Functional evaluation of paraplegin mutations by a yeast complementation assay

Authors

  • Florian Bonn,

    1. Institute of Genetics, Center for Molecular Medicine (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
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  • Krishna Pantakani,

    1. Institute of Human Genetics, University of Goettingen, Goettingen, Germany
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  • Moneef Shoukier,

    1. Institute of Human Genetics, University of Goettingen, Goettingen, Germany
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  • Thomas Langer,

    1. Institute of Genetics, Center for Molecular Medicine (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
    2. Max-Planck-Institute for Biology of Aging, Cologne, Germany
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  • Ashraf U. Mannan

    Corresponding author
    1. Institute of Human Genetics, University of Goettingen, Goettingen, Germany
    2. DFG-Research Center Molecular Physiology of the Brain, Goettingen, Germany
    • Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, D-37073, Goettingen, Germany
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  • Communicated by Peter J. Oefner

Abstract

An autosomal recessive form of hereditary spastic paraplegia (AR-HSP) is primarily caused by mutations in the SPG7 gene, which codes for paraplegin, a subunit of the hetero-oligomeric m-AAA protease in mitochondria. In the current study, sequencing of the SPG7 gene in the genomic DNA of 25 unrelated HSP individuals/families led to the identification of two HSP patients with compound heterozygous mutations (p.G349S/p.W583C and p.A510V/p.N739KfsX741) in the coding sequence of the SPG7 gene. We used a yeast complementation assay to evaluate the functional consequence of novel SPG7 sequence variants detected in the HSP patients. We assessed the proteolytic activity of hetero-oligomeric m-AAA proteases composed of paraplegin variant(s) and proteolytically inactive forms of AFG3L2 (AFG3L2E575Q or AFG3L2K354A) upon expression in m-AAA protease-deficient yeast cells. We demonstrate that the newly identified paraplegin variants perturb the proteolytic function of hetero-oligomeric m-AAA protease. Moreover, commonly occurring silent polymorphisms such as p.T503A and p.R688Q could be distinguished from mutations (p.G349S, p.W583C, p.A510V, and p.N739KfsX741) in our HSP cohort. The yeast complementation assay thus can serve as a reliable system to distinguish a pathogenic mutation from a silent polymorphism for any novel SPG7 sequence variant, which will facilitate the interpretation of genetic data for SPG7. Hum Mutat 31:1–5, 2010. © 2010 Wiley-Liss, Inc.

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