The spectrum of KIAA0196 variants, and characterization of a murine knockout: implications for the mutational mechanism in hereditary spastic paraplegia type SPG8

BACKGROUND: The hereditary spastic paraplegias (HSPs) are rare neurodegenerative gait disorders which are genetically highly heterogeneous. For each single form, eventual consideration of therapeutic strategies requires an understanding of the mechanism by which mutations confer pathogenicity. SPG8 is a dominantly inherited HSP, and associated with rather early onset and rapid progression. A total of nine mutations in KIAA0196, which encodes the WASH regulatory complex (SHRC) member strumpellin, have been reported in SPG8 patients so far. Based on biochemical and cell biological approaches, they have been suggested to act via loss of function-mediated haploinsufficiency. METHODS: We generated a deletion-based knockout allele for E430025E21Rik, i.e. the murine homologue of KIAA0196. The consequences on mRNA and protein levels were analyzed by qPCR and Western-blotting, respectively. Motor performance was evaluated by the foot-base angle paradigm. Axon outgrowth and relevant organelle compartments were investigated in primary neuron cultures and primary fibroblast cultures, respectively. A homemade multiplex ligation-dependent probe amplification assay enabling identification of large inactivating KIAA0196 deletion alleles was applied to DNA from 240 HSP index patients. RESULTS: Homozygous but not heterozygous mice showed early embryonic lethality. No transcripts from the knockout allele were detected, and the previously suggested compensation by the wild-type allele upon heterozygosity was disproven. mRNA expression of genes encoding other SHRC members was unaltered, while there was evidence for reduced SHRC abundance at protein level. We did, however, neither observe HSP-related in vivo and ex vivo phenotypes, nor alterations affecting endosomal, lysosomal, or autophagic compartments. KIAA0196 copy number screening excluded large inactivating deletion mutations in HSP patients. The consequences of monoallelic KIAA0196/E430025E21Rik activation thus differ from those observed for dominant HSP genes for which a loss-of-function mechanism is well established. CONCLUSIONS: Our data do not support the current view that heterozygous loss of strumpellin/SHRC function leads to haploinsufficiency and, in turn, to HSP. The lethality of homozygous knockout mice, i.e. the effect of complete loss of function, also argues against a dominant negative effect of mutant on wild-type strumpellin in patients. Toxic gain-of-function represents a potential alternative explanation. Confirmation of this therapeutically relevant hypothesis in vivo, however, will require availability of appropriate knockin models. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13023-015-0359-x) contains supplementary material, which is available to authorized users.