Mouse models for methylmalonic aciduria

Abstract

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.

Figure 1. Diagram of pBAC_MMA showing the size of the insert and the relation of the methylmalonyl-CoA mutase locus to flanking upstream and downstream sequence (A).

(B) Two intact genes are contained within pBAC_MMA. The methylmalonyl-CoA mutase and centromere protein Q (Cenpq) loci are divergently transcribed and share a putative 200 bp CpG island.

Figure 2. Fluorescent in situ hybridisation study on fibroblasts probed with digoxigenin labelled BAC RPCI-11-463L20.

Yellow arrows indicate where the transgene (pink spots) has been detected. A–F: 0.6-, 1-, 2-(Mouse C), 2-(Mouse E), 3- and 6-copy mouse founder lines respectively. The presence of only one signal per diploid number establishes the presence of a single integration site of the mutase transgene. G: The 0.6-copy line (Mouse A) did not have a signal in all cells of the interphase spread, suggesting the possibility of mosaicism. H: Metaphase spread from a 2-copy homozygous mouse (0.6-copy founder line) showing two sites where the transgene was detected.

Figure 3. Relative gene expression analysis of the human MUT transgene compared to mouse Mut gene in different tissues from the four independent transgenic progeny mouse lines with various copy numbers of the human MUT gene.

Data presented as mean ± SEM.

Figure 4. Comparison of weights of the 2-copy and 4-copy mouse lines.

Body weight over two years (A) female mice and (B) male mice. Organ weight of four month old mice (C) actual weight and (D) organ weight relative to body weight, where the mean body weight for four month old Mut⁺ controls (Mut^+/− or Mut^+/+) was 30.0±6.4 g and for hemizygous 2-copy partial rescue was 15.4±1.6 g. Data presented as mean ± SEM. *p<0.05 hemizygous 2-copy partial rescue vs Mut ⁺ mouse tissue.

Figure 5. Kaplan Myer curve comparing survival rates for rescue mice relative to control mice over a 30 month period.

Rescue mice have a higher earlier loss in the first 6 months, which then plateaus and follows the loss seen in control mice.

Figure 6. Comparison of metabolite levels from Mut⁺ controls (Mut^+/− or Mut^+/+ ), 2-copy hemizygous rescue and 2-copy homozygous rescue mice at various ages.

(A) Methylmalonic acid levels in urine (B) C3 carnitine levels from dried blood spots and (C) methylmalonic acid levels in plasma. Data presented as mean ± SEM. Rescue mice significantly higher (p<0.05) in metabolite levels for each sample compared to Mut⁺ controls.

Figure 7. LC-MSMS analysis of methylmalonic acid concentrations in various tissues.

(A) liver, (B) kidney, (C) cortex, (D) cerebellum and (E) muscle. Data presented as mean ± SEM.

Figure 8. Ratio of incorporation of ¹⁴C propionate relative to ³H phenylalanine incorporation for each mouse cell line.

The activity of methylmalonyl-CoA mutase was measured in fibroblast cell lines developed from the mouse models. Homozygous knockout mice with no mouse mutase have a very low phenylalanine to propionate ratio compared to wild type mice. The rescue mouse lines have increasing amounts of enzyme activity compared to the knockout mouse, however do not attain normal levels. Data presented as mean ± SEM.

Figure 9. Comparison of mutase expression in the liver, kidney and brain of the 2- and 4-copy mouse models.

Expression was normalised to human beta-actin. Data are expressed as mean (± SEM) fold change of mutase expression in each sample relative to wild type mouse mutase expression levels.