each group. Information was obtained from questionnaires completed by the patients' physicians, published reports, unpublished communications, and personal experience. The most common signs and symptoms at the onset of clinical dif®culty were lethargy, failure to thrive, recurrent vomiting, dehydration, respiratory distress, and muscular hypotonia (Table 94-1). Little interclass difference was observed for these major clinical manifestations or for such less common ones as developmental retardation, hepatomegaly, or coma. Patients in the mut 0 class, however, presented earlier than those in the other groups (Fig. 94-9). Whereas 80 percent of children in the mut0 class became ill during the ®rst week of life, Table 94-1 Clinical Presentation in 45 Patients wth Methylmalonic Acidemia Fig. 94-9 Age at clinical onset in 45 patients with methylmalonic acidemia. Inset numbers denote percentages of patients in each group. (Reprinted with permission from Matsui SM, Mahoney MJ, Rosenberg LE: The natural history of the inherited methylmalonic acidemias. New Engl J Med 308:857, 1983.) less than half the children in the three other groups presented during this interval. Furthermore, clinical onset occurred in 90 percent of mut0 patients before the end of the ®rst month, whereas onset beyond the ®rst month was observed in an appreciable fraction of patients in each of the other groups. A survey of 20 mut patients has reached similar conclusions.265 The laboratory ®ndings in affected patients at the time that methylmalonic acidemia (with or without aciduria) was ®rst documented are shown in Table 94-2. As expected, serum Cbl concentrations were routinely normal. Metabolic acidosis, with blood pH values as low as 6.9 and serum bicarbonate concentrations as low as 5 mEq/L, was observed in the majority of patients in all four groups. Ketonemia or ketonuria was found in 80 percent of patients, with hyperammonemia being only slightly less common, occurring in 70 percent of affected patients. Leukopenia, thrombocytopenia, and anemia were the only other manifestations that were noted in 50 percent or more of this group of patients. Earlier case reports266 reported that hypoglycemia occurs in about 40 percent of affected patients. Inadvertently, this parameter was not assessed in this survey. It should be mentioned that mutase de®ciency is not always associated with serious clinical consequences. Ledley et al.267 reported eight children, between the ages of 18 months and 13 years, who had methylmalonate accumulation in blood and urine, Table 94-2 Laboratory Findings in 45 Patients with Methylmalonic Acidemia Mutant Class Signs and Symptoms at Onset cblA cblB mut 2 mut 0 Total Lethargy Failure to thrive Recurrent vomiting Dehydration Respiratory distress Muscular hypotonia Developmental retardation Hepatomegaly Coma 78 75 58 64 89 44 36 11 50 83 86 86 86 67 57 33 67 29 100 40 80 100 50 33 25 0 40 85 77 77 62 55 91 65 57 38 84 73 73 71 67 63 47 41 40 Numerical values represent percentages of patients in each group. Reprinted with permission from reference 264. Mutant Class Findings at Clinical Onset cblA cblB mut 2 mut 0 Total Normal serum cobalamin Metabolic acidosis Ketonemia/ketonuria Hyperammonemia Hyperglycinemia/glycinuria Leukopenia Anemia Thrombocytopenia 100 100 78 50 70 70 10 75 100 88 67 83 83 45 45 45 100 100 100 80 40 60 0 40 100 85 85 75 70 62 58 40 100 92 81 71 68 60 55 50 Numerical values represent percentages of patients in each group. Reprinted with permission from reference 264. Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM but had no symptoms. Presumably, these apomutase-de®cient patients have an enzyme defect so ``leaky'' that homeostasis is not compromised. At least some of these individuals continued to be symptom free 7 years after the initial report.268 Another report determined that patients, initially ascertained by a newborn screening program with methylmalonic aciduria urine levels of less than 1400 mmoles/mmole creatinine, had normal somatic and cognitive outcomes.269 Conversely, another group of patients appear to have methylmalonic acidemia without a demonstrable defect in mutase activity, at least as measured in cultured cells. Although the elevations of methylmalonate are relatively mild as compared with mutase-de®cient patients, they are chronic and usually are discovered on laboratory workup for failure to thrive or developmental retardation.270 Because Cbl metabolism also appears to be normal in this group, the cause of the disease remains an enigma. Roe et al. have described a patient with psychomotor delay, methylmalonic aciduria without episodes of metabolic acidosis, and methylmalonic semialdehyde dehydrogenase de®ciency.271 It is possible that others in this group have the same defect. Chemical Abnormalities In Vivo. Large amounts of methylmalonic acid have been found in the urine or blood of all reported patients. Whereas normal children and adults excrete less than 0.04 mmole (5 mg) methylmalonate daily, children with isolated methylmalonic acidemia have excreted from 2.1 to 49 mmoles (240 to 5700 mg) in a 24-h period. Their plasma concentrations of methylmalonate, almost undetectable in normal subjects, have ranged from 0.22 to 2.9 mM (2.6±34 mg/dl). In the few patients in whom it was measured, the cerebrospinal ¯uid concentration of methylmalonate equaled that of plasma (for references to early case reports, the reader is referred elsewhere266). It is important to note that patients with mild, late-onset, or ``benign''267 disease may have much lower levels, particularly when clinically asymptomatic.267,268 No relationship between the quantities of methylmalonate accumulated in body ¯uids and the etiology of mutase de®ciency (i.e., apoenzyme vs. coenzyme de®ciency) has been reported. Methylmalonate is surely the major, but not the only, abnormal metabolite found in body ¯uids of these patients. Because propionyl CoA carboxylation is reversible, propionate and some of its precursors (butanone) or metabolites (b-hydroxypropionate and methylcitrate) also accumulate in blood and urine,8,92,93,272,273 although their amounts are small compared with that of methylmalonate. Several groups have studied the relationship between protein or amino acid loading and methylmalonate accumulation in these patients. Without exception, administration of protein or amino acids known to be precursors of propionate and methylmalonate, such as methionine, threonine, valine, or isoleucine, has resulted in augmented methylmalonate accumulation and, in some instances, ketosis or acidosis.8,10±12 When Cbl-responsive patients are given supplements of this vitamin, such augmentation by methylmalonate precursors is lessened considerably.274 All these ®ndings suggest that patients with discrete defects at the mutase step have a major block in the utilization of methylmalonyl CoA that is expressed as methylmalonate accumulation. Localization of Enzymatic Defects. Because the conversion of propionate to succinate is blocked in each of the methylmalonic acidemias, an early screening test for these disorders measured the ability of intact peripheral blood leukocytes or cultured ®broblasts to oxidize [14C]propionate to 14CO2 and compared this with the oxidation of [14C]succinate to 14CO2.261 By including estimation of [14C]methylmalonate oxidation as well, this test can distinguish between de®ciency of propionyl CoA carboxylase and of methylmalonyl CoA mutase. Incorporation of [14C]propionate into trichloroacetic acid±precipitable material by intact cultured cells has replaced the more cumbersome 14CO2 evolution technique.230,275 Further discrimination among the methylmalonic Table 94-3 Methylmalonyl CoA Mutase Activity in Liver Homogenates from Patients with Methylmalonic Acidemia Enzymatic Activity* Subjects Controls (3) Patients 1 2 3 4 Without Added AdoCbl 535±866 1 8 3 80 With Added AdoCbl (4 3 1025 M) 799±1058 3 33 7 1368 *Assayed by measuring conversion of D L -[3H]methylmalonyl CoA to [3H]succinyl CoA. Values expressed as picomoles of succinate formed per milligram protein per 30 min. Reprinted with permission from reference 276. acidemias has depended on studies of Cbl uptake and AdoCbl formation by intact cultured ®broblasts, on assays of mutase activity in cell extracts, and on genetic complementation studies with cultured cell heterokaryons. Mutase Apoenzyme De®ciency. Morrow and colleagues276 provided the ®rst evidence in vitro for apoenzyme abnormalities and for biochemical heterogeneity among the methylmalonic acidemias. In four patients who had died, they studied mutase activity in liver homogenates by measuring the conversion of DL[3H]methylmalonyl CoA to [3H]succinyl CoA (Table 94-3). Activity was barely detectable in three and showed no response when AdoCbl was added at concentrations suf®cient to saturate the normal enzyme. In the fourth, mutase activity was restored to control values by AdoCbl. These ®ndings were interpreted as evidence for a mutase apoenzyme defect in the ®rst three patients and for defective AdoCbl synthesis in the fourth. These ®ndings were con®rmed subsequently in studies with cultured ®broblasts.277 Cells from the ®rst three patients synthesized AdoCbl normally but had much reduced mutase activity in extracts regardless of the amount of AdoCbl added; cells from the fourth had a distinct defect in AdoCbl synthesis. Subsequently, it has become clear that two general types of apomutase defects exist. In one type, designated mut 0 and constituting about two thirds of the mut complementation group, mutase activity in extracts of cultured ®broblasts is undetectable ( < 0.1 percent of control), even when assayed in the presence of AdoCbl concentrations greatly in excess of that normally required to saturate the enzyme.277±279 When CRM was sought by radioimmunoassay under steady-state conditions in cell lines from 21 such patients, 12 had no immunologically identi®able mutase protein (CRM ), whereas 9 had reduced amounts of CRM ranging from 1 to 40 percent of that found in control extracts.280 In a follow-up study,281 cells from this group of patients were pulse labeled to determine how amounts of newly synthesized mutase protein, detected by speci®c immunoprecipitation, compared with the CRM values obtained under steady-state conditions. As expected, all CRM mutants had easily detectable newly synthesized mutase. Of 11 CRM lines, however, 5 had amounts of newly synthesized mutase ranging from barely detectable to nearly half that seen in controls. Thus, some apomutase mutations lead to the synthesis of unstable mutase proteins, which are rapidly degraded intracellularly. One other result of this study bears mention. Using a pulse-chase experimental protocol, mitochondrial import and cleavage of the apomutase precursor were studied in control lines and in 38 lines from mut mutants that synthesized mutase protein. In one of the 38 mutant lines, an N-terminal deletion resulted in failure of the mutant mutase to be taken up by mitochondria.281,282 All the others underwent normal mitochondrial uptake and processing. 2179 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2180 PART 9 / ORGANIC ACIDS Fig. 94-10 Schematic representation of the secondary structure of human methylmalonyl CoA mutase, predicted by homology modeling based on the radiographic structure of the P. shermanii mutase a subunit. Helices are denoted by rectangles, b strands by bold arrows, and coils or turns by a thin line. For each of the two main domains, the ba pairs are labeled. The AdoCbl is represented by the horizontal thick line labeled Cbl (the position of the corrin ring) attached to an almost vertical thick line indicating the DMB side chain as it extends into the Cbl-binding domain. The approximate positions of a number of characterized missense mutations determined in mut patients are indicated on the structure by small ellipses. Although many mutations affecting AdoCbl binding are located in the Cbl-binding domain, several, including R93H, Y231N, and R369H, are in the N-terminal (ba)8 barrel domain. (Redrawn with permission from Thoma NH, Leadley PF: Homology modelling of human methylmalonyl-CoA mutase: A structural basis for point mutations causing methylmalonic aciduria. Protein Sci 5:1922, 1996.) The second type, designated mut , involves a structurally abnormal mutase apoenzyme. The mutant apoenzymes in extracts of these cells retain maximally 2 to 75 percent of control activity, have a Km for AdoCbl approximately 200 to 5000 times normal, show a normal Km for methylmalonyl CoA, and exhibit increased thermolability relative to control enzyme.278,279,283 By radioimmunoassay, the amount of immunologically reactive mutase protein in these extracts ranges from 20 to 100 percent of control.280 Because pairwise crosses between mut 0 and mut generally yield noncomplementing heterokaryons and because there are affected individuals who appear to be mut 0 /mut compound heterozygotes, both mutant types re¯ect abnormalities of the locus coding for the apomutase structural gene.278,279 The identi®cation of nonsense and missense mutations within the mutase gene that lead to absent enzyme or abnormal enzyme kinetics have ®rmly established this conclusion. Ledley and his colleagues ®rst described the molecular changes in mut patients. They surveyed a number of patient ®broblasts and found no evidence for gross rearrangements at the genomic level, but reductions in mRNA in some lines.284 A number of point mutations were described, including nonsense changes that lead to a mut 0 phenotype,282 missense mutations that also generate a mut 0 phenotype,285,286 and a missense mutation that generates a mut phenotype.287 Each of these changes was con®rmed as a mutation causing de®ciency of mutase activity by expressing the variant protein, either in a mut 0 ®broblast line285,286 or in Saccharomyces cerevisiae cells.287 Interestingly, one of the mut 0 lines was able to complement a number of the other mut 0 and mut lines, although not all,286 an example of interallelic complementation. To date, 28 mutations and 2 benign sequence changes have been identi®ed, some of which have been characterized by expression in cultured cells or in E. coli.68,288,289 A common mutation (G717V) was found in ®ve black patients of African and African-American ancestry,290,291 and in a series of patients from Japan, six patients carried the same mutation (E117X).292 Figure 94-10 is a linear representation of the structure of human mutase,86 based on the crystal structure of the P. shermanii homologue.87 On it are indicated the locations of a number of the missense mutations in mutase identi®ed so far. The effects of some of these on mutase activity have been rationalized in terms of the predicted three-dimensional structure of the human enzyme.68,86,289,293 The easiest to explain are the mut0 mutations G630E and G703R, affecting residues that line the binding pocket for the DMB side-chain of AdoCbl. Although both change ¯exible glycine residues to charged ones, the main effect is to introduce bulky side chains into the narrow binding pocket, effectively blocking access to it and preventing AdoCbl binding.293 Some changes in the Cbl-binding domain (G623R, G626C) appear to affect the positioning of His627, whose side-chain provides the essential bottom ligand to the bound AdoCbl,86,293 whereas others likely affect the position or interaction of the ba strands that form the Cbl-binding domain. One of these, G717V, results in a highly unstable protein, in addition to modifying AdoCbl binding.68 Mutations in the N-terminal TIM barrel, particularly those producing a mut phenotype, are less easily explained. W105R appears to affect the substrate channel in the TIM barrel, whereas A377E, V368D, and R369H are in the likely dimer interface. The interaction between dimerization or dimer stability and AdoCbl binding, revealed by the mut phenotype of R369H,68 remains unexplained. Even less apparent is an explanation of the interallelic complementation supported by two mutations in the N-terminus of mutase, R93H and G94V. R93H was identi®ed in homozygous form in a cell line that showed interallelic complementation with a Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM AdoCbl normally when incubated with OH-[57Co]Cbl, ATP, and a reducing system designed to bypass the Cbl reductases (Fig. 94-8). Subsequent biochemical167 and genetic complementation295±297 studies have differentiated two mutant classes among patients defective in AdoCbl synthesis. One class, which contains the index Cbl-responsive patient, is designated cblA, and may be due to a de®ciency of one of the mitochondrial Cbl reductases, perhaps an NADPH-linked aquacobalamin reductase.298 Complementation has been demonstrated between several cblA lines, raising the possibility of interallelic complementation in this disorder as well.299 The second, designated cblB, has been shown to result from a speci®c de®ciency of cob(I)alamin adenosyltransferase.168 Fig. 94-11 Model for interallelic complementation between pccB and pccC mutations causing propionic acidemia. The schematic shows a pair of b subunits aligned head to toe so that two functional domains are produced, with the carboxybiotin site coming from one subunit and the propionyl CoA binding site coming from the other. In the case in which each b subunit contains complementing mutations Ð near the N-terminus of one subunit for dupKICK140 and near the C-terminus of the other subunit for R410W Ð the outcome is that only one of the two functional sites is inactivated. The second functional site retains activity despite the presence of a mutation on each subunit. number of other mut lines,286 and G94V was found in heterozygous form in a cell line that appeared to show interallelic complementation with the G717V mutation in vivo.68 Subsequent in vitro experiments with mutant proteins expressed in E. coli have con®rmed its ability to complement a range of other mut cell lines (J. Janata and W. Fenton, unpublished observations). This region of mutase is the least homologous between the human and P. shermanii enzymes. In the bacterial enzyme, this region forms a long element that wraps around the TIM domain of the other subunit, and thus appears to be involved with subunit±subunit interactions. How this relates to AdoCbl binding (G94V is mut )68 and how these mutations modify the effects of mutations in the distant Cbl-binding domain to produce interallelic complementation are questions that may only be answered by crystal structures of the human enzyme and these mutant versions. It seems clear from the available structures, however, that complementation is not simply a matter of bringing together unaffected regions from each subunit of a mutase dimer to form one ``normal'' active site,293 as may be the case for the b subunits of PCC (Fig. 94-11). The active site of mutase is clearly contained in a single subunit, with no possibility for sharing elements of it between subunits.87 Moreover, the combined active site model predicts that the kinetic parameters of the restored site should be the same as wild type. This is not what is observed in vivo for the complementing G94V/G717V pair, where the ``complemented,'' presumably heterodimeric, enzyme has a Km at least 10-fold lower than either of its homodimeric parents, but 100-fold higher than wild type.68 Similar results have been obtained in vitro by combining the individually expressed mutant enzymes (J. Janata and W. Fenton, unpublished observations), suggesting a more complex mechanism for restoration of activity upon interallelic complementation between mutase mutants. Finally, it should be mentioned that the only patient thus far reported to have methylmalonyl CoA racemase de®ciency294 has been restudied and shown conclusively to be a mut mutant biochemically and genetically.17 Defective Synthesis of AdoCbl. A series of observations by Rosenberg,14 Mahoney,15 and their colleagues on the ®broblasts of the index patient with Cbl-responsive methylmalonic acidemia led to the demonstration of a primary defect in AdoCbl synthesis in intact cells, resulting in a de®ciency of mutase activity. Although intact cells were unable to convert OH-[57Co]Cbl to Ado[57Co]Cbl, cell-free extracts from this line synthesized Pathophysiology. All studies in vivo and in vitro in patients with methylmalonic acidemia due to speci®c methylmalonyl CoA mutase de®ciency indicate that the primary block in the conversion of methylmalonyl CoA to succinyl CoA explains the accumulation of methylmalonate in blood and urine; the augmentation of methylmalonate excretion and the precipitation of ketosis by protein, amino acids, or propionate; and the excretion of longchain ketones formed in the catabolism of branched chain amino acids. However, the primary block does not explain several important physiologic disturbances: the acidosis, hypoglycemia, hyperglycinemia, and hyperammonemia. Oberholzer et al.10 pointed out that the concentration of methylmalonate in the blood (no more than 3 mM) could not alone explain the acidosis and suggested other possibilities. They proposed that an accumulation of CoA, ``trapped'' intracellularly as methylmalonyl CoA, could lead to an insuf®ciency of this widely utilized coenzyme and, secondarily, to impaired carbohydrate metabolism and subsequent acidosis. Alternatively, they suggested that an excess of methylmalonyl CoA, a known inhibitor of pyruvate carboxylase,300 could interfere with gluconeogenesis and lead directly to hypoglycemia and indirectly to excessive catabolism of lipid, with ketosis and acidosis. Halperin et al.301 showed that methylmalonate inhibited the transmitochondrial shuttle of malate and argued that impairment of this key step in gluconeogenesis could lead to hypoglycemia. As discussed earlier for de®ciencies of b-ketothiolase and propionyl CoA carboxylase, the mechanism of the hyperglycinemia and hyperammonemia so often observed in children with any one of these disorders probably re¯ects inhibition of the intramitochondrial glycine cleavage enzyme and of CPS I, respectively, by the accumulated organic acids or their CoA esters.208,209,211± 215 Thus, as shown in Fig. 94-12, each of the major secondary biochemical abnormalities in the propionic and methylmalonic acidemias can be explained satisfactorily by inhibition of speci®c intramitochondrial processes by the accumulated organic acids and esters. As a further consideration, about half the reported patients with isolated methylmalonic acidemia also show pancytopenia.264 One report suggests that methylmalonate inhibits growth of marrow stem cells in a concentration-dependent fashion.302 By comparing and contrasting the ®ndings in patients with isolated mutase de®ciency with those in patients with Cbl de®ciency (as in classic pernicious anemia), it has been possible to shed some light on the mechanism responsible for the hematologic and neurologic abnormalities in the latter disorder. Thus, the absence of megaloblastic anemia in any patient with isolated mutase de®ciency militates against any involvement of this enzyme in the typical megaloblastosis seen in Cbl de®ciency. Similarly, the cerebellar and posterior column abnormalities so often encountered in Cbl-de®cient patients have never been observed in patients with methylmalonic acidemia due to speci®c mutase dysfunction. Therefore, the notion that neurologic dysfunction in pernicious anemia re¯ects aberrant incorporation of odd-chain or branched-chain fatty acids into myelin because of a block in the propionate pathway has little to recommend it. It appears likely, then, that abnormalities in the Cbl-dependent methionine synthase account for the hematologic and neurologic 2181 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2182 PART 9 / ORGANIC ACIDS Fig. 94-12 Proposed mechanisms of hypoglycemia, hyperglycinemia, and hyperammonemia in patients with inherited de®ciencies of b-ketothiolase (see Chapter 93), propionyl CoA carboxylase, or methylmalonyl CoA mutase. Inhibitory effects of the enlarged intramitochondrial pools of acyl CoA esters (such as propionyl CoA) or their respective free acids on selected mitochondrial functions are shown by the numbered dashed lines corresponding to the following enzymatic or shuttle-mediated reactions: (1) pyruvate carboxylase; (2) the transmitochondrial malate shuttle; (3) the glycine cleavage enzyme; (4) carbamyl phosphate synthetase I; and (5) N -acetylglutamate synthetase. abnormalities in Cbl-de®cient patients. This matter will be discussed further when we consider that group of patients with methylmalonic acidemia and homocystinuria. Genetic Considerations. Each of the four etiologic bases for speci®c methylmalonyl CoA mutase de®ciency (mut 0, mut , cblA, and cblB) is almost certainly inherited as an autosomal-recessive trait. This conclusion is based on the following ®ndings. First, approximately equal numbers of affected males and females are encountered in each group.264 Second, no instance of vertical transmission from affected parent to affected child has been reported. Third, interclass heterokaryons formed between cell lines from different etiologic groups (i.e., mut 0 cblA) complement each other, whereas intraclass heterokaryons (i.e., mut 0 mut 0) generally do not (with the exception of interallelic complementation); thus, each mutant class behaves as a recessive in culture.295±297 Fourth, cell lines from heterozygotes for the mut0, mut , and cblB mutations show partial mutase apoenzyme de®ciency279 and partial adenosyltransferase de®ciency,168 respectively. And ®fth, among a large group of mut mutants studied, some have inherited a genetically different mutant allele from each parent, thereby being compound heterozygotes rather than true homozygotes.279,284,285 It is not possible to de®ne with any precision the prevalence of these disorders in the general population. A survey of newborns in Massachusetts has suggested that methylmalonic acidemia may occur in 1:48,000 infants.303 A similar survey in Quebec yielded 1:61,000 infants.304 Because this study screened urines from infants 3 to 4 weeks of age and because it is known that many children with methylmalonic acidemia die in the ®rst week of life from ketoacidosis or hyperammonemia or both, the true prevalence must be greater. Diagnosis, Treatment, and Prognosis. Because simple colorimetric assays for urinary methylmalonate and more complex gas chromatography±mass spectrometry assays for serum and urinary methylmalonate are now available, it should no longer be dif®cult to make a diagnosis of methylmalonic acidemia once this condition is considered. Other sources of neonatal or infantile ketoacidosis must be ruled out. If excessive amounts of methylmalonate are found in the urine, Cbl de®ciency can be excluded by direct measurement of serum Cbl concentration. Con®rmation and etiologic designation (i.e., mut or cbl) depend on studies with cultured cells and extracts therefrom.17,305 Prenatal detection of methylmalonic acidemia has been accomplished in two ways: by measurement of methylmalonate in amniotic ¯uid and maternal urine at mid-trimester306,307 and by studies of mutase activity and Cbl metabolism in cultured amniotic ¯uid cells.295,307,308 Assays of [14C]propionate utilization230 in uncultured chorionic villous biopsy specimens have proven unsatisfactory, however.309 Mutase apoenzyme307,308 and AdoCbl synthesis230,306 de®ciencies have been identi®ed prenatally. Two treatment regimens for children with methylmalonic acidemia exist and should be used in tandem. A diet restricted in protein (or a special formula restricted in amino acid precursors of methylmalonate) should be instituted as soon as life-threatening problems such as ketoacidosis, hypoglycemia, or hyperammonemia have been addressed; and supplementary Cbl (1±2 mg CNCbl or, preferably, OH-Cbl intramuscularly daily for several days) should be given as soon as the diagnosis of methylmalonic acidemia is made (or even seriously considered). Such measures should decrease the circulating concentrations of methylmalonate and propionate. Even Cbl-unresponsive children with delayed development have been shown to improve markedly when treated with careful dietary protein restriction.310,311 As discussed above for patients with propionyl CoA carboxylase de®ciency, Roe and associates235,312,313 have pointed out that L-carnitine supplements may be a useful therapeutic adjunct in patients with methylmalonic acidemia, presumably by repleting intracellular and extracellular stores of free carnitine that are depleted in affected patients because of exchange with excess methylmalonyl CoA and propionyl CoA. Likewise, oral antibiotic therapy may prove useful here as well. Thompson and his colleagues report that three patients showed subjective improvement in alertness and appetite following brief metronidazole therapy237; longer treatment periods have resulted in signi®cant improvements in other patients, including decreased number and severity of acidotic episodes, increased appetite, decreased vomiting, growth acceleration, and improved behavior.29,314 Total parenteral nutrition also has been used in at least one reported case.233 The previously mentioned survey264 suggests that both the response to Cbl supplements and the long-term outcome in affected patients depend considerably on the nature of the biochemical lesion causing the methylmalonic acidemia. As shown in Figure 94-13, essentially none of the children designated mut 0 or mut responded to Cbl supplements with a distinct decrease in blood or urinary methylmalonate, whereas over 90 percent of the cblA and about 40 percent of the cblB patients showed such a response. Given the complete absence of mutase activity in cells from the mut 0 group, it is not surprising that they Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM Fig. 94-13 Biochemical response to cobalamin supplementation in 45 patients with methylmalonic acidemia. MMA refers to the concentration of methylmalonate. The supplementation protocol generally used 1 mg CN-Cbl parenterally daily for 7 to 14 days. Inset numbers denote the percentage of patients in each group. (Reprinted from Matsui SM, Mahoney MJ, Rosenberg LE: The natural history of the inherited methylmalonic acidemias. New Engl J Med 308:857, 1983.) Fig. 94-14 Long-term outcome in 45 patients with methylmalonic acidemia. The ages of the patients surveyed ranged from a few weeks to 14 years. (Reprinted with permission from Matsui SM, Mahoney MJ, Rosenberg LE: The natural history of the inherited methylmalonic acidemias. New Engl J Med 308:857, 1983.) were regularly Cbl-unresponsive in vivo. The disappointing absence of response in four mut patients presumably means that even parenteral Cbl supplements could not drive tissue concentrations of AdoCbl suf®ciently high to increase signi®cantly their mutase holoenzyme activity. The fraction (60 percent) of cblB patients unresponsive to Cbl supplements presumably has such complete adenosyltransferase de®ciency that AdoCbl synthesis cannot be augmented by Cbl supplements, as it apparently can in the cblB patients with leaky mutations that permit responsiveness in vivo. Patients in the cblA group were uniformly responsive, suggesting either that the responsible mutations are generally leaky, thereby allowing mass action to result in more AdoCbl synthesis, or that alternative pathways of Cbl reduction that require high substrate concentrations exist in cells. It should be emphasized that clinical responsiveness in vivo does not mean complete correction of mutase de®ciency. Even in a patient whose clinical improvement is dramatic, Cbl administration only reduces, rather than eliminates, methylmalonate excretion. Studies with cultured cells from a variety of patients with methylmalonic acidemia17,297 suggest that raising holomutase activity to only 10 percent of normal values by supplementing the growth medium with OH-Cbl results in distinct augmentation of propionate pathway activity (or, conversely, in a distinct decrease in the magnitude of the metabolic block). Some patients in the cblB group, unresponsive to CN-Cbl or OH-Cbl in vivo, might be expected to respond to AdoCbl itself, but published reports on two patients suggest that this logical alternative is ineffective.315,316 The long-term outlook for affected patients is revealing. As noted in Fig. 94-14, the mut0 group has the poorest prognosis, with 60 percent deceased and 40 percent distinctly impaired developmentally at the time of the survey. Shevell et al. reported similar ®ndings.265 In sharp contrast, the cblA patients (i.e., the group biochemically most responsive to Cbl supplements) had the best outcome: 70 percent were alive and well at ages up to 14 years. The cblB and mut groups were intermediate, with about equal fractions in each group being found in the alive and well, the alive and impaired, or the deceased category. It is interesting, albeit anecdotal, that the index patient in the cblA group (now over 30 years old) discontinued Cbl supplements at age 9 years despite advice to the contrary. In the ensuing years, his development and general health have remained excellent despite the high concentration of methylmalonate in his blood and his continued excretion of very large amounts of methylmalonate. Perhaps, as in some other inherited metabolic disorders, treatment of methylmalonic acidemia is most critical during the early years of life, making expert clinical management in the early weeks or months of life most important. There have been several reports of ``metabolic stroke'' in patients following episodes of metabolic decompensation.317 ±319 Three of the patients317,318 belonged to the Cbl-responsive cblA group, but were not being treated at the time. Extrapyramidal signs, particularly dystonia, were accompanied by bilateral lucencies of the globus pallidus and persisted after the acute crisis had passed. In one case, the dystonia has been gradually progressive over a period of 7 years without visible progression of the neurologic lesions.319 Another complication of long-term survival of some methylmalonic acidemia patients is chronic renal failure.320 One report has indicated that 8 of 12 non±Cbl-responsive patients (1±9 years of age) had a reduced glomerular ®ltration rate, with ®ve severely affected.321 In one of these, ``greatly improved metabolic control'' over a period of 18 months led to increased, but still impaired, renal function.321 Signi®cantly, the index cblA patient referred to above returned for treatment of moderate renal dysfunction due to biopsy-proven interstitial nephritis. We do not yet know what impact better metabolic control and Cbl supplementation may have in this and similar cases. A 7-year-old boy with mut methylmalonic aciduria developed persistent lactic acidosis and multiorgan failure. He was shown to have glutathionine de®ciency and responded to treatment with high-dose ascorbate.322 The feasibility of prenatal therapy with Cbl supplements also has been demonstrated. Ampola et al.307 showed that administration of Cbl supplements to a woman carrying a Cbl-responsive, affected fetus resulted in signi®cant reduction in maternal excretion of methylmalonate. Other cases also have been reported.323,324 However, the value of this regimen over one in which therapy is instituted immediately postnatally remains to be established. Because of the poor prognosis of early-onset severe methylmalonic aciduria, liver transplantation has been attempted in a limited number of patients.325± 328 Although liver transplantation appears to protect against acute metabolic decompensation, biochemical correction is incomplete and it is not certain that 2183 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2184 PART 9 / ORGANIC ACIDS there will be complete protection against the renal and neurologic complications. Finally, it must be noted that preliminary steps have been taken toward somatic gene therapy for mutase de®ciency.329,330 Besides all the usual questions of safety and longterm stability of response that surround somatic gene therapy, two important issues remain unanswered for mutase: How much activity must be restored in vivo to normalize the biochemical hallmarks of the disease? And, does correction of the defect in the liver, for example, lead to reversal or amelioration of the pathologic changes in other organ systems, such as the kidneys, and overall clinical improvement? Much work remains to be done before we can address these problems. these children suffered from a defect in cellular metabolism of Cbl such that both Cbl-dependent enzyme activities (mutase and methionine synthase; see Fig. 94-7) were de®cient. The methylmalonic aciduria in these children is distinctly less severe than that encountered in children with isolated mutase de®ciency. It is important to note that elevations of homocystine may be unremarkable in some cblC patients, even when acutely ill. One of the cblF patients had no detectable homocystinuria despite a cellular de®cit in methionine synthase activity,344 although all the others have shown homocystinuria.345±347 Moreover, neither hyperglycinemia nor hyperammonemia has been reported in any of the cblC, cblD, or cblF patients. Combined De®ciency of Methylmalonyl CoA Mutase and Methionine Synthase. Clinical and Laboratory Presentations. Many patients with inherited combined methylmalonic acidemia and homocystinuria have been the subject of individual case reports.263,331±347 Cells from these children comprise three biochemically and genetically distinct complementation groups, designated cblC, cblD, and cblF.167,295,296,348 Clinical ®ndings have varied widely among the more than 100 known patients in the cblC group, including some who have been diagnosed only in adult life. In a review of 50 patients, 44 had onset in the ®rst year of life and 6 had onset after 6 years of age.349 The median age of onset was 1 month, and the range was from birth to 14 years. Thirteen of the early-onset patients died, with a mean age of death at 9.6 months and a range of 1 to 47 months. The early-onset patients had feeding dif®culties, hypotonia, failure to thrive, seizures, microcephaly, developmental delay, cortical atrophy, hydrocephalus, nystagmus, pigmentary retinopathy, and decreased visual acuity. Blood ®ndings included megaloblastic anemia, thrombocytopenia, leukopenia, and neutropenenia. In some patients there was renal failure, sometimes with a hemolyticuremic syndrome. The later-onset patients presented in childhood or adolescence with acute neurologic ®ndings, which included decreased cognitive performance, confusion, dementia, delirium, myelopathy, and tremor. Only one late-onset patient in this series had pigmentary retinopathy. Hematologic abnormalities were seen in half the late-onset patients. Signi®cantly, serum Cbl and folate concentrations are generally normal in cblC patients. Neither of the two brothers in the cblD group263 had any clinically signi®cant problems until later in life. The older brother came to medical attention because of severe behavioral pathology and moderate mental retardation at 14 years of age. He also had a poorly de®ned neuromuscular problem involving his lower extremities. His then 2-year-old brother was asymptomatic, although biochemically affected. No hematologic abnormalities were noted in either sib. Five patients have been reported in the cblF group. The ®rst two (both female) were small for gestational age and had methylmalonic aciduria, poor feeding, growth retardation, and persistent stomatitis.344,345 One had minor facial anomalies, dextrocardia, and abnormal Cbl absorption from the gut. The other had a persistent rash, macrocytosis, and elevated homocysteine and died suddenly despite a good biochemical response to Cbl treatment. A male cblF patient had recurrent stomatitis in infancy, arthritis at age 4, and confusion, disorientation, and a pigmentary dermatitis at age 10.347 Another boy had aspiration pneumonia at birth, hypotonia, lethargy, hypoglycemia, thromobocytopenia, and neutropenia. A native Canadian girl was diagnosed at 6 months of age because of anemia, failure to thrive, developmental delay, recurrent infections, low serum Cbl, and Cbl malabsorption.346 As a group, the cblF patients have responded well to treatment with Cbl. Localization of Defective Cellular Metabolism of Cbl. It has long been clear that patients in the cblC and cblD groups have a defect in cellular metabolism of Cbl. This conclusion is based on the following data: total Cbl content of liver, kidney, and cultured ®broblasts is markedly reduced262,332,350,351; the ability of cultured cells to retain 57Co-labeled CN-Cbl352 or to convert 57Co-labeled CN-Cbl or OH-Cbl to AdoCbl and MeCbl is markedly impaired15,296; activity of methylmalonyl CoA mutase and methionine synthase in cultured cells is de®cient, such de®ciency being improved by supplementation of the growth medium with OH-Cbl296,297,353; and the mutase and methionine synthase apoenzymes in cells from affected patients appear to be normal.262,263,296,297,354 The precise nature of the metabolic defect in the cblC and cblD classes remains elusive, but some progress has been made. Because these mutant cells demonstrate normal receptor-mediated adsorptive endocytosis of the TC II±Cbl complex and normal intralysosomal hydrolysis of TC II,17,153,154,296 perusal of Fig. 94-8 makes it clear that the defects in the cblC and cblD cells affect some step or steps subsequent to cellular uptake, common to the synthesis of both coenzymes, and prior to the binding of the Cbl coenzymes to their respective apoproteins. Signi®cantly, cblC (and, to a lesser extent, cblD) cells use CN-Cbl less well than OH-Cbl353,355 and are unable to convert CN-Cbl to OH-Cbl, a step shown in normal cells to be a metabolic prerequisite for the synthesis of both AdoCbl and MeCbl.355 The latter results have been interpreted as evidence for a defect in a cytosolic cob(III)alamin reductase, which is required for reducing the trivalent cobalt of Cbl prior to alkylation.355 In both cblC and cblD ®broblast extracts, partial de®ciencies of CN-Cbl b-ligand transferase and microsomal cob(III)alamin reductase have been described by Pezacka.356,357 Watanabe and colleagues have described a partial de®ciency of a mitochondrial NADH-linked aquacobalamin reductase in cblC ®broblast extracts.298 The suggestion that glutathionyl Cbl may be an intermediate in the reductive pathway358 provides another potential site for the mutation in one of these groups. Finally, it should be pointed out that the distinction between the cblC and cblD classes is based ®rst and foremost on complementation studies that de®ne the two classes as unique,296 although essentially only one example of cblD exists. The biochemical differences between them appear to be quantitative rather than qualitative, with the cblC group having more severe metabolic derangements (and, at the same time, more severe clinical involvement) than the sibs designated cblD. Thus, the possibility must be considered that cblD is an allele of cblC that shows interallelic complementation. Studies using cultured ®broblasts from two patients in the cblF group are of particular interest. As with cells from cblC and cblD patients, both mutase and methionine synthase activities were impaired, and AdoCbl and MeCbl contents were reduced. In contrast to the cblC and cblD mutants, however, the cblF cells accumulated unmetabolized, non±protein-bound CN-Cbl in lysosomes.359 These ®ndings indicate that cblF cells are de®cient in the mediated process by which Cbl vitamers exit from lysosomes after being taken up by receptor-mediated endocytosis. The biochemical features of patient ®broblasts that distinguish the various methylmalonic acidemias are summarized in Table 94-4. Chemical Abnormalities In Vivo. In addition to the methylmalonic aciduria and homocystinuria that characterize the cblC, cblD, and cblF groups of patients, some have shown hypomethioninemia and cystathioninuria. This constellation of chemical abnormalities, plus the normal serum Cbl values, led to the proposal262,331 that Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. CHAPTER 94 / DISORDERS OF PROPIONATE AND METHYLMALONATE METABOLISM Table 94-4 Salient Biochemial Features of Cultured Fibroblasts from Patients with the Various Methylmalonic Acidemias Mutant Class Biochemical Parameter mut 0 mut2 cblA cblB Studies with intact cells [14C]propionate oxidation [14C]MeH4F ®xation MeCbl synthesis AdoCbl synthesis Conversion of CN-Cbl to OH-Cbl Lysosomal ef¯ux of free Cbl Enzyme activities in cell extracts* Mutase holoenzyme Mutase total enzyme Met synthase holoenzyme Met synthase total enzyme Cob(I)alamin adenosyltransferase cblC cblD cblF nt nt *Holoenzyme is defined as that enzyme activity measured in the absence of added cofactor; total enzyme is that activity measured in the presence of saturating concentrations of cofactor. Abbreviations: normal; markedly deficient or undetectable; partially deficient; nt not tested; MeCbl methylcobalamin; AdoCbl adenosylcobalamin; MeH4F N 5-methyltetrahydrofolate; CN-Cbl cyanocobalamin; OH-Cbl hydroxocobalamin. Pathophysiology. The megaloblastic anemia so commonly observed in the cblC patients almost surely re¯ects the enzymatic disturbance of methionine synthase. This can be stated with some assurance because patients with isolated methylmalonyl CoA mutase de®ciency (mut 0, mut , cblA, cblB) more severe than that encountered in the cblC patients exhibit no such hematologic dysfunction. The early and severe central nervous system abnormalities encountered in the cblC group probably re¯ect the methionine synthase abnormality as well, in that such patients do not have the severe metabolic ketoacidosis that probably accounts for the neurologic problems in patients with mutase de®ciency only. Thus, patients with severe, inherited dysfunction in the Fig. 94-15 Summary scheme of inherited defects of propionate and methylmalonate metabolism. The circled numbers and their key signify the nine general sites at which abnormalities have been identi®ed. Abbreviations: PCC 5 propionyl CoA carboxylase; MCC 5 b-methylcrotonyl CoA carboxylase; PC 5 pyruvate carbox- ylase; ACC 5 acetyl CoA carboxylase; MUT 5 methylmalonyl CoA mutase; CblIII 5 cob(III)alamin (e.g., OH-Cbl); CblI 5 cob(I)alamin; AdoCbl 5 adenosylcobalamin; MeCbl 5 methylcobalamin; MS 5 methionine synthase. 2185 Downloaded from OMMBID - The Online Metabolic & Molecular Bases of Inherited Disease (www.ommbid.com). Copyright 2001 The McGraw-Hill Companies. All rights reserved. Any use is subject to the Terms of Use as given on the Web site. 2186 PART 9 / ORGANIC ACIDS synthesis of both Cbl coenzymes resemble closely patients with exogenous Cbl de®ciency, both groups having prominent hematologic and neurologic manifestations resulting from the blocked methionine synthase system. Genetic Considerations. Because equal numbers of affected males and affected females exist in the cblC group, because females have been as seriously affected as males, and because cells from affected patients behave as recessives in complementation studies,295 it seems safe to predict that this disorder is inherited as an autosomal-recessive trait. The mode of inheritance of the cblD and the cblF mutations cannot yet be de®ned with certainty, because of the paucity of known patients. Identi®cation of heterozygotes for cblC, cblD, or cblF has not yet been accomplished. One additional contribution of the somatic cell genetic studies used to characterize these disorders deserves mention. The locus coding for the human methionine synthase structural gene was originally mapped to chromosome 1 using human±hamster hybrids.354 This assignment has been con®rmed with the cloning of the gene.360±363 Diagnosis, Treatment, and Prognosis. The combination of methylmalonic aciduria, homocystinuria, and normal serum Cbl concentrations is the triad needed to distinguish patients in the cblC, cblD, or cblF groups from those with isolated mutase de®ciency, with other causes of homocystinuria such as cystathionine synthase de®ciency or N5,10-methylenetetrahydrofolate reductase de®ciency, with Cbl de®ciency, or with the cblE and cblG mutations affecting only methionine synthase (see Chapters 88 and 155). Such distinctions, easily con®rmed by cell studies, are critical because appropriate therapy depends on them. Whereas exogenous Cbl de®ciency will respond dramatically to physiologic amounts of Cbl and certain forms of homocystinuria will respond to supplements of pyridoxine or folate, successful treatment of cblC, cblD, or cblF patients may demand administration of very large amounts (up to 1 mg daily) of OH-Cbl.263,333,335 ±337,344 Such treatment has resulted in dramatic decreases in urinary methylmalonate (and less dramatic decreases in urinary homocystine) in patients who have received it.364 Supplementation with betaine can reduce homocysteine levels and restore methionine, although the clinical effects of this treatment are unclear.365 Early diagnosis and prompt institution of therapy with Cbl supplements (and betaine) may be the only way to change the outcome of these patients, which, at least in the case of the cblC group, has been dismal thus far.366 Documentation of experience with such treatments will be particularly important in assessing the clinician's ability to modify the natural history of these disorders. 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