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Originally published In Press as doi:10.1074/jbc.M310533200 on October 10, 2003

J. Biol. Chem., Vol. 278, Issue 52, 52909-52913, December 26, 2003
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A Knock-out Mouse Model for Methylmalonic Aciduria Resulting in Neonatal Lethality*

Heidi Peters{ddagger}§, Mikhail Nefedov{ddagger}, Joseph Sarsero{ddagger}, James Pitt{ddagger}, Kerry J. Fowler{ddagger}||, Sophie Gazeas{ddagger}, Stephen G. Kahler{ddagger}**, and Panayiotis A. Ioannou{ddagger}||{ddagger}{ddagger}

From the {ddagger}Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria 3052, Australia and the ||Department of Pediatrics, University of Melbourne, Royal Children's Hospital, Melbourne, Australia

Received for publication, September 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality. Mice appear phenotypically normal at birth and are indistinguishable from littermates. By 15 h of age, they develop reduced movement and suckle less. This is followed by the development of abnormal breathing, and all of the mice with a null phenotype die by 24 h of age. Urinary levels of methylmalonic and methylcitric acids are grossly increased. Measurement of acylcarnitines in blood shows elevation of propionylcarnitine with no change in the levels of acetylcarnitine and free carnitine. Incorporation of [14C]propionate in primary fibroblast cultures from mut-/- mice is reduced to approximately 6% of normal level, whereas there is no detectable synthesis of mut mRNA in the liver. This is the first mouse model that recapitulates the key phenotypic features of mut0 methylmalonic aciduria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylmalonic aciduria (MMA,1 MIM 251000 [OMIM] ) is an autosomal recessive inborn error of organic acid metabolism. The true incidence of the disorder has been difficult to determine, and variable figures have been reported. A recent review of newborn screening data indicates an incidence of isolated MMA (mutase and cobalamin A/B) in the order of 1 in 140,000 in Australia (1). The condition results from a functional defect in the enzyme methylmalonyl-CoA mutase (MCM, EC 5.4.99.2 [EC] ) either due to a defect in the mutase (mut) gene itself (designated muto or mut-) or from a defect in the metabolism of the cofactor adenosylcobalamine. MCM is a nuclear-encoded mitochondrial enzyme that catalyzes the conversion of L-methylmalonyl-CoA to succinyl-CoA, which then enters the citric acid cycle. L-Methylmalonyl-CoA is predominantly derived from the catabolism of branched chain amino acids in the diet, odd chain fatty acids via propionyl-CoA, and propionate synthesized from gut flora.

Following normal pregnancy and delivery-affected individuals with the mut0 form typically present in the newborn period with overwhelming illness consisting of acidosis, vomiting, poor feeding, hypotonia, and lethargy. Untreated, there is progression to coma and death. The mut0 form may have a less severe phenotype. Aggressive supportive therapy results in survival. However, any intercurrent illness or "metabolic stress" may lead to metabolic instability characterized by repeat recurrent life-threatening episodes of metabolic decompensation.

Currently, the mainstay of treatment for MMA is strict dietary restriction and drug manipulation. The aim of such treatment is to control methylmalonate (MM) and propionate production by limiting dietary intake of precursors and endogenous production of substrate by catabolism. Despite such treatment, morbidity and mortality remain high (2). There are many long term complications such as poor appetite and growth, pancreatitis, cardiomyopathy, and metabolic stroke resulting in permanent neurological damage. Individuals that survive infancy develop renal failure later in childhood and adolescence (3). The underlying pathophysiology leading to these complications is poorly understood.

Liver and combined liver/kidney transplantations have been attempted with the aim of replacing the deficient enzyme activity, thus preventing the need for diet and avoiding the long term complications. The outcome of such treatment has been successful in allowing patients to relax the dietary restrictions and preventing acute metabolic decompensations (4). However, the natural history of the renal failure and propensity to metabolic stroke remain unclear at this stage (5). Furthermore, liver transplantation is restricted because of the risks associated with it as well as the limitation of suitable donors. A better understanding of the pathophysiology of the disease is therefore needed to enable the development of an effective therapy for this disorder. However, progress has been limited by the lack of animal models for this disorder.

The human mutase gene is ubiquitously expressed and encodes for a 750 amino acid precursor protein including a 32 amino acid mitochondrial leader sequence (6, 7). The leader sequence is cleaved within the mitochondria, and a mature enzyme is formed. Three functional domains have been proposed as follows: (i) the C terminus (residues 578-750), which comprises the cobalamin binding domain; (ii) the ({beta}/{alpha})8 barrel (residues 87-416) to which the CoA of methylmalonyl-CoA binds; and (iii) the N terminus with a region involved in the dimerization of the two MCM monomers (residues 32-87). Cloning of the mouse locus revealed a 94% homology to the human amino acid sequence (8). Furthermore, the mouse cDNA was shown to complement patient cell lines, thus confirming its homology at a functional level.

To develop a mouse model for mut0 MMA, we have disrupted the mouse mutase locus by gene targeting. We have chosen to disrupt the mouse locus within the CoA binding domain by replacement of exon 3 with an antibiotic selection marker by homologous recombination. This is the first mouse model to be developed for the mut-/- form of MMA disease. Here we describe the observed phenotype and the biochemical characterization of these mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Targeting Cassette—The primers 2aF, 5'-TGGGAGTATTCATGCTTCAC-3', and 2aR, 5'-CTGCATACTGACGGATGGTC-3', were designed to amplify a 349-bp product encompassing mouse mutase exon 2. Similarly, the primers 13F, 5'-ATGGATCCTCAGCCAGACC-3', and 13R, 5'-TGTATACAGATCAGCGTGTTTC-3', were designed to amplify a 280-bp product encompassing mouse exon 13. The mouse exon 2 and exon 13-specific PCR products were used as probes to screen the RPCI-23 mouse BAC library (BACPAC Resources, Oakland, CA). Among the clones identified, the BAC clone 38D7 (RPCI-23; 38D7, 100 kb) appeared to contain the entire mouse mutase locus. The targeting vector was constructed by subcloning an 11.2-kb NheI/BamHI restriction digestion fragment containing exons 2-5 of the mouse mutase locus from BAC clone 38D7 into the tetracycline resistance gene of the pBR322 plasmid. Insertion of this fragment disrupted the tetracycline resistance gene and produced pBR_mutA (data not shown). A 1468-bp fragment containing exon 3 and flanking intronic sequence was deleted from pBR_mutA by NcoI restriction digestion and replaced with an antibiotic selection cassette to disrupt the CoA binding domain (Fig. 1A). The 1.55-kb neomycin/kanamycin antibiotic selection cassette was amplified by PCR from the plasmid pEGFP-N22 (9) using primers LNK-F, 5'-AATATTTAAATAACTTCGTATAGCATACATTATACGAAGTTATATTCTAAATACATTCAAATAT-3', and LNK-R, 5'-AATATTTAAATATAACTTCGTATAATGTATGCTATACGAAGTTATGAACAAACGACCCAACA-3', with attached loxP sites (underlined sequence). The selection cassette was gel-purified and cloned into the NcoI sites of pBR_mutA using TA cloning to produce pBR_mutB (Fig. 1B). Clones containing the selection cassette in the reverse orientation were used for targeting into embryonic stem (ES) cells. This construct when homologously recombined into the mouse mutase locus should cause deletion of exon 3 (Fig. 1C). If the selection cassette is spliced out by splicing between exons 2 and 4, it should produce a frameshift with premature protein truncation and total loss of enzyme function.



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  		FIG. 1.
Targeted disruption of mouse mutase gene. A, diagram of the mouse mutase protein showing the CoA and cobalamin binding domains. Our targeting construct was designed to delete a region of the CoA binding domain between amino acids 126 and 251 that is involved in substrate binding. B, a restriction map of the mutase gene between exons 2 and 6. Exons are denoted as boxes. The gene replacement construct where the selectable marker cassette consists of a neomycin/kanamycin antibiotic marker in the reverse orientation with flanking loxP sites is shown below. C, the mutase locus after gene disruption. The position of probes used in Southern blot analysis is shown as filled squares, and the expected size of restriction fragments are marked. Oligonucleotides used in screening and genotyping are also marked. Restriction enzymes used were SacI (S), EcoRI (E), PstI (P), NheI (N), BamHI (B), and NcoI (Nc).

 
Generation and Microinjection of Targeted Embryonic Stem Cells—Mouse ES cells from a W9.5 line were used for transfection. Cells (5 x 107) were electroporated with 30 µg of pBR_mutB DNA, which had been linearized by restriction digestion with EcoRV. Settings were 0.8 kV, 3 microfarads, and maximum {Omega} using a Bio-Rad gene pulser (Bio-Rad). Transfected ES cells were grown on STO NeoR feeder cells using standard ES medium containing leukemia inhibitory factor (Chemicon International, Temecula, CA) and {beta}-mercaptoethanol. G418 (Invitrogen) antibiotic selection at 200 µg/ml was added after 24 h and continued for 7 days. Resistant colonies were picked from day 6 to day 11. Cell lines were established and stored at -70 °C. An aliquot of each line was seeded onto gelatin plates and grown to confluency. DNA was extracted and used for PCR screening to detect correct recombinants. Correctly targeted cell lines were initially determined by PCR using the primers sF, 5'-CAATAGAAGCCTTCCCAGAATG-3', and sR, 5'-TTTTCTGGATTCATCGACTGT-3', to give a product of 3 kb (Fig. 1C).

Targeted cell lines were karyotyped and confirmed by fluorescent in situ hybridization and Southern blot analysis to contain a single targeting event (data not shown). Standard methods were used to microinject the targeted ES cell line into C57BL/6 blastocysts (10). These were transferred into pseudopregnant Swiss HSDOla-recipient mice. Chimeric mice were detected by coat color and mated with C57BL/6 mice to produce heterozygotes. Heterozygous progeny were interbred to produce homozygous knock-out mice.

Genotyping and Southern Analysis of Mice—DNA was extracted from mouse tail by standard methods and used for PCR genotyping (8). Primers used for screening were as follows: neoF, 5'-CAACAGACAATCGGCTGCTC-3'; neoR, 5'-GTCACGACGAGATCCTCGC-3' (485 bp); expF, 5'-GGACCATATCCTACCATGT ATAC-3'; and expR, 5'-ACAGTGTCAATAGCAACTCCAG-3' (1243 bp). Southern blot analysis was performed on genomic DNA restriction digested with EcoRI and PstI and probed with a 250-bp exon 2 PCR fragment to confirm correct targeting of the 5' end of the construct. Correct targeting was confirmed at the 3' end by SacI digestion and probing with a 200-bp exon 6 PCR probe (Fig. 1C).

RT-PCR Analysis—Total RNA was extracted from liver using the RNeasy kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. First strand synthesis was carried out with SuperScriptTM RNase H reverse transcriptase kit (Invitrogen) using a random primer. PCR was carried out using primers 2F, 5'-CTTATATGGCACACCCCAGAA-3', and 6R, 5'-TCCTGAATGATGATTCGTGTG-3', for exons 2-6 (product length 1.2 kb) of the mouse mutase gene. Control RT-PCR for the mouse Frda mRNA was carried out with primers FATGCN-F, 5'-GCTCTCTAGAYGAGAC-3', and FATGCN-R, 5'-CCCAAARGAGACATC-3' (product length 125 bp).

[14C]Propionate Incorporation Assay—Fibroblast cultures were set up from skin biopsies. Cells were grown to confluence in 25-cm2 flasks before trypsinization, splitting one-third, and incubation in a depleted medium (minimum Eagle's medium with added L-glutamine, non-essential amino acids, and fetal bovine serum) for 3 days. Cells were washed and incubated with labeled medium (Earl's balanced salt solution containing 200 nmol/ml propionate and 18 nmol/ml (1.0 mCi/ml) [14C]propionate) for 18 h. Mutase activity was determined using the method of [14C]propionate incorporation into trichloroacetic acid-precipitable material (11). Incorporation was normalized with respect to protein content. Protein was determined using the Lowry method.

Analysis of Urine and Blood Metabolites—A 2-15-µl urine sample was collected from each newborn pup and at various time points after birth within the first 24 h and was frozen until analysis. Organic acids were extracted with ethyl acetate, converted to trimethylsilyl derivatives, and analyzed by gas chromatography-mass spectrometry using an Agilent 5971 system (Palo Alto, CA) (12). Selected ion monitoring at 287 m/z (methyl citric acid) and 247 m/z (methylmalonic acid) was performed.

For measurement of urine MM levels by electrospray tandem mass spectrometry, a 2-µl volume of urine was mixed with 50 µl of 100 µM 2H3-MM (MSD Isotopes, Montreal, Canada) and 220 µl of 50% acetonitrile:water (v:v). A 30-µl aliquot of this mixture was analyzed by flow injection into 80 µl/min of 50% acetonitrile:water (v:v) infused into the ion source of a Quattro LC tandem mass spectrometer (Micromass, Manchester, United Kingdom) with multiple reaction monitoring for 117 > 73 m/z (MM) and 120 > 76 m/z (2H3-MM).

For the measurement of acylcarnitines by tandem mass spectrometry, blood was collected from newborn mice at various time points and spotted onto absorbent cotton fiber paper (Guthrie) cards. After drying, 3-mm spots were excised from the cards, extracted with methanol, and butylated in microtiter plates according to standard methods (13). The same flow analysis parameters as above were used for the measurement of acylcarnitines, including the levels of C3, C2, and C0 carnitines. multiple reaction monitoring transitions (M + H > 85 m/z) were used to measure the acylcarnitines.

Histopathology—Tissue samples were collected from culled mouse pups. Kidney, liver, and the intact brain were dissected and placed in 10% formalin. Coronal sections were taken from each organ of wild type mice, heterozygous mice, and homozygote knock-out mice at various times after birth and stained with hemotoxylin and eosin (H&E) or periodic acid Schiff reagent.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The targeting construct, pBR_mutB (Fig. 1B), was designed to replace exon 3 of the mouse mutase locus with a neomycin/kanamycin selection cassette, running in the reverse orientation, flanked by loxP sites. The exon 3 region is critical for CoA binding, and its disruption should result in a loss of function. Furthermore, even if the selection cassette was deleted by alternative splicing between exons 2 and 4, the deletion should lead to premature termination. A low targeting frequency in the order of 1 in 1000 was obtained between the targeting construct and homologous sequences in mouse ES cells. One correctly targeted ES cell line was identified by PCR screening, Southern blot analysis, and fluorescent in situ hybridization analysis (data not shown). However, the first chimeric animals generated after injection of this cell line into C57Bl/6 blastocysts were inexplicably lost over the first weeks after birth along with non-chimeric animals. No cause could be established for the loss of these animals. The injection was repeated and resulted in two germ line chimeras. These mice were crossed with C57BL/6 to produce heterozygous progeny and establish a healthy colony of heterozygous knock-out animals. Heterozygous knock-out animals are phenotypically normal and have normal growth and fertility. Heterozygous mice were intercrossed to produce homozygous progeny. Among 76 offspring from 12 littermates from such crosses, we identified by PCR screening 17 animals that had the homozygous knock-out genotype (Fig. 2A), a result consistent with the expected Mendelian frequency. Southern blot analysis for the 5' and 3' ends of the targeting cassette gave the expected size fragments (Fig. 2, B and C), also confirming homozygosity for the knock-out allele in affected neonates.



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  		FIG. 2.
Molecular characterization of knockout mice. A, PCR screening of mouse tail DNA for the neomycin/kanamycin selection cassette and the region between exons 2 and 3. Lane X, molecular weight marker X; lane 1, negative control; lane 2, wild type mouse (Mut+/+); lane 3, mouse heterozygous for the exon 3 deletion (Mut+/-); Lane 4, neonate mouse homozygous for the exon 3 deletion (Mut-/-), giving a PCR product only with the neomycin/kanamycin primers. B, Southern blot analysis of genomic DNA from homozygous (Mut-/-) (lane 1), heterozygous (Mut+/-) (lane 2) and wild type (Mut+/+) (lane 3) mice after EcoRI/PstI restriction digestion and probing with an exon 2 PCR product. The wild type locus corresponds to the 3024-bp band, whereas the knock-out allele corresponds to the 5712-bp band. C, as for B after digestion with SacI and probing with an exon 6 PCR product, giving a 8567-bp band with the wild type allele and a 13,782-bp band with the knock-out allele. D, analysis of mRNA expression in knock-out mice. Shown is RT-PCR of total RNA extracted from liver of (lane 1) wild type, heterozygous (lane 2), and homozygous (lane 3) knock-out animals using primers amplifying between exons 2 and 6 of the mouse mutase locus together with control primers for the mouse Frda locus.

 
Homozygous null mice were born normally and were indistinguishable from their normal littermates at birth. They were of comparable birth weight and initially were active, vigorous, and suckled normally as evidenced by the observation of large milk spots in the stomach shortly after birth. By 15-18 h of age, homozygous null mice gradually became less active and stopped suckling (observation of pups behavior along with evidence of reduced/absent milk spots). Within a few hours, homozygous pups developed intermittent gasping respirations and had to be humanely sacrificed. Homozygous null mice did not survive beyond 24 h of age.

The mutase expression was examined in total RNA extracted from liver tissue of wild type, heterozygous, and homozygous knock-out mice. RT-PCR was performed using primers flanking the targeting cassette. No product was observed in homozygous mice. Control primers from the widely expressed Frda locus were used to confirm integrity of the RNA (Fig. 2D).

Biochemical Characterization—Enzyme activity was assessed indirectly using [14C]propionate incorporation in cultured fibroblasts. Measurements were performed in duplicate, giving incorporation of 635 pmol propionate/mg protein/18 h in wild type mice compared with 39 pmol propionate/mg protein/18 h in homozygous knock-out mice. The residual enzyme activity detected in homozygous knock-out animals corresponded to only approximately 6% of the activity relative to wild type mice.

MM and methylcitric acid were grossly increased in urine as assessed by gas chromatography-mass spectrometry (Fig. 3A). Measurement of urinary MMA levels collected at various time points post-delivery and measured using tandem mass spectrometry identified grossly elevated MM levels immediately after birth with a sequential increase over time (Fig. 3B). There was no significant difference between levels in heterozygotes and wild type mice, although the method used may not have adequate sensitivity to detect small increases in heterozygotes.



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  		FIG. 3.
Analysis of urine metabolites in homozygous knock-out animals. A, gas chromatography-mass spectrometry-selected ion-monitoring chromatograms showing increase in MM and methylcitric (MC) acid levels in homozygous knock-out mice (KO) compared with wild type control mice (C). Methylcitric acid gives two peaks due to the formation of diastereoisomers. B, methylmalonic acid was measured by tandem mass spectrometry in urine samples of homozygous knock-out animals at various time points after birth. Accumulation of urinary MM in homozygous knock-out mice with time was observed.

 
Analysis of urine by dipstick showed a urinary pH of 6 with none of the urine samples tested (n = 15), showing evidence of ketonuria. This included testing of urine samples immediately prior to death and with greatly elevated MM levels.

Analysis of acylcarnitines identified the mean of propionylcarnitine (C3) levels to be approximately 6 times greater than mean levels in heterozygous and wild type mice (Table I). There was no significant difference between knock-out and wild type mice for the other acylcarnitines. A comparison of the ratio of propionylcarnitine to free carnitine (C3:C0) and to acetylcarnitine (C3:C2) showed that these ratios were approximately 7-fold higher in homozygous mice when compared with unaffected controls (Table I). These ratios have similarly been shown to be increased in human MMA subjects (14).


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  		TABLE I
Measurement of acylcarnitines by tandem mass spectrometry in mouse blood

The free carnitine (C0), acetylcarnitine (C2), and propionylcarnitine (C3) values were measured in blood samples from five homozygous knock-out mice (mut-/-) and four control mice. The C3 carnitine levels (in µm) (mean ± S.D.) are shown as well as the C3:C0 and C3:C2 ratios. Only propionylcarnitine values are elevated (p = 0.002) in homozygous knock-out animals.

 
Histology of the kidney showed no identifiable abnormality in homozygous mice. Sections were taken through five regions of the brain, and no gross histological abnormalities were detected on H&E stain (data not shown). Examination of liver samples taken within 12 h of birth was normal in homozygous knock-out animals in contrast to samples collected at around 20 h of age, which showed evidence of moderate fatty change in liver parenchyma (Fig. 4).



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  		FIG. 4.
Histological analysis of liver sections in homozygous knock-out mice. H&E stain of liver tissue collected immediately post-culling from wild type mouse and homozygous knock-out mice at <12 h and ~20 h of age, showing moderate fatty change in sample at ~20 h.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mice homozygous for the knock-out allele demonstrate a phenotype similar to that of the human mut0 MMA. As frequently occurs with the human MMA disorder, they are born normally and develop disease in the newborn period once they are removed from the maternal circulation and commence ingestion of protein. The maternal circulation in this model also appears to be protective of the fetus, at least to the extent that mice develop normally in utero and are born healthy. Nevertheless, levels of MMA in the urine are already significantly elevated even with the first sample collected straight after birth. This suggests the maternal circulation does not completely "detoxify" the fetus. This finding is in agreement with the observed elevation of odd-numbered long-chain fatty acids in fetuses in disorders of propionate metabolism, reflecting the ongoing in utero elevation of propionyl-CoA despite the maternal circulation (15). The significance of this to the health of the developing fetus is unclear at this stage.

Studies of RNA by RT-PCR in these mice failed to show any mutase product in homozygotes. The targeting cassette was designed such that if the antibiotic marker was alternatively spliced the resulting product would result in a frameshift with a premature termination codon. We postulate that no product was produced as a result of unstable RNA and nonsense-mediated decay. In keeping with the null genotype was the [14C]propionate incorporation, indicating a marked reduction in MCM activity.

Biochemically, these mice demonstrate the characteristic biochemical changes observed in MMA. They have elevated urinary levels of methylmalonic and methylcitric acids and an elevation in blood of C3 or propionylcarnitine in the range observed with human disease (12, 14). The C3:C2 and C3:C0 ratios, which are used as another indicator for the identification of the organic acid disorders MMA and propionic acidemia (PA) in newborn-screening programs, are also elevated in our animal model.

Massive ketosis with hyperglycinuria was first reported in the early 1960s in an infant suffering from a disorder in the pathway of propionate metabolism that was later confirmed to be PA (16). Since that time, there have been many reports of individuals with PA or MMA describing them as disorders with massive ketoacidosis. Therefore, it is interesting that our mouse model does not have evidence of ketosis on dipstick testing of the urine. Although there may be differences between human and mouse metabolism, this is not likely to be the (only) explanation, given the recent report of a knock-out mouse model for propionic acidemia in which ketosis was readily identified by strip-testing the urine of homozygous knock-out mice as early as 10 h of age (17). In this context, it is interesting that a study on two children with MMA reported that they were not particularly ketotic during illnesses (18). However, one patient demonstrated an elevation of ketones following loading with protein, L-valine, propionate, and L-leucine. It was postulated that MM-CoA may be toxic to citrate synthase, leading to an increase in acetyl-CoA used for ketone body production. This could be exacerbated by hypoglycemia, an observed secondary affect of elevated MM-CoA, leading to utilization of fat and therefore further ketone body production. In another infant with B12-responsive MMA, a rise in ketones prior to the increase in methylmalonic acid was observed, which did not occur until 12-24 h into the illness (19). This finding has prompted the clinical practice of measuring urinary ketones in MMA patients with the onset of intercurrent infections to detect impending instability in MMA. A review of the literature for other case reports of MMA indicates that the occurrence of ketosis in MMA is variable with a significant number of patients not developing ketosis. Although individuals with propionic acidemia may have a greater propensity to develop ketosis, a patient has also been described in whom this was not a consistent finding (20), suggesting that a more rigorous evaluation of the occurrence and triggers for ketosis in these disorders may be warranted.

Histopathology on the liver shows that our homozygous knock-out mice rapidly develop fatty changes with increasing levels of methylmalonic acid. Similar findings were also reported in homozygous knock-out mice for PA (17). Variable hepatomegaly has been described in a number of cases of MMA, whereas fatty change has also been reported as a histological finding (21). This is probably because of a secondary effect of elevated propionyl-CoA or other metabolites on fatty acid synthesis (22).

The MMA knock-out model described in this report is the first mouse model for this disease. The accurate recapitulation of the main phenotypic features of the disease should make this mouse model an invaluable tool for further investigations into the pathophysiology of MMA, particularly in relation to the neurological complications of this disease as well as for the development of much needed novel therapies for this disorder.


   FOOTNOTES
 
* The work was also partially supported by a grant from the Brock-hoff Foundation. This work was approved by the Royal Children's Hospital Animal Ethics Experimentation Committee (Project Number A459). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a postgraduate scholarship from the National Health & Medical Research Council of Australia. Back

Present address: BACPAC Resources, Oakland Children's Hospital Research Institute, Oakland, CA. Back

** Present address: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins Hospital, Blalock 1008, Baltimore, MD 21287-4922. Back

{ddagger}{ddagger} To whom correspondence should be addressed: CAGT Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Rd., Parkville, Victoria 3052, Australia. Tel.: 61-3-8341-6232; Fax: 61-3-8341-6212; E-mail: ioannoup{at}cryptic.rch.unimelb.edu.au.

1 The abbreviations used are: MMA, methylmalonic aciduria; MCM, methylmalonyl-CoA mutase; MM, methylmalonate; ES, embryonic stem; RT, reverse transcriptase; PA, propionic acidemia. Back


   ACKNOWLEDGMENTS
 
We are grateful for the support of Dr. Lucille Voullaire for the fluorescent in situ hybridization analysis of the targeted mouse ES cell line. We also thank Dr. Guy Besley and the staff at Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester, United Kingdom for their help with the [14C] propionate assay.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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