Congenital bovine spinal dysmyelination is caused by a missense mutation in the SPAST gene - PubMed (original) (raw)

Congenital bovine spinal dysmyelination is caused by a missense mutation in the SPAST gene

Bo Thomsen et al. Neurogenetics. 2010 May.

Abstract

Bovine spinal dysmyelination (BSD) is a recessive congenital neurodegenerative disease in cattle (Bos taurus) characterized by pathological changes of the myelin sheaths in the spinal cord. The occurrence of BSD is a longstanding problem in the American Brown Swiss (ABS) breed and in several European cattle breeds upgraded with ABS. Here, we show that the disease locus on bovine chromosome 11 harbors the SPAST gene that, when mutated, is responsible for the human disorder hereditary spastic paraplegia (HSP). Initially, SPAST encoding Spastin was considered a less likely candidate gene for BSD since the modes of inheritance as well as the time of onset and severity of symptoms differ widely between HSP and BSD. However, sequence analysis of the bovine SPAST gene in affected animals identified a R560Q substitution at a position in the ATPase domain of the Spastin protein that is invariant from insects to mammals. Interestingly, three different mutations in human SPAST gene at the equivalent position are known to cause HSP. To explore this observation further, we genotyped more than 3,100 animals of various cattle breeds and found that the glutamine allele exclusively occurred in breeds upgraded with ABS. Furthermore, all confirmed BSD carriers were heterozygous, while all affected calves were homozygous for the glutamine allele consistent with recessive transmission of the underlying mutation and complete penetrance in the homozygous state. Subsequent analysis of recombinant Spastin in vitro showed that the R560Q substitution severely impaired the ATPase activity, demonstrating a causal relationship between the SPAST mutation and BSD.

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Figures

Fig. 1

Fig. 1

Clinical and pathological characteristics of bovine spinal dysmyelination. a Phenotypic appearance of a calf affected with bovine spinal dysmyelination. b Whole slide digital microscopic image of cervical spinal cord stained with Luxol fast blue. Lack of myelin is seen as reduced blue staining in the ascending gracile funiculus (a), the ascending dorsolateral spinocerebellar tracts (b), and the descending sulcomarginal tracts (c)

Fig. 2

Fig. 2

a Physical map of the bovine spinal dysmyelination locus between the linked microsatellite markers BP38 and BMS1953. The location and order of the genes are shown according to the cattle genome assembly Btau_4.0. b Expression analysis by reverse transcription-polymerase chain reaction of SPAST. Tissues analyzed are SC1 spinal cord 1 (cervical), SC2 spinal cord 2 (lumbar), CE cerebellum, FC frontal cortex, HE heart, MG mammary gland, and LI liver

Fig. 3

Fig. 3

Multiple sequence alignment of Spastin homologues. Key domains in Spastin are color marked: The AAA domain ATPase associated with diverse cellular activities (yellow), MIT microtubule interacting and trafficking domain (purple), Walker A and B motifs (green), and arginine finger and second region of homology (blue). The arginine residue at position 560 is evolutionary conserved from mammals to insects (red)

Fig. 4

Fig. 4

Sequence of the missense mutation causing the R560Q substitution. The chromatograms show the sequence of a genomic fragment spanning the putative disease-causing mutation. Wild-type and affected individuals were homozygous for arginine and glutamine, respectively, whereas individuals with bovine spinal dysmyelination carrier status were heterozygous

Fig. 5

Fig. 5

a Coomassie blue stain of a sodium dodecyl sulfate-polyacrylamide gel. Recombinant wild-type and mutant Spastin fused to glutathione _S_-transferase (GST) were expressed in Escherichia coli and purified by affinity chromatography using glutathione sepharose columns. Lane 1, molecular size marker. Lane 2, wild-type GST-Spastin fusion protein. Lane 3, mutant GST-Spastin protein. b ATPase activity of wild-type and mutated Spastin were measured as a function of time. ATP hydrolysis was measured spectrophotometrically using a coupled enzyme assay. Wild-type Spastin denatured by boiling served as a control

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