Abstract for presentation at 11th International Congress of Human Genetics

Upregulation of gene expression is a potential therapeutic strategy for methylmalonic aciduria

  • Dr Mahmoud Khaniani, Paediatric Department, Melbourne University,Tabriz University of Medical Science, Iran, Australia
  • Dr Joseph Sarsero, Murdoch Children Research Institute, Royal Children Hospital, Melbourne, Australia, Australia
  • Dr Jean-Francois Benoist, Deportment of Biochemistry and Hormonology, Robert Debry Hospital, 75019 Paris, France, France
  • Ms Leonie Wood, Murdoch Children Research Institute, Royal Children Hospital, Melbourne, Australia, Australia
  • Dr Heidi Peters, Murdoch Children Research Institute, Royal Children Hospital, Melbourne, Australia, Australia

    • Methylmalonic aciduria (MMA) is a metabolic autosomal recessive disorder resulting from either deficiency of the methylmalonyl-CoA mutase (MCM) enzyme encoded by methylmalonyl-CoA mutase (MUT) locus or defect in the adenosylcobalamin (AdoCbl). A number of mutations leading to MMA do not abolish completely normal gene expression. It is postulated that increasing the expression of the MCM enzyme gene in MMA patients with low-level of residual activity of the normal MCM enzyme could potentially be therapeutic for such individuals. Lower levels of induction may still be clinically beneficial in decreasing disease progression and severity.
    A bacrteial artificial chromosome (BAC) clone containing the entire human MUT functional genomic sequence was identified and an in-frame fusion between the enhanced green fluorescent protein (EGFP) reporter gene and the human genomic MUT locus was constructed by homologous recombination. Insertion of EGFP-Kan/Neo cassette into the correct targeted position in the BAC was initially confirmed by PCR analysis. High-resolution mapping of the recombinant clone was performed with frequent-cutting enzymes BamHІ, NheІ, SpeІ and NcoI. The linearised genomic fragment from the modified clone was transfected into HeLa cells by using Lipofectamine 2000, based on the flow cytometry data, single positive clones were isolated. A number of stable HeLa cell lines containing the MUT-EGFP genomic reporter fusion were produced.
    In PCR analysis and high-resolution mapping no detectable differences between the recombinant clone and the parent clone were observed, other than those generated by the insertion of the EGFP-Kan/Neo cassette into the targeted region. Cisplatin, butyric acid, propionic acid, lactate and wheat grass at 2% ethanol concentration, increased MUT-EGFP expression.
    The development of the cellular genomic reporter demonstrates the utility of using this construct to monitor MCM enzyme expression from the human locus, and for screening various metabolites for their effects on MCM gene expression. This assay should also facilitate high throughput screening for pharmacological compounds that can modulate the expression of the MUT gene in a clinically relevant manner.

    Conference Organiser - ICMS Pty Ltd