title: Functional evaluation of paraplegin mutations by a yeast complementation assay.
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 (AFG3L2(E575Q) or AFG3L2(K354A)) 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.
authors.affiliation: Institute of Genetics, Center for Molecular Medicine, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Bonn
authors.forename: Krishna
authors.initials: K
authors.lastname: Pantakani
authors.forename: Moneef
authors.initials: M
authors.lastname: Shoukier
authors.forename: Thomas
authors.initials: T
authors.lastname: Langer
authors.forename: Ashraf U
authors.initials: AU
authors.lastname: Mannan
mesh_terms: D049069:ATP-Dependent Proteases; D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics*; D011110:Polymorphism, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D015419:Spastic Paraplegia, Hereditary / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism
chemical_list: D049069:ATP-Dependent Proteases; C120699:AFG3L2 protein, human; D008666:Metalloendopeptidases; C112889:SPG7 protein, human; C114583:m-AAA proteases; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities