Development of Transgenic Mice Containing an Introduced Stop Codon on the Human Methylmalonyl-CoA Mutase Locus

The mutation R403stop was found in an individual with mut⁰ methylmalonic aciduria (MMA) which resulted from a single base change of C→T in exon 6 of the methylmalonyl-CoA mutase gene (producing a TGA stop codon). In order to accurately model the human MMA disorder we introduced this mutation onto the human methylmalonyl-CoA mutase locus of a bacterial artificial chromosome. A mouse model was developed using this construct.The transgene was found to be intact in the mouse model, with 7 copies integrated at a single site in chromosome 3. The phenotype of the hemizygous mouse was unchanged until crossed against a methylmalonyl-CoA mutase knockout mouse. Pups with no endogenous mouse methylmalonyl-CoA mutase and one copy of the transgene became ill and died within 24 hours. This severe phenotype could be partially rescued by the addition of a transgene carrying two copies of the normal human methylmalonyl-CoA mutase locus. The “humanized” mice were smaller than control litter mates and had high levels of methylmalonic acid in their blood and tissues.This new transgenic MMA stop codon model mimics (at both the phenotypic and genotypic levels) the key features of the human MMA disorder. It will allow the trialing of pharmacological and, cell and gene therapies for the treatment of MMA and other human metabolic disorders caused by stop codon mutations.     

Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning.

Genetic defects in the enzyme methylmalonyl CoA mutase cause a disorder of organic acid metabolism termed "mut methylmalonic acidemia." Various phenotypes of mut methylmalonic acidemia are distinguished by the presence (mut-) or absence (mut0) of residual enzyme activity. The recent cloning and sequencing of a cDNA for human methylmalonyl CoA mutase enables molecular characterization of mutations underlying mut phenotypes. We identified compound heterozygous mutations in a mut0 fibroblast cell (MAS) line by cloning the methylmalonyl CoA mutase cDNA by using the polymerase chain reaction (PCR), sequencing with internal primers, and confirming the pathogenicity of observed mutations by DNA-mediated gene transfer. Both mutations alter amino acids common to the normal human, mouse, and Propionibacterium shermanii enzymes. This analysis points to evolutionarily preserved determinants critical for enzyme structure or function. The application and limitation of cDNA cloning by PCR for the identification of mutations are discussed.     

Mouse models for methylmalonic aciduria

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.     

In-depth phenotyping reveals common and novel disease symptoms in a hemizygous knock-in mouse model (Mut-ko/ki) of mut-type methylmalonic aciduria

Isolated methylmalonic aciduria (MMAuria) is primarily caused by deficiency of methylmalonyl-CoA mutase (MMUT or MUT). Biochemically, MUT deficiency results in the accumulation of methylmalonic acid (MMA), propionyl-carnitine (C3) and other metabolites. Patients often exhibit lethargy, failure to thrive and metabolic decompensation leading to coma or even death, with kidney and neurological impairment frequently identified in the long-term. Here, we report a hemizygous mouse model which combines a knock-in (ki) missense allele of Mut with a knock-out (ko) allele (Mut-ko/ki mice) that was fed a 51%-protein diet from day 12 of life, constituting a bespoke model of MMAuria. Under this diet, mutant mice developed a pronounced metabolic phenotype characterized by drastically increased blood levels of MMA and C3 compared to their littermate controls (Mut-ki/wt). With this bespoke mouse model, we performed a standardized phenotypic screen to assess the whole-body impairments associated with this strong metabolic condition. We found that Mut-ko/ki mice show common clinical manifestations of MMAuria, including pronounced failure to thrive, indications of mild neurological and kidney dysfunction, and degenerative morphological changes in the liver, along with less well described symptoms such as cardiovascular and hematological abnormalities. The analyses also reveal so far unknown disease characteristics, including low bone mineral density, anxiety-related behaviour and ovarian atrophy. This first phenotypic screening of a MMAuria mouse model confirms its relevance to human disease, reveals new alterations associated with MUT deficiency, and suggests a series of quantifiable readouts that can be used to evaluate potential treatment strategies.     

Mapping of human methylmalonyl CoA mutase (MUT) locus on chromosome 6.

Methylmalonyl CoA mutase (MCM) catalyzes an essential step in the degradation of several branch-chain amino acids and odd-chain fatty acids. Deficiency of this apoenzyme causes the mut form of methylmalonic acidemia, an often fatal disorder of organic acid metabolism. An MCM cDNA has recently been obtained from human liver cDNA libraries. This clone has been used as a probe to determine the chromosomal location of the MCM gene and MUT locus. Southern blot analysis of DNA from human-hamster somatic-cell hybrid cell lines assigned the locus to region q12-p23 of chromosome 6. In situ hybridization further localized the locus to the region 6p12-21.2. A highly informative RFLP was identified at the MCM gene locus which will be useful for genetic diagnostic and linkage studies.     

Homology modeling of human methylmalonyl-CoA mutase: a structural basis for point mutations causing methylmalonic aciduria.

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.     

The cblD defect causes either isolated or combined deficiency of methylcobalamin and adenosylcobalamin synthesis

Intracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism.     

Phenotype of disease in three patients with identical mutations in methylmalonyl CoA mutase

Summary We have previously identified a mutation in the gene for methylmalonyl CoA mutase in a patient with the mut^- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.     

Clustering of mutations in methylmalonyl CoA mutase associated with mut- methylmalonic acidemia.

Mutations have been described in human methylmalonyl CoA mutase (MCM) that exhibit partial defects in enzyme activity, including cobalamin-dependent (i.e., mut-) or interallelic complementation. This work describes mutations in cells from four patients, three of whom exhibit a cobalamin-dependent phenotype and all four of whom exhibit interallelic complementation. Four novel mutations (R694W, G648D, G630E, and G626C) are identified that cluster near the carboxyl terminus of the protein, a region close to another mut- mutation (G717V). Each of these mutations was shown to express a phenotype congruent with that of the parental cell line, after transfection into mut0 fibroblasts, and each exhibits interallelic complementation in cotransfection assays with clones bearing a R93H mutation. The activity of mutant enzymes expressed in Saccharomyces cerevisiae parallels the residual activity of the parental cell lines and exhibits novel sensitivities to pH and salt. The clustering of these mutations identifies a region of MCM that most likely represents the cobalamin-binding domain. The location of this domain, as well as the pattern of sequence preservation between the homologous human and Probiono-bacterium shermanii enzymes, suggests a mechanism for interallelic complementation in which the cobalamin-binding defect is complemented in trans from the heterologous subunits of the dimer.     

MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA. In humans, deficiencies in the mutase lead to methylmalonic aciduria, a rare disease that is fatal in the first year of life. Such inherited deficiencies can result from mutations in the mutase structural gene or from mutations that impair the acquisition of cobalamins. Recently, a human gene of unknown function, MMAA, has been implicated in methylmalonic aciduria (Dobson, C. M., Wai, T., Leclerc, D., Wilson, A., Wu, X., Dore, C., Hudson, T., Rosenblatt, D. S., and Gravel, R. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15554-15559). MMAA orthologs are widespread in bacteria, archaea, and eukaryotes. In Methylobacterium extorquens AM1, a mutant defective in the MMAA homolog meaB was unable to grow on C(1) and C(2) compounds because of the inability to convert methylmalonyl-CoA to succinyl-CoA (Korotkova N., Chistoserdova, L., Kuksa, V., and Lidstrom, M. E. (2002) J. Bacteriol. 184, 1750-1758). Here we demonstrate that this defect is not due to the absence of adenosylcobalamin but due to an inactive form of methylmalonyl-CoA mutase. The presence of active mutase in double mutants defective in MeaB and in the synthesis of either R-methylmalonyl-CoA or adenosylcobalamin indicates that MeaB is necessary for protection of mutase from inactivation during catalysis. MeaB and methylmalonyl-CoA mutase from M. extorquens were cloned and purified in their active forms. We demonstrated that MeaB forms a complex with methylmalonyl-CoA mutase and stimulates in vitro mutase activity. These results support the hypothesis that MeaB functions to protect methylmalonyl-CoA mutase from irreversible inactivation.     

Linkage relationships of the human methylmalonyl CoA mutase to the HLA and D6S4 loci on chromosome 6

The human methylmalonyl CoA mutase (MCM) cDNA has been used to localize the MUT locus on the short arm of chromosome 6 proximal to the glyoxalase locus in 6p deletion cell lines. A HindIII polymorphism identified by the MCM cDNA was used to study linkage relationships of MUT to HLA (A-B-DQ-DR) and D6S4 in the reference CEPH families. The maximum lod score for MUT versus HLA was 3.04 at a recombination fraction of 0.28. The maximum lod score for MUT versus D6S4 was 22.93 at a recombination fraction of 0.01. These data suggest that MUT and D6S4 loci are tightly linked and may be used as one locus in a haplotype form for linkage studies on proximal 6p and diagnostic analysis of pedigrees with mut methylmalonic acidemia.     

Regulation by SREBP-2 defines a potential link between isoprenoid and adenosylcobalamin metabolism

Mevalonate kinase (MVK) catalyses an early step in cholesterol biosynthesis converting mevalonate to phosphomevalonate. Cob(I)alamin adenosyltransferase (MMAB) converts cob(I)alamin to adenosylcobalamin, functionally required for mitochondrial methylmalonyl-CoA mutase activity and succinyl-CoA formation. These two synthenic genes are found in a head-to-head formation on chromosome 12 in man and chromosome 5 in mouse. The 330bp intergenic region showed several conserved NF-Y sites indicative of potential bidirectional regulatory SREBP synergism. Both MVK and MMAB appear to be regulated in a similar manner, to a large extent by SREBP-2, since their tissue expression pattern was similar and both genes were suppressed by an excess of cholesterol as well as SREBP-2 knockdown. Statin treatment in mice upregulated both Mvk and Mmab mRNA levels indicating that this treatment may be useful in inborn errors of cblB complementation associated with methylmalonic aciduria as well as hyper IgD and periodic fever syndrome (HIDS).     

Failure of lysosomal release of vitamin B12: a new complementation group causing methylmalonic aciduria (cblF).

A patient has been described with methylmalonic aciduria because of an inability to release free vitamin B12 from lysosomes. Complementation analysis was performed using fibroblasts from this patient and those from patients having previously described mutations causing methylmalonic aciduria (mut, cblA, cblB, cblC, and cblD). Incorporation of label from [1-14C]propionate into acid-precipitable material was elevated in heterokaryons formed by polyethylene glycol (PEG) treatment of mixed cultures of cells from the patient and all other complementation groups as compared to the incorporation in parallel cultures not treated with PEG. These results indicate that complementation occurred in all cases and support the assignment of the patient to a new complementation group that has been designated cblF.     

Crystal structure and mutagenesis of the metallochaperone MeaB: insight into the causes of methylmalonic aciduria

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B(12)-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central alpha/beta-core G domain that is typical of the GTPase family, as well as alpha-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP- and GTP-bound forms of this protein. Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.     

Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.     

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.     

Absence of Diabetes and Pancreatic Exocrine Dysfunction in a Transgenic Model of Carboxyl-Ester Lipase-MODY (Maturity-Onset Diabetes of the Young)

Background

CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas.

Methods

We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure.

Results

Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation.

Conclusions

In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein.     

Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening

Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography-mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient's urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.     

Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.