|Home | About | Journals | Submit | Contact Us | Français|
Methylmalonic acidemia (MMAemia) is the biochemical hallmark of a group of genetic metabolic disorders that share a common defect in the ability to convert methylmalonyl-CoA into succinyl-CoA. This disorder is due to either a mutant methylmalonyl-CoA mutase apoenzyme or impaired synthesis of adenosylcobalamin, the cofactor for this enzyme. In this article, we will provide an overview of the pathways disrupted in these disorders, discuss the known metabolic blocks with a particular focus on molecular genetics, and review the use of selected model organisms to study features of methylmalonic acidemia.
The clinical foundation for understanding the phenotype of methylmalonic acidemia derives from a series of reports published in the late 1960s [1, 2, 3]. Oberholtzer et al. described two unrelated patients with methylmalonic acidemia . The first affected individual was initially suspected to have a renal tubular dysfunction syndrome. This patient had frequent attacks of dehydration and acidosis, and perished during a decompensation at age 2 years. On postmortem examination, a fungal pneumonia was noted and a peculiar histopathological appearance of the kidneys was documented. Specifically, the kidneys were shrunken; the tubules were diminutive and had increased interstitial tissue with a lymphocytic infiltration. The second child, a 6 year-old female, displayed a similar phenotype with respect to acid-base instability. Classical analytic chemical methods, such as paper chromatography, melting point analysis, and reactivity with diazotized p-nitroaniline, were used to demonstrate that she produced large amounts of methylmalonic acid in her urine, blood and cerebral spinal fluid (CSF). Of note, she had a CSF: plasma methylmalonic acid (MMA) gradient, with concentrations of MMA equal to 1.55 mM in her plasma and 1.575 mM in her CSF. The authors also recognized that the metabolic acidosis was only partly explained by the plasma MMA levels. A propensity toward ketosis was demonstrated, with an exquisite sensitivity to oral propionic acid. The child had problems with growth and motor skills in the early years, but when assessed at age 5 years, had a normal IQ.
The same year Stokke et al. studied the third child born to a family that had two infants perish in the newborn period with overwhelming acidosis and coma . Using a newer method of GC/MS, they demonstrated that the patient produced MMA in enormous amounts. Whole body metabolism was studied in the index case with C14-valine and H3-MMA. The patient did not respond to parenteral cobalamin but did demonstrate a clinical improvement when treated with a simple hyperalimentation consisting of elemental amino acids and glucose given IV, and fats and carbohydrates administered by nasogastric feeding, prior to perishing from an intercurrent infection. In the next year, a patient with a similar phenotype of intermittent ketoacidosis and severe methylmalonic aciduria was proven to respond to vitamin B12, firmly establishing a role for the vitamin in human intermediary metabolism [3, 4]. The early studies on patients with methylmalonic acidemia generated theories to explain the metabolic perturbations seen in these individuals, demonstrated fundamental precursor relationships, described a renal lesion seen in the patients, demonstrated that MMA is likely produced de novo or concentrated in the nervous system, showed that the disorder could be treated with precursor restriction and possibly hyperalimentation, proved that the block was located at the methylmalonyl-CoA mutase (MCM, EC 18.104.22.168) step and was co-factor responsive in some patients. These papers provided the foundation of current therapies for methylmalonic acidemia.
Scientific studies of methylmalonic acidemia have provided a paradigm for the importance of human genetics and the investigation of rare disorders as a means to elucidate fundamental aspects of metabolism. Over the past three decades, great progress has been made in understanding and treating this group of disorders. However, the challenges faced by physicians caring for the early patients, such as the propensity toward metabolic decompensation, growth and feeding problems, renal disease and premature death, still exist [5, 6, 7, 8]. The use of model systems to study methylmalonic acidemia may help guide the development and testing of newer therapies for this devastating disorder. In this review, we will update the reader on the molecular genetics of isolated methylmalonic acidemia and highlight the use of model systems to study MMAemia and cobalamin metabolic disorders.
An overview of the major pathway for propionyl-CoA metabolism into the Krebs cycle is depicted in Figure 1 and the location of the intracellular steps of cobalamin metabolism are displayed in Figure 2. Minor metabolic pathways for propionyl-CoA metabolism exist [9, 10], are activated in patients with blocks in the pathway and are likely to be more significant in methylmalonic acidemia than in propionic acidemia . The biotin-dependent propionyl-CoA carboxylase reaction may be the least likely reaction in the sequence to be reversed because of the consumption of ATP by the reaction and the direction of the equilibrium in vivo . Methylmalonic acidemia is always associated with secondary perturbations of propionyl-CoA metabolism. These perturbations include elevated propionylcarnitine, propionylglycine, 2-methylcitrate, and increased concentrations of propionate in the blood. Methylmalonic acidemia also shares many features with propionic acidemia, both clinically and metabolically. The source of bicarbonate in the propionyl-CoA fixation reaction is not clearly defined and may represent a way to modulate the regional production of methylmalonic acid in certain body compartments. The propionyl-CoA carboxylase reaction and propionic acidemia will not be discussed in this review.
D-methylmalonyl-CoA (D-MMCoA) is formed as a product of the propionyl-CoA carboxylase reaction. D-MMCoA requires racemization prior to becoming a substrate for the methylmalonyl-CoA mutase reaction and a deficiency of D-methylmalonyl-CoA racemase (MCR, EC 22.214.171.124) has long been postulated as a potential etiology of hereditary methylmalonic acidemia. The one patient suspected of having a defect at this step was later shown to have a lesion at the methylmalonyl-CoA mutase locus . The enzyme has been purified from a number of sources and shown to have enzymatic activity on D-MMCoA. The human MCR homologue had eluded direct characterization until several years ago. Bobik and Rasche pioneered a novel approach to identify this gene  that others have also successfully used to find other cobalamin metabolic genes [14, 15]. This genomic method relies on the operon principle in that bacteria tend to organize genes involved in a metabolic pathway or specialized enzymatic reaction in the same transcriptional unit. After analyzing the genomic sequences of several species of bacteria, conserved genes near the methylmalonyl-CoA mutase enzyme were noted . The authors hypothesized that one of the genes was a MCR and proved this by demonstrating the MCR gene product catalyzed the conversion of D-MMCoA to L-MMCoA. They also then identified the human homologue of MCR and demonstrated enzymatic activity in an in vitro assay. The use of model organisms and genomic principles has been demonstrated to be a powerful method of finding conserved genes involved in methylmalonyl-CoA metabolism.
The exact role MCR may have in human metabolism remains to be defined. One theory has suggested that the enzymatic function of the racemase reaction can be bypassed by a “free” MMA shunt and offers an explanation as to why there have been no patients with methylmalonic acidemia that harbor genetic lesions at this step identified to date . The formal resolution of the role that this enzyme plays in human metabolism may require the identification of patients who harbor mutations in the MCR gene. A linked assay to assess the function of this enzyme has been described . If such patients can be found and are asymptomatic, the mutant alleles they harbor will need to be directly assayed for activity. These individuals will need to be carefully studied and stressed in vivo by substrate loading before dismissing the role that this enzyme may have in methylmalonyl-CoA metabolism.
An enzyme deficiency at this step may also be studied in model organisms as an alternative and supporting means of examining the importance of the reaction. The gene has been identified in C. elegans and studied in a deletion mutant, which is viable and relatively resistant to oxidative stress . A murine homologue is predicted to exist and construction of a mouse model may also be useful. Alternatively, deficiency of this enzyme may be lethal and not observed in living patients or its function may be redundant. The striking conservation of this enzyme across the phyla suggests it may have an important function, which has yet to be formally studied in man.
The generation of free MMA requires hydrolysis of the methylmalonyl-CoA thioester into free coenzyme A and methylmalonic acid. This reaction has received some study in previous years. Kovachy, Copley and Allen purified the enzyme, D-methylmalonyl coenzyme A hydrolase, which hydrolyzes D-methylmalonyl coenzyme A to free methylmalonic acid and coenzyme A . The kinetic constants for this enzyme were defined experimentally. Several years later, Shimomura et al. purified and characterized 3-hydroxyisobutyryl-coenzyme A hydrolase, an enzyme that acts in the terminal portion of valine metabolism . These authors noted that 3-hydroxyisobutyryl-coenzyme A hydrolase had activity toward D-MMCoA and commented on the similarities in the molecular weight and pH optima for the two enzymes. The gene encoding a specific D-methylmalonyl coenzyme A hydrolase has not been identified in humans. However, if overlapping activities for the 3-hydroxyisobutyryl-coenzyme A hydrolase and D-methylmalonyl coenzyme A hydrolase exist, the former may represent a functional modifier in methylmalonic acidemia.
Bacterial operon-based gene finding and studies on this enzyme in other model organisms may help elucidate the exact role that this protein has in D-methylmalonyl coenzyme A metabolism. Of note, a succinate to propionate conversion pathway has been described in E. coli .The pathway is encoded in an operon organized as follows: a methylmalonyl-CoA mutase, an MMAA-like protein, a methylmalonyl-CoA decarboxylase and finally a propionyl-CoA:succinyl-CoA transferase protein. This may indicate that bacteria use an alternative metabolic pathway to catabolize the thioester once it is formed. Studies in higher organisms are necessary to determine if such a hydrolase pathway is present in humans.
Bobik and Rasche described two genes that clustered with methylmalonyl-CoA mutase in bacterial operons . One was proven to be an MCR; the other was considered to function as an accessory protein needed for the methylmalonyl-CoA mutase enzyme. Dobson et al. used bioinformatics and orthology relationships to independently identify the same latter gene as a candidate for cblA deficiency . To prove causation, they characterized this gene, called MMAA, and sequenced the DNA from patients with cblA lesions at this locus. Mutations in a number of patients were identified, including premature terminations and non-conservative missense changes. Dobson et al. speculated that the gene product could function as part of a mitochondrial pore, allowing cobalamin to enter into the mitochondria. A mitochondrial leader sequence was suggested by informatic analysis but the gene product has not been formally proven to localize to the mitochondrial inner space or to the mitochondrial membranes. At this point, a role for MMAA has not been defined and there are no direct functional or biochemical assays for this protein. However, a bacterial homologue of MMAA, meaB, has been shown to associate with the methylmalonyl-CoA mutase enzyme in vitro . These authors have suggested that the function of meaB, and by inference MMAA, is to protect the MCM enzyme from inactivation during catalytic cycles. Such a role would certainly be feasible for the human gene product. Determining the function of this gene might be accomplished by a number of biochemical and genetic approaches, such as direct purification, targeted over-expression or ablation in mice. Multiple mutations in various regions of the gene have been identified, which will help guide future structure and function studies.
In cblB class methylmalonic acidemia the defective gene is MMAB . It encodes an ATP:cob(I)alamin adenosyltransferase of the pduO family . It should be noted that multiple enzymatic solutions to this reaction occur in nature  and that humans and mice only appear to possess a pduO-like family. Two approaches were used to identify the gene defective in cblB deficiency. Dobson et al. used informatics to identify the human homologue of the pduO gene and then directly sequenced the DNA from patient cell lines of the cblB class . Leal et al. performed direct complementation of a bacterial pduO mutant with a bovine cDNA expression library, isolated the bovine homologue and then the human gene homologue which they called ATP: cob(I)alamin adenosyltransferase (ATR) . Recently, methionine synthase reductase (MSR), an enzyme thought to have a primary role in the reductive reactivation of methionine synthase, has been demonstrated to localize to the mitochondria and associate with the MMAB gene product in vitro . A model for MSR involvement in the ATP:cob(I)alamin adenosyltransferase reaction has been proposed. A bacterial homologue of MMAB has been crystallized and homology considerations have afforded insight into the nature of mutations observed at this locus .
The MCM step has received the most attention of any of the steps in this pathway. The enzyme has been studied extensively in a variety of species . The P. shermani MCM has been crystallized and the structure solved at 2 Angstroms, allowing enzymatic and mutational mechanisms to be inferred in the human condition . The human MCM gene was identified by Ledley et al., who screened an expression library with mutase antibodies to isolate the first human cDNA . Over the last 10 years a number of studies have described the spectrum of mutations observed at the mutase locus in human patients [29, 30]. Several assays exist to measure the function of this enzyme, including an automated HPLC assay .
The mutase enzyme exists as a homodimer, possibly with unequivalent active sites . The enzyme has several functional domains from N- to C- terminus including: a mitochondrial importation signal, a dimerization domain (inferred from studies with mutant alleles), a coenzyme-A ester binding pocket, a spacer or linker region, and a cobalamin binding motif. Multiple mutational mechanisms operate at the MCM locus including premature stop codons [30, 33, 34], missense mutations [35, 36, 37], splice site mutations , mRNA stability mutations , deletions  and isodisomy . Kinetic properties of mutant enzymes historically had been studied using cell lines and tissues from affected patients. Few groups have undertaken direct biochemical characterization of mutant alleles. Crane and Ledley examined selected mutant cDNAs in a yeast over-expression system  while others have used bacterial hosts to produce the enzyme [40, 41]. The advantage of the yeast system is that the enzyme can be expressed with very high specific activities under standard growth conditions and can be processed and targeted to the mitochondria as it would in a vertebrate . Kinetic analysis can be performed with either approach, although purified enzyme is always the preferred starting material for Km and Vmax determination. Detailed biochemical characterization of mutant alleles at this locus will be important because most patients are allelic compounds. Interallelic interactions between mutant subunits can occur and create an intermediate or mut- deficiency state that might otherwise be unexpected from de novo prediction algorithms [40, 43]. This phenomenon makes correlations between genotype and phenotype possible for only a subset of missense alleles or when patients are homozygous.
At least two other genetic entities in the above pathway can be associated with isolated methylmalonic acidemia, cblD (variant 2) deficiency  and cblH deficiency . The biochemical and genetic etiology of these disorders has not yet been determined.
Studies on bacterial systems have defined B12 transport, synthetic pathways and enzymatic functions of dependent enzyme systems and have been extensively reviewed in the past . Below, we will focus on uses and potential applications of widely used eukaryotic model organisms.
S. cervisiae may be useful to model certain aspects of methylmalonic acidemia and disordered propionate metabolism. Yeast do not possess a methylmalonyl-CoA mutase gene and use a cobalamin-independent methionine synthase. Furthermore, yeast use the methylcitrate cycle to metabolize propionyl-CoA . In this pathway, 2-methylcitrate is formed from propionyl-CoA and oxaloacetate. A dehydration of 2-methylcitrate generates methylisocitrate that is subsequently acted upon by a methylisocitrate lyase to yield succinate and pyruvate. The enzymatic details of these steps have not yet been fully elicudated. The cycle may be viewed as enabling an organism to oxidize propionate to pyruvate. Yeast harboring mutations in the pdh gene, a putative methylcitrate dehydratase, are sensitive to exogenous propionate . The exact basis for this sensitivity is not known but may be relevant to the understanding of the targets of propionyl-CoA toxicity in mammalian systems. For example, in the aforementioned mutant, increased methylcitrate production may be causing the growth defect observed in the pdh mutant.
The method of chemogenomic profiling may be employed to study the sensitivity of yeast to propionate and methylmalonate [49, 50]. This method should allow the identification of yeast genes that confer heightened sensitivity or resistance to an exogenously added chemical or metabolite such as MMA. Yeast strains harboring gene(s) that confer resistance or sensitivity to MMA should be easily identified by whole genome parallel analysis. These strains might then be studied to determine whether they have mammalian homologues that may be targets of MMA toxicity.
Because yeast do not harbor B12 synthetic or metabolic capabilities, they may be used to reconstitute the mutase pathway in a controlled fashion in the mitochondria. Yeast can produce a functional MCM apoenzyme when the gene is expressed from an inducible promoter . Other than MCM, genes in the pathway have not been assayed for function using yeast overexpression systems to date. Specific mutations may be easier to study in yeast and supramolecular complexes between the methylmalonyl-CoA mutase and other genes, such as MMAA might also be produced, detected and studied. Alternatively, yeast genes suspected of participating in the methylcitrate cycle might be expressed in mutant cell lines derived from patients or knock-out mice in an attempt to “engineer” a new propionate pathway in a different organism. Metabolic pathway re-engineering has been used with success in other disorders , such as PKU, and may represent a novel therapeutic approach for patients with propionate spectrum metabolic disorders.
C. elegans is an attractive model to use because of easy maintenance, a short life span, extensive biological characterization, and the ability to easily knock genes down by RNA interference (RNAi). In addition, there are numerous research tools available for C. elegans including a centralized, comprehensive database (www.wormbase.org) that has not only sequence information, but a wide spectrum of genomic and genetic data, such as RNAi experiments, microarray data and global expression clustering, mutants and mutant phenotypes for each gene and large scale two-hybrid data sets . Whole genome RNAi experiments have been performed on wild-type and sensitized backgrounds, a centralized mutant stock center exists, and an effort to create deletion mutants of every gene is underway.
We have performed an analysis of the C. elegans genome to identify putative homologues of genes involved in propionate and cobalamin metabolism. Highly conserved homologues of all the genes known to be involved in the conversion of propionate to succinate - PCCA, PCCB, MCR, MCM, MMAA and MMAB (Table 1) - are predicted to exist, as well as homologues of methionine synthase reductase and methionine synthase. Each potential C. elegans homologue has a protein-to-protein BLAST score of less than e-35 and a reverse BLAST analysis indicates the existence of only one homologue of each gene in the human database . The existence of such highly conserved homologues in C. elegans is strong evidence that this primitive animal possesses the same pathway for the conversion of propionate to succinate as is found in humans. Furthermore, the existence and functional demonstration of MCR enzymatic activity in C. elegans supports this hypothesis .
Functional verification of the pathway was assessed after substrate loading wild-type animals with 10 mM propionic acid. The nematodes were removed by centrifugation and the supernatant was analyzed by GC/MS. After propionate loading, the worms showed a 15-fold increase in methylmalonate production from baseline . This increase in MMA production was not observed in negative controls consisting of the bacterial feeding strain incubated with 10 mM propionate; demonstrating the increase in MMA was not due to the bacterial feeding strains. RNAi directed against PCCA, PCCB, MCR, MCM, MMAA and MMAB produce no visibly detectable phenotype. Typically, RNAi experiments in C. elegans are measured by the appearance of visually observable phenotypes such as egg laying defects or lack of motor coordination. Given the severe phenotype that these mutations cause in humans, the lack of a visual mutant phenotype is surprising. The absence of a phenotype may be explained by the nematodes ability to expel the methylmalonate into its surrounding environment, either by diffusion or secretion, or by low-penetrance of the RNAi-dependent phenotype. Substrate loading after RNAi against known and suspected genes involved in propionate metabolism, followed by metabolite analysis, may allow for rapid characterization of gene(s) suspected to play a role in cobalamin utilization. C. elegans appears to possess a MCM and a cobalamin-dependent methionine synthase as well as all other known cobalamin metabolic enzymes, such as MSR, MMAA and MMAB. Therefore homologues of the genes mutated in cbl deficiency syndromes, such as cblC, cblD (variant 1 and variant 2), cblH and cblF, may be present in C. elegans and may cause a chemical phenotype when knocked down or out in wild type animals. C. elegans may be a valuable tool in determining the underlying genetic defect in these disorders. Finally, suspected enhancers and suppressors of the pathway may be studied in a similar manner. Community-wide efforts are currently underway to create knock-outs of all the genes in the C. elegans genome, which should facilitate the use of this versatile animal in the study of MMA as well as other human inborn errors of metabolism where homologues are known or suspected to exist.
Two mouse models of mut-class methylmalonic acidemia have been created [54, 55]. The phenotype of the affected animals mirrors the most extreme end of the human syndrome, with 100% fatality in the affected animals by 24–36 hours of life. The animals perish with massive elevations of MMA in the urine, plasma and tissues. The mice prove that the pathway of propionyl-CoA metabolism operates in a manner similar to humans. The existence of a functionally equivalent pathway had been previously suspected based on homology between the mouse and human genes and the observation that the mouse gene could correct the propionate incorporation defect seen in human cells by transfection .
The mouse models have afforded a description of some aspects of disordered metabolism seen in methylmalonic acidemia. For example, the animals do not develop severe ketonuria as assessed by dipstick analysis . Immediately after birth, the animals have significant methylmalonic aciduria suggesting that MMA accumulates prenatally. When measured on embryonic day 16, the mut- mice have plasma MMA concentrations that exceed 100 uM . These metabolite levels argue against the theory that the placenta provides complete protection (detoxification) from MMA in utero. The MMA tissue content has been followed during the life of the mut- miceand suggests that a substantial amount of MMA may derive from skeletal muscle as well as the liver, kidney and brain . The production of MMA by the muscle may provide one possible explanation of why liver transplantation and combined liver-kidney transplantations does not completely alleviate aberrant metabolite production in this disorder.
The murine models created to date have disease-related phenotypes but are too severe to study the long-term effects of methylmalonic acidemia. Over-expression of a well characterized mutant or synthetic MCM allele via a transgenic construct, either as a BAC or transgene driven by a heterologous promoter, may rescue the lethal phenotype. Alternatively, transgenic rescue with a wild-type gene under the control of an inducible or a tissue-specific promoter may prove useful in creating a conditional-on model to study the effects of MMA on certain organs. For instance, it may be possible to simulate a liver transplantation by transgenic rescue using a liver specific promoter regulating MCM production or assessing the metabolic capacity of the skeletal muscle to restore homeostasis by driving gene expression only in that location. Conditional–off alleles may also be useful to examine organ specific contributions to metabolism. Finally, the knocking-in of selected human mutation(s) into the MCM locus, such as those that participate in interallelic complementation or mutations that have predominately cobalamin Km effects, may allow for a versatile model of a partial deficiency state to be developed. The current models may be useful for proof of principle gene and stem cell therapy experiments and possibly, in utero correction.
Studies on methylmalonic acidemia began with clinical investigations and there still is a great deal to learn about the disorders by continued study of the existing patient populations. Before new and novel therapies are considered, careful clinical characterization of the patients with methylmalonic acidemia, particularly the adult and transplanted individuals, should be pursued. Varied regimens to treat the condition have been employed, including renal, hepato - and combined hepato-renal transplantation . These therapies may be viewed as precursors to cell based therapies and patients who have undergone transplantation should be carefully studied prior to and after the procedures. Others have advocated that a registry of such patients be created so longer-term outcomes and complications can be described . Liver and combined liver/kidney transplantation can completely eliminate the inherent tendency toward ketoacidotic crises but the patients are not cured of the biochemical phenotype and can still exhibit progression of the disease after transplant. For example, one patient who received a liver transplant subsequently developed renal failure requiring transplantation  and another had a metabolic stroke 5 years after transplantation . Transplant patients may enable the study of organ specific contributions to whole body MMA metabolism. A formal consideration might also be given to the potential of damaging a transplanted kidney in the setting of liver-kidney and kidney transplants due to the magnitude of the MMA elevations. Long-term outcome studies on the patients will be required to determine the significance of these metabolic perturbations. The renal disease of methylmalonic acidemia and the propensity of the patients to develop metabolic strokes also require further investigation and may benefit from animal models that replicate the mut- state.
As mentioned above, precise correlations between genotype and enzymatic activity have yet to be undertaken for most mutations seen in isolated methylmalonic acidemia. This activity will be an important undertaking in an era of tandem mass spectrometry based newborn screening that promises to identify patients with MMA by virtue of increased propionylcarnitine in the newborn blood spot. Continued studies on patients will be of utmost importance in making such correlations and organized efforts to form registries will be needed to assist these endeavors. Human genetic studies may also aid in the localization and identification of genes involved in the known cobalamin disorders, particularly cblC, cblD (and variants), cblF and cblH, as well as other syndromes that can feature elevated MMA levels, such as the combined malonic-methylmalonic acidemia syndrome , and transient methylmalonic aciduria .
As a greater diversity of genomes becomes sequenced, so will the opportunities to study cobalamin pathways and enzymes in other species. Database analysis indicates that Zebrafish, Fugu, Canis, Bos Taurus, Swine, Great Apes, Gallus, and Rattus appear to harbor a functional methylmalonyl-CoA mutase. Any of these organisms may provide a natural model of MMA or in the future, be used to develop one.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.