Novel Mouse Models of Methylmalonic Aciduria Recapitulate Phenotypic Traits with a Genetic Dosage Effect.

Methylmalonic aciduria (MMAuria), caused by deficiency of methylmalonyl-CoA mutase (MUT), usually presents in the newborn period with failure to thrive and metabolic crisis leading to coma or even death. Survivors remain at risk of metabolic decompensations and severe long term complications, notably renal failure and neurological impairment. We generated clinically relevant mouse models of MMAuria using a constitutive Mut knock-in (KI) allele based on the p.Met700Lys patient mutation, used homozygously (KI/KI) or combined with a knockout allele (KO/KI), to study biochemical and clinical MMAuria disease aspects. Transgenic Mut(ki/ki) and Mut(ko/ki) mice survive post-weaning, show failure to thrive, and show increased methylmalonic acid, propionylcarnitine, odd chain fatty acids, and sphingoid bases, a new potential biomarker of MMAuria. Consistent with genetic dosage, Mut(ko/ki) mice have lower Mut activity, are smaller, and show higher metabolite levels than Mut(ki/ki) mice. Further, Mut(ko/ki) mice exhibit manifestations of kidney and brain damage, including increased plasma urea, impaired diuresis, elevated biomarkers, and changes in brain weight. On a high protein diet, mutant mice display disease exacerbation, including elevated blood ammonia, and catastrophic weight loss, which, in Mut(ki/ki) mice, is rescued by hydroxocobalamin treatment. This study expands knowledge of MMAuria, introduces the discovery of new biomarkers, and constitutes the first in vivo proof of principle of cobalamin treatment in mut-type MMAuria.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Correction of methylmalonic aciduria in vivo using a codon-optimized lentiviral vector.

Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector-mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut(-/-)MUT(h2)) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut(-/-)MUT(h2) and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder.

A founder mutation in PET100 causes isolated complex IV deficiency in Lebanese individuals with Leigh syndrome.

Leigh syndrome (LS) is a severe neurodegenerative disorder with characteristic bilateral lesions, typically in the brainstem and basal ganglia. It usually presents in infancy and is genetically heterogeneous, but most individuals with mitochondrial complex IV (or cytochrome c oxidase) deficiency have mutations in the biogenesis factor SURF1. We studied eight complex IV-deficient LS individuals from six families of Lebanese origin. They differed from individuals with SURF1 mutations in having seizures as a prominent feature. Complementation analysis suggested they had mutation(s) in the same gene but targeted massively parallel sequencing (MPS) of 1,034 genes encoding known mitochondrial proteins failed to identify a likely candidate. Linkage and haplotype analyses mapped the location of the gene to chromosome 19 and targeted MPS of the linkage region identified a homozygous c.3G>C (p.Met1?) mutation in C19orf79. Abolishing the initiation codon could potentially still allow initiation at a downstream methionine residue but we showed that this would not result in a functional protein. We confirmed that mutation of this gene was causative by lentiviral-mediated phenotypic correction. C19orf79 was recently renamed PET100 and predicted to encode a complex IV biogenesis factor. We showed that it is located in the mitochondrial inner membrane and forms a ∼300 kDa subcomplex with complex IV subunits. Previous proteomic analyses of mitochondria had overlooked PET100 because its small size was below the cutoff for annotating bona fide proteins. The mutation was estimated to have arisen at least 520 years ago, explaining how the families could have different religions and different geographic origins within Lebanon.

Typical and atypical phenotypes of PNPO deficiency with elevated CSF and plasma pyridoxamine on treatment.

Pyridox(am)ine phosphate oxidase (PNPO) deficiency causes severe early infantile epileptic encephalopathy and has been characterized as responding to pyridoxal-5'-phosphate but not to pyridoxine. Two males with PNPO deficiency and novel PNPO mutations are reported and their clinical, metabolic, and video-electroencephalographic (EEG) findings described. The first child showed electro-clinical responses to pyridoxine and deterioration when pyridoxine was withheld. At last review, he has well-controlled epilepsy with pyridoxal-5'-phosphate monotherapy and an autism spectrum disorder. The second child had a perinatal middle cerebral artery infarct and a myoclonic encephalopathy. He failed to respond to pyridoxine but responded well to pyridoxal-5'-phosphate. At the age of 21 months he has global developmental delay and hemiparesis but is seizure-free with pyridoxal-5'-phosphate monotherapy. Plasma and cerebrospinal fluid pyridoxamine levels were increased in both children during treatment with pyridoxine or pyridoxal-5'-phosphate. These observations indicate that differential responses to pyridoxine and pyridoxal-5'-phosphate treatment cannot be relied upon to diagnose PNPO deficiency.

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 mut(0) 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.

Treatment of a methylmalonyl-CoA mutase stopcodon mutation.

There are limited treatment options for the metabolic disorder methylmalonic aciduria. The disorder can be caused by nonsense mutations within the methylmalonyl-CoA mutase gene, resulting in the production of a truncated protein with little or no catalytic activity. We used a genomic reporter assay and mouse primary cell lines which carry a stop-codon mutation in the human methylmalonyl-CoA mutase gene to test the effects of gentamicin and PTC124 for stop-codon read-through potential. Fibroblast cell lines were established from methylmalonic aciduria knockout-stop codon mice. Addition of gentamicin to the culture medium caused a 1.5- to 2-fold increase in mRNA expression of the human methylmalonyl-CoA mutase gene. Without treatment the cells contained 19% of the normal levels of methylmalonyl-CoA mutase enzyme activity which increased to 32% with treatment, suggesting a functional improvement. Treatment with PTC124 increased the amount of human methylmalonyl-CoA mutase gene mRNA by 1.6±0.3-fold and a trend suggesting increased enzyme activity. The genomic reporter assay, BAC_MMA(∗)EGFP, expresses enhanced green fluorescent protein when read-through of the stop codon occurs. Using flow cytometry, RT-real-time PCR and enzyme assay, read-through was measured. Treatment with PTC124 at 20µmol/L resulted in a significant increase in enhanced green fluorescent protein, a 2-fold increase in mRNA expression and a trend to a slight increase in enzyme activity. The clinical relevance of these effects may be tested in mouse models of MMA carrying nonsense mutations in the methylmalonyl-CoA mutase gene. Pharmacological approaches have the advantage of providing a broader effect on multiple tissues, which will benefit many different disorders with similar nonsense mutations.

Fetal progenitor cell transplantation treats methylmalonic aciduria in a mouse model.

Methylmalonic aciduria is a rare disorder caused by an inborn error of organic acid metabolism. Current treatment options are limited and generally focus on disease management. We aimed to investigate the use of fetal progenitor cells to treat this disorder using a mouse model with an intermediate form of methylmalonic aciduria. Fetal liver cells were isolated from healthy fetuses at embryonic day 15-17 and intravenously transplanted into sub-lethally irradiated mice. Liver donor cell engraftment was determined by PCR. Disease correction was monitored by urine and blood methylmalonic acid concentration and weight change. Initial studies indicated that pre-transplantation sub-lethal irradiation followed by transplantation with 5 million cells were suitable. We found that a double dose of 5 million cells (1 week apart) provided a more effective treatment. Donor cell liver engraftment of up to 5% was measured. Disease correction, as defined by a decrease in blood methylmalonic acid concentration, was effected in methylmalonic acid mice transplanted with a double dose of cells and who showed donor cell liver engraftment. Mean plasma methylmalonic acid concentration decreased from 810 ± 156 (sham transplanted) to 338 ± 157 µmol/L (double dose of 5 million cells) while mean blood C3 carnitine concentration decreased from 20.5 ± 4 (sham transplanted) to 5.3 ± 1.9 µmol/L (double dose of 5 million cells). In conclusion, higher levels of engraftment may be required for greater disease correction; however these studies show promising results for cell transplantation biochemical correction of a metabolic disorder.

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.

Stop codon read-through of a methylmalonic aciduria mutation.

A stop codon defect in methylmalonyl-CoA mutase (resulting in a truncated unstable protein) accounts for up to 14% of mutations identified as causes of Methylmalonic aciduria. There are currently limited treatment regimes for patients with this inherited condition. We aimed to investigate the use of stop codon read-through drugs in a genomic reporter assay cell line with a defect in the mutase gene. A single C-T base change was introduced into exon 6 of the human MUT sequence in the BAC clone RP11-463L20 resulting in an arginine residue being replaced with a TGA stop codon. An enhanced green fluorescent protein reporter gene was introduced in-frame with exon 13 of the MUT gene. The construct was transfected into HeLa cells to produce the genomic reporter assay cell line. To test the suppression of nonsense mutations, cells were incubated in the presence of different compounds for a period of 72 h then analysed by flow cytometry. Treatment of the cells with gentamicin resulted in a 1.6-fold increase in reporter protein, whilst G418 treatment resulted in no change, however the two drugs together acted synergistically to increase the production of methylmalonyl-CoA mutase 2.0-fold (confirmed by mRNA, flow cytometry and enzyme activity). Zidovudine, adefovir and cisplatin were also found to have some activity in the stop codon read-through genomic reporter assay. These results encourage further testing of compounds as well as follow up animal studies. This is the first study to demonstrate the use of stop codon read-through drugs for the potential treatment of Methylmalonic aciduria.

Gene induction for the treatment of methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria is an autosomal recessive inborn error of the propionate metabolic pathway. One form of this disorder is caused by mutations in methylmalonyl-coenzyme A mutase (MCM), resulting in reduced levels of enzyme activity. The pharmacological up-regulation of residual mutase activity is one approach to advance treatment strategies for individuals affected by this disorder. We describe the construction, characterization and use of a cellular genomic reporter assay for MCM expression that will potentially identify therapeutic pharmacological agents for methylmalonic aciduria treatment. METHODS: Homologous recombination was used to insert an enhanced green fluorescent protein (EGFP) cassette inframe before the last codon of exon 13 of the MCM gene (MUT) in a BAC clone. The construct was used to generate stable HeLa cell lines. EGFP expression was measured by flow cytometry and the real-time reverse transcriptase-polymerase chain reaction was used to quantify changes in MUT gene mRNA levels. RESULTS: The genomic reporter assay used to screen a selection of compounds. Cisplatin, zidovudine and adefovir were found to increase the levels of MCM mRNA and EGFP expression, providing support for the possible efficacy of these pharmacological compounds in treating methylmalonic aciduria. CONCLUSIONS: This assay has the potential of being used in high-throughput screening of chemical libraries for the identification of novel compounds that specifically modulate the expression of MCM.

Three-year audit of the hyperphenylalaninaemia/phenylketonuria spectrum in Victoria.

AIMS: To determine the prevalence, the types and severity of hyperphenylalaninaemia (including phenylketonuria (PKU)) in Victoria and to report on a new treatment modality of PKU. METHODS: We reviewed the medical records of all patients diagnosed with high blood phenylalanine levels by newborn screening between November 2001 and October 2004. RESULTS: We identified 17 newborn babies with high levels of blood phenylalanine (total samples: 190,835). Dihydrobiopterin reductase deficiency was excluded in all babies. Five babies had persistent phenylalanine levels of 200-300, and do not receive any dietary or pharmaceutical therapy. One baby was diagnosed as having pyruvoyl tetrahydro-pterin synthase deficiency. Following reports of tetrahydrobiopterin (BH(4))-responsive PKU, we have performed a BH(4) load (20 mg/kg, 6R-5,6,7,8-tetrahydro-L-biopetrin dehydrochloride; Schricks Laboratories, Jona, Switzerland) in 10 newborn babies with PKU (one baby with a phenylalanine level of 2600 micromol/L was started on diet without prior load). Three babies had a significant response to BH(4) (>35% decrease in phenylalanine level). Protein restriction (1.2 g/kg/day) and introduction of phenylalanine-free formula, in addition to BH(4) treatment, were necessary in one patient. The other patients maintain good metabolic control with BH(4) treatment only (at approximately 11 mg/kg/day) and an intake of 2-3 g protein per day. Of the nine babies who are on a full PKU diet, three have high phenylalanine tolerance (consistently >40 mg/kg/day). CONCLUSION: There is a spectrum of severity of hyperphenylalaninaemia in the population. The detection of BH(4)-responsive PKU patients offers them a less restrictive dietary regimen and an improved quality of life, and may enable near normal life-style in adolescence.

POLG mutations and Alpers syndrome.

Alpers-Huttenlocher syndrome (AHS) an autosomal recessive hepatocerebral syndrome of early onset, has been associated with mitochondrial DNA (mtDNA) depletion and mutations in polymerase gamma gene (POLG). We have identified POLG mutations in four patients with hepatocerebral syndrome and mtDNA depletion in liver, who fulfilled criteria for AHS. All were compound heterozygous for the G848S and W748S mutations, previously reported in patients with progressive external ophtalmoplegia or ataxia. We conclude that AHS should be included in the clinical spectrum of mtDNA depletion and is often associated with POLG mutations, which can cause either multiple mtDNA deletions or mtDNA depletion.

Molecular studies in mutase-deficient (MUT) methylmalonic aciduria: identification of five novel mutations.

Mutase-deficient (MUT) methylmalonic aciduria (MMA) is an autosomal recessive inborn error of organic acid metabolism, resulting from a functional defect in the nuclear encoded mitochondrial enzyme methylmalonyl-CoA mutase (MCM) (EC.5.4.99.2). The enzyme requires 5'-deoxyadenosylcobalamin as a cofactor. Isolated MMA results from either apoenzyme or cofactor defects, and is classified into several genotypic classes and complementation groups. These are designated mut(-) or mut(0) (together termed mut), depending on minimal or no apoenzyme activity respectively and cobalamin A or B (cbl A/B) for cofactor defects. To date various studies have identified over 53 disease-causing mutations from patients with mut(0/-) MMA. These are predominantly missense/nonsense nucleotide substitutions. In this study, we report the genotype analysis on 7 patients diagnosed with mut MMA. Five novel mutations were identified (R403stop, 497delG, P615T, 208delG and R467stop) and one novel polymorphism (c712A->G). The previously reported R228Q mutation was found in one patient, who is a compound heterozygote for this mutation and the R467stop mutation. A recently reported N219Y mutation was found in one patient. The 497delG mutation was detected as a homozygous deletion. The remaining mutations were observed in compound heterozygotes, with the second mutation yet to be identified. Many of the unidentified mutations may occur within the promotor or intronic regions.