Open Access Research article

Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle

Randy J Chandler1, Jennifer Sloan1, Hong Fu2, Matthew Tsai1, Sally Stabler3, Robert Allen3, Klaus H Kaestner2, Haig H Kazazian2 and Charles P Venditti1*

Author Affiliations

1 Genetic Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda MD 20892 USA

2 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19114, USA

3 Department of Medicine, University of Colorado School of Medicine, Denver CO 80206, USA

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BMC Medical Genetics 2007, 8:64  doi:10.1186/1471-2350-8-64

Published: 15 October 2007

Additional files

Additional file 1:

Genotype and screening assays. (A). A 2% agarose gel showing the results of the genotyping reaction across the 5' loxP site. Lanes 1–4, 6, and positive control are from heterozygote animals and exhibit two bands: 190 bp (wild-type) and 225 bp, which contains the flank sequences as well as the loxP site. Lane 5, homozygous for the wild-type Mut locus, has a single wild-type band while lane 7, a homozygous Mut knock-out, has a single loxP site. Para-nitroanaline (PNA) reactivity of 3.5 μl of urine from each animal yields an emerald green positive reaction for the Mut mutant. The bottom of the panel shows the standards for the PNA reaction: A-10 mM, B-100 mM, C-1 mM, D-blank. The affected animal (number 7) appears to have a urinary MMA concentration between 10 and 100 mM.

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Additional file 2:

Primers for PCR and RT-PCR

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Additional file 3:

Western analysis of wild-type and Mut embryonic fibroblast extracts. Western blotting using anti-mutase antisera reveals a band of ~80 kd in the wild-type extracts that is completely absent from the Mut null MEF line. Recombinant murine methylmalonyl-CoA mutase expressed in yeast served as a positive control (Y) and is located next to the marker (M) lane. The sizes of the molecular weight standards in kilodaltons are indicated. Anti-actin antibodies were used to control for the amount of protein loaded per well.

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Additional file 4:

Methylmalonic acid fold change by tissue type. The values are averages from prenatal [embryonic day 19] (n = 3), neonatal [8–12 hour] (n = 3) and metabolic crisis [20–24 hour] (n = 2) affected animals. The fold change is plotted for each time point compared to control littermates (n = 2).

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Additional file 5:

2-Methylcitrate I/II Ratios in Mouse Organs

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Additional file 6:

Tissue distribution of murine methylmalonyl-CoA mutase. Western analysis of tissue extracts prepared from a wild-type mouse. 10 μg of total protein were loaded in each lane and probed with anti-mutase antibodies or anti-actin antibodies. A recombinant mouse methylmalonylCoA mutase protein (labeled yeast) served as the positive control. The marker lane (M) and the sizes of the molecular weight standards in kilodaltons are indicated.

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