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

Jahic A, Khundadze M, Jaenisch N, Schüle R, Klimpe S, Klebe S, Frahm C, Kassubek J, Stevanin G, Schöls L, Brice A, Hübner C, Beetz C

Orphanet Journal of Rare Diseases 2015, 10 :147 (16 November 2015)

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

Amir Jahic¹, Mukhran Khundadze², Nadine Jaenisch³, Rebecca Schüle⁴⁵⁶, Sven Klimpe⁷,Stephan Klebe⁸, Christiane Frahm³, Jan Kassubek⁹, Giovanni Stevanin¹⁰, Ludger Schöls⁴⁵, Alexis Brice¹⁰, Christian A. Hübner² and Christian Beetz¹^*

*Corresponding author: Christian Beetz christian.beetz@med.uni-jena.de

Author Affiliations

¹Department of Clinical Chemistry and Laboratory Medicine, Jena University Hospital, Jena, Germany

²Institute of Human Genetics, Jena University Hospital, Jena, Germany

³Hans Berger Department of Neurology, Jena University Hospital, Jena, Germany

⁴Hertie-Institute for Clinical Brain Research, Department of Neurodegenerative Diseases, University of Tübingen, Tübingen, Germany

⁵German Research Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany

⁶Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics, Miami, FL, USA

⁷Department of Neurology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany

⁸Department of Neurology, University Hospital, Freiburg, Germany

⁹Department of Neurology, University of Ulm, Ulm, Germany

¹⁰INSERM U1127, Sorbonne Universités, UPMC Univ Paris 06 UMR_S1127, CNRS UMR7225, EPHE, Institut du Cerveau et de la Moelle épinière, Paris, France

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Orphanet Journal of Rare Diseases 2015, 10:147 doi:10.1186/s13023-015-0359-x

The electronic version of this article is the complete one and can be found online at:http://www.ojrd.com/content/10/1/147

Received:	3 July 2015
Accepted:	19 October 2015
Published:	16 November 2015

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

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 ofKIAA0196. 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 monoallelicKIAA0196/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.