WO2013156785A1

WO2013156785A1 - Diagnostic gene

Info

Publication number: WO2013156785A1
Application number: PCT/GB2013/050988
Authority: WO
Inventors: Cathryn Suzanne Mellersh; Oliver Paul FORMAN
Original assignee: Animal Health Trust
Priority date: 2012-04-20
Filing date: 2013-04-19
Publication date: 2013-10-24
Also published as: GB201206946D0

Abstract

The invention relates to the use of the β-III spectrin gene (SPTBN2) as a biomarker for the in vitro diagnosis of cerebellar cortical degeneration, to in vitro methods of assessing the cerebellar cortical degeneration status in a canine mammal and to primers and diagnostic kits for use in said method.

Description

DIAGNOSTIC GENE

FIELD OF THE INVENTION

The invention relates to the use of the β-ΙΙΙ spectrin gene (SPTBN2) as a biomarker for the in vitro diagnosis of cerebellar cortical degeneration, to in vitro methods of assessing the cerebellar cortical degeneration status in a canine mammal and to primers and diagnostic kits for use in said method.

BACKGROUND OF THE INVENTION

Cerebellar cortical degeneration, also known as cerebellar abiotrophy, is a disease characterised by clinical signs of cerebellar dysfunction, such as ataxia- dysmetria, broad based stance, loss of balance and intentional tremors.

Cerebellar cortical degeneration has been described in several canine breeds [1- 15], classified as neonatal through to adult onset forms, with breed specific rates and extents of disease progression. In Beagles the development of neurological signs is first noticed when affected dogs start to ambulate at around three weeks of age. The affected puppies exhibit wide based-stance, loss of balance and dysmetric gait with inability to regulate rate and range of movement. Progression of the clinical signs has been reported to be minimal [2, 10] . The main histopathological lesions characterising the disease in Beagle dogs are extensive degeneration to loss of Purkinje cells and secondary lesions in the molecular and granular layers [10] . A previous case report suggested an autosomal recessive mode of inheritance for neonatal cerebellar cortical degeneration (NCCD) in the Beagle [2] . Neonatal cerebellar cortical degeneration has also been reported in other canine breeds, including the Rhodesian Ridgeback, Samoyed and Irish Setter [1, 9, 15], with retrotransposon disruption of GRMl associated with neonatal cerebellar ataxia in Coton De Tulear dogs [16] .

There is therefore a great need to identify the causal genetic variant responsible for NCCD in canines.

SUMMARY OF THE INVENTION According to a first aspect of the invention there is provided the β-ΙΙΙ spectrin gene (SPTBN2) for use as a biomarker for the in vitro diagnosis of cerebellar cortical degeneration in a canine mammal. According to a second aspect of the invention, there is provided an in vitro method of assessing the cerebellar cortical degeneration status in a canine mammal, the method comprising the step of detecting genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2). According to a further aspect of the invention, there is provided a primer pair for use in a method of assessing the cerebellar cortical degeneration status in a canine mammal, wherein said primers are capable of amplifying all or part of the β-ΙΙΙ spectrin gene (SPTBN2), wherein the amplified region is less than 500 nucleotides in length, such as less than 300 nucleotides in length, and wherein the primers are as defined herein.

According to a further aspect of the invention, there is provided a kit for use in a method of assessing the cerebellar cortical degeneration status in a canine mammal, wherein said kit comprises:

(a) a primer pair, wherein said primers are capable of amplifying all or part of the β-ΙΙΙ spectrin gene (SPTBN2), wherein the amplified region is less than 500 nucleotides in length, such as less than 300 nucleotides in length, and wherein the primers are as defined herein; and

(b) means for providing a test sample from the canine mammal .

According to a further aspect of the invention, there is provided a method of treating cerebellar cortical degeneration in a canine mammal, which method comprises assessing the cerebellar cortical degeneration status of a canine mammal by use of a method as defined herein and if the canine mammal is identified as affected by cerebellar cortical degeneration, treating said canine mammal to prevent or reduce the onset of cerebellar cortical degeneration.

According to a further aspect of the invention, there is provided a method of treating cerebellar cortical degeneration in a canine mammal, which method comprises increasing the level of non-mutant, wild-type β-ΙΙΙ spectrin gene (SPTBN2) expression and/or β-ΙΙΙ spectrin gene (SPTBN2) product activity in the canine mammal . BRIEF DESCRIPTION OF THE FIGURES

Figure 1: (A) Cerebellar folia of four-week old Beagle puppy with NCCD . Loss of occasional Purkinje cells (grey arrows) . The black arrow identifies a degenerating Purkinje cell with hypereosinophilic cytoplasm and a condensed nucleus. lOOx magnification .

(B) . Cerebellar folia of eight-week old full-sibling Beagle puppy with

NCCD . Marked loss of Purkinje cells (grey arrows) . Associated granular cell layer depletion secondary to Purkinje cell loss (white double headed arrows) . lOOx magnification . Figure 2: Subacute loss of Purkinje cells (PC) is documented by so- called "empty baskets" (EB) that have been visualised by Bielschowsky ^' s impregnation technique. The baskets (white arrows) synapse to the PC perikarya and in order to inhibit PC activity. ML: molecular layer; GL: granule cell layer; scale bar: 40 μηι.

Figure 3: Spatial characteristics of cerebellar cortical degeneration in the β-ΙΙΙ spectrin deficient beagle after calbindin-immunohistochemistry and haematoxylin counterstain . (A) Navigator figure depicting the sampled areas. (B) The ventral aspects of the vermis show least numeric loss of calbindin- positive (brown) Purkinje cells (PC), a narrow subarachnoid space (SAS) and remnants of the external germinative cell layer (EGL) . Furthermore, the granule layer (GL) is well populated and its histoarchitecture is preserved . Degenerative changes are restricted to dystrophic dendrites (white arrowhead) . Purkinje cell loss becomes increasingly evident in the dorsal vermis (C) and the ansiforme lobulus of the cerebellar hemispheres (D). Resident PC show thickening and abnormal arborisation (C, white arrowhead) of the dendrites. Concomitantly the EGL is cytodepleted and the granular layer becomes mildly disorganised .

Calbindin-staining in the most affected hemispheres is restricted to scattered axons, loosely bundled in the foliary white matter (D, black arrowhead) . PC perikarya in many folia are completely missing while the Purkinje cell layer features a moderate Bergmann ^'s gliosis. Scale bar: 0.7 cm for A ; 130 μηη for B,C,D. Figure 4: Results of the sequencing experiments. (A) Reads from the mRNA-seq experiment aligned across the deletion and visualised in IGV. Reads are represented by grey bars, with the deletion indicated with a black horizontal line in reads. A single nucleotide polymorphism (c.5580T>C) is also located 18 bp downstream of the deleted sequence in the NCCD case, and is highlighted by the black rectangle. (B) Sanger sequencing to confirm the 8 bp deletion in the case, the sire of the case (obligate heterozygote) and a wild-type individual (sibling). The 8 bp sequence upstream of the deletion is identical to the deleted sequence. Figure 5: Location of the canine 8 bp SPTBN2 mutation in the β-ΙΙΙ spectrin protein. Protein domains: ABD, actin binding domain. 4.1, protein 4.1 binding domain. AN K, ankyrin binding domain. PH, pleckstrin homology domain. MAD1 ; MAD2, membrane associated domains. Adapted from Bauer et a/ [35] . Figure 6: Pedigree of the NCCD Beagle family. Squares and circles represent male and female individuals respectively. Shaded symbols represent NCCD cases (all deceased). The distribution and number of affected individuals is consistent with an autosomal recessive mode of inheritance. Homozygous wild- type individuals are represented as wt/wt, heterozygotes as wt/del, and mutant homozygotes as del/del.

Figure 7: Western blot analysis of wild-type and NCCD affected cerebellum tissue homogenates. Full length β-ΙΙΙ spectrin was confirmed in wild- type cerebellum tissue (wt/wt), but no full length or truncated β-ΙΙΙ spectrin could be identified in cerebellum tissue from the NCCD affected Beagle (del/del). Mouse brain extract (M) was used a positive control. Beta-actin was used as a loading control .

DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of the invention there is provided the β-ΙΙΙ spectrin gene (SPTBN2) for use as a biomarker for the in vitro diagnosis of cerebellar cortical degeneration in a canine mammal. According to a second aspect of the invention, there is provided an in vitro method of assessing the cerebellar cortical degeneration status in a canine mammal, the method comprising the step of detecting genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2). The present inventors have identified a genetic mutation in the canine β-ΙΙΙ spectrin (SPTBN2) gene that is associated with cerebellar cortical degeneration in canines, such as Beagles.

In addition, the inventors have developed a genotyping-based diagnostic test that can be used to determine whether a dog is clear, affected by, or a carrier of cerebellar cortical degeneration. This can be used, inter alia, in selective breeding to avoid affected offspring.

Furthermore, the inventors have devised a diagnostic genotyping assay that determines the presence or absence of mutation in the canine β-ΙΙΙ spectrin (SPTBN2) gene in canine DNA.

SPTBN2 is a gene associated with spinocerebellar type 5 (SCA5) in humans [26], that fully segregates with the disease providing a strong candidate variant for NCCD in the Beagle. Spectrins are a family of cytoskeletal proteins, with tetrameric structures comprising two a and two β subunits, with diversity and specialization of function. Spectrins are important structural components of the plasma membrane and play a significant role in restricting and stabilizing membrane spanning proteins within specific subdomains of the plasma membrane. The spectrin cytoskeleton was first discovered in erythrocytes and has since been identified in a variety of cells [27] . β-ΙΙΙ spectrin is primarily expressed in the nervous system and the highest levels of expression are found in Purkinje cell soma and dendrites [28]. β-ΙΙΙ spectrin has been shown to stabilize the glutamate transporter EAAT4 at the plasma membrane of the Purkinje cells [29], facilitate protein trafficking by linking the microtubule motor to vesicle-bound cargo [30] and maintain a high density of sodium channels within the soma and dendrites of Purkinje cells [31] . β-ΙΙΙ spectrin is critical for development of Purkinje cells [32] .

In humans, three mutations in SPTBN2 have been shown to cause autosomal dominant SCA5. The identified causal mutations include two in-frame deletions of 39 and 15 bp which alter the structure of the 3^rd of 17 spectrin repeats, and a single base pair substitution causing an amino acid change (L253P) in a highly conserved region of the calponin homology domain [26] . The consequence of the two inframe deletions in p- III spectrin is predicted to be disruption of the highly ordered triple alpha helical structure of the spectrin repeat, causing conformational changes in the tetrametric α-β spectrin complex [26] . Studies suggest the resulting mutant protein may affect the localization of EAAT4 and GluR52, one possible outcome of which is glutamate signalling abnormalities and Purkinje cell death [26] . The L253P missense mutation has been shown to result in loss of interaction with the Arpl subunit of the dynactin-dynein complex, affecting the role of β-ΙΙΙ spectrin in vesicle trafficking, preventing transport of both β-ΙΙΙ spectrin and EAAT4 to the cell membrane from the Golgi apparatus in Purkinje cells causing cell dysfunction and death. [33]

Experimentally induced β-ΙΙΙ spectrin deficiency in mice from two independent studies resulted in phenotypes that resemble NCCD in Beagle dogs [31, 34] . One β-ΙΙΙ spectrin deficient strain was produced by targeting replacement of exon 3 to 6 of SPTBN2 with the neomycin-resistance gene, resulting in a frameshift and a premature stop codon in exon 7. As a result no full length β-ΙΙΙ spectrin is produced in -β-ΙΠ^7" mice, although a low level of near full length protein is produced due to novel exon 1 (rather than exon 2) to exon 7 splicing [31] . Homozygous β-ΙΙΙ spectrin deficient mice develop characteristics of progressive cerebellar ataxia from a few weeks of age with cerebellar atrophy and Purkinje cell loss. In the parallel study the β-ΙΙΙ spectrin deficient mouse strain is the result of βgeo insertion between exons 25 and 26 resulting in premature termination in spectrin repeat 14, which is closer to the position to the Beagle mutation, although results in the loss of the ankyrin binding domain [34] . The β- III spectrin deficient mice from this study display a mild non progressive ataxia by 6 months and a myoclonic seizure disorder by one year [34] . It is apparent that onset of ataxia is later for the β-ΙΙΙ spectrin deficient mouse in comparison to Beagle NCCD cases, with mice not showing significant signs of ataxia until six months of age (past sexual maturity). This is more comparable to the human disease, though the differences in the modes of inheritance suggest different mutational effects. Deficient mice also show only a mild ataxia and remain ambulatory, while the dogs described are more severe both in terms of degree of ataxia (astasia) and Purkinje cell loss. In the study by Stankewich et al., no Purkinje cell loss was documented by 18 months of age, only atrophy of the dendritic arbor. Disparity in phenotype between species may suggest differences in cerebellar development, function, and potentially the involvement of β-ΙΙΙ spectrin. Further understanding of canine cerebellar function would be required to shed light on the described differences and common principles.

It has been shown that heterozygous mice, generated by exon 2-6 replacement, do not display any characteristics of cerebellar ataxia [33], in common with heterozygous dogs in the Beagle population, suggesting that SCA5 in heterozygous humans is caused by dominant negative effects of mutant β-ΙΙΙ spectrin, rather than haploinsufficiency. Histopathologic examination in heterozygous mice revealed normal size and morphology of the cerebellum and immunostaining studies showed no changes on Purkinje cell morphology. These histopathological findings cannot be correlated with heterozygous Beagle dogs as none underwent post-mortem examination and all of them are currently alive and clinically unaffected. Interestingly, slight motor impairments were reported for heterozygous mice generated by pgeo insertion between exons 25 and 26, perhaps indicating that the truncated protein is having a slight dominant negative effect, and illustrates how disease progression is dependent on the positioning of SPTBN2 mutations.

Cerebellar cortical degeneration in dogs is an autosomal recessive condition. Thus the cerebellar cortical degeneration status may be selected from : clear of cerebellar cortical degeneration, affected by (i.e. having or likely to develop) cerebellar cortical degeneration, or a carrier of cerebellar cortical degeneration. The individual animal tested may or may not be entirely symptomless and\or may be considered to be at risk from cerebellar cortical degeneration (based on pedigree etc.) . In one embodiment, the canine mammal is a dog . In a further embodiment, the canine mammal is a dog which is a breed selected from the list consisting of : Beagle, Rhodesian Ridgeback, Samoyed and Irish Setter. In a yet further embodiment, the canine mammal is a Beagle. In one embodiment, the method of the invention comprises the steps of:

(i) providing a sample of nucleic acid from the canine mammal;

(ii) detecting genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2) in the nucleic acid sample; and

(iii) correlating the result from (ii) with the cerebellar cortical degeneration status of the canine mammal .

In one embodiment, the nucleic acid comprises genomic DNA.

The method of the invention may optionally comprise, in addition to detecting genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2), the assessment from the same sample of other markers which are linked or associated with other canine disorders. Thus, in one embodiment, the sample is assessed for one or more other markers which are linked or associated with canine disorders. In one aspect of the invention, the method may include the step of screening a canine mammal for its cerebellar cortical degeneration status as described herein, and if the animal is identified as a carrier, selecting it for breeding with an animal which is not a carrier of cerebellar cortical degeneration (i .e. is clear of cerebellar cortical degeneration and homozygous for the non-mutant, wild-type allele). The ability to identify carriers for breeding purposes is of great importance because cerebellar cortical degeneration is a debilitating disease which requires euthanasia at a young age. Thus, in one embodiment, the method of the invention additionally comprises the step of establishing whether or not the canine mammal is heterozygous or homozygous for the genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2) . It will be appreciated that if the canine mammal is homozygous for the genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2), it is diagnosed as a canine mammal suffering from cerebellar cortical degeneration .

It will be appreciated that ein if the canine mammal is heterozygous for the genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2), it is selected as being suitable for breeding with a canine mammal of the same breed which is homozygous for the wild-type β-ΙΙΙ spectrin gene (SPTBN2) .

It will be appreciated that if the canine mammal is homozygous for the wild-type β-ΙΙΙ spectrin gene (SPTBN2), it is selected as being suitable for breeding with a canine mammal of the same breed which is homozygous or heterozygous for the wild-type β-ΙΙΙ spectrin gene (SPTBN2) .

In one embodiment, the cerebellar cortical degeneration is neonatal cerebellar cortical degeneration (NCCD) .

Particular methods of detecting markers in nucleic acid samples are described in more detail hereinafter. Nucleic acid sample

The sample from the canine mammal may be prepared from any convenient sample, for example from blood or skin tissue. In one embodiment, DNA is extracted from blood or from buccal (cheek) cells on a swab. The DNA sample analysed may be all or part of the sample being obtained. Methods of the present invention may therefore include obtaining a sample of nucleic acid obtained from the canine mammal . Alternatively, the assessment of the β-ΙΙΙ spectrin gene (SPTBN2) may be performed or based on an historical DNA sample, or information already obtained therefrom e.g. by assessing the β- III spectrin gene (SPTBN2) in DNA sequences which are stored on a databank.

It will be appreciated that the assessment may be performed using mRNA (or cDNA), rather than genomic DNA.

Genetic Variations

It will be appreciated that the genetic variations include any variation in the native, non-mutant or wild type genetic code of the β-ΙΙΙ spectrin gene

(SPTBN2) from said canine mammal under analysis. Examples of such genetic variations include : mutations (e.g. point mutations), substitutions, deletions, single nucleotide polymorphisms (SNPs), haplotypes, chromosome

abnormalities, Copy Number Variation (CNV), epigenetics and DNA inversions. References herein to the term "single-nucleotide polymorphism (SNP)" is intended to refer to DNA sequence variation occurring when a single nucleotide in the β-ΙΙΙ spectrin gene (SPTBN2) differs between members of a species or between paired chromosomes in an individual.

In one embodiment the genetic variation is a functional mutation i.e. one which is causative of cerebellar cortical degeneration. Mutations may be functional in that they affect amino acid encoding, or by disruption of regulatory elements (e.g., which may regulate gene expression, or by disruption of sequences - which may be exonic or intronic - involved in regulation of splicing). However it will be appreciated that other markers showing association with cerebellar cortical degeneration, may also have diagnostic utility and could be used in combination with the assessment of the invention.

In one embodiment the genetic variation is an insertion mutation which causes a frameshift in the β-ΙΙΙ spectrin gene (SPTBN2). This may cause premature termination.

In one embodiment, the genetic variation is within exon 29 of the β-ΙΙΙ spectrin gene (SPTBN2). In one particular embodiment, the genetic variation comprises a deletion mutation within the β-ΙΙΙ spectrin gene (SPTBN2). In a further embodiment, the deletion mutation comprises an 8 bp deletion between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (as identified in the current whole genome sequence assembly (CanFam 2.0 : http://www.ensembl .org/Canis fami Maris/).

In one embodiment, the primers used to detect the β-ΙΙΙ spectrin gene (SPTBN2) comprise:

Forward : 5'-TACTGGACACCACGGACAAGT-3' (SEQ ID NO : 1); and

Reverse: 5 '- G G C CTCTATCTCTG C CTTG AT- 3 ' (SEQ ID NO : 2).

These give fragment amplification of:

Normal, non-mutant, wild-type = 268bp

Mutant = 260bp

In one embodiment, assessment of the β-ΙΙΙ spectrin gene (SPTBN2) will establish whether or not the individual animal is heterozygous or homozygous for the specific length variant in this region.

Accordingly, in one embodiment the method of the present invention comprises detecting genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2) in a genomic DNA sample obtained from the canine mammal as described above e.g. a deletion mutation between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (as identified in the current whole genome sequence assembly (CanFam 2.0 : http://www.ensembl .org/Canis fami Maris/). Use of other polymorphisms

The genetic variation may be one which is in linkage disequilibrium with the hereinbefore mentioned deletion mutation - this may for example be a microsatellite repeat polymorphism or a single nucleotide polymorphism (SN P), which may be in an intron, exon or promoter sequence of the β-ΙΙΙ spectrin gene (SPTBN2), or located sufficiently close to the β-ΙΙΙ spectrin gene (SPTBN2) to be in linkage disequilibrium with the mutation .

In one embodiment, any such polymorphism will be a common polymorphism (allele frequency >0.05) . As is understood by the person skilled in the art, linkage disequilibrium is the non-random association of alleles. Further details may be found in Kruglyak (1999) Nature Genetics, Vol 22, page 139 and Boehnke (2001) Nature Genetics 25 : 246-247). For example, results of recent studies indicate significant linkage disequilibrium may extend to around 2 M B depending on the breed of dog (400-700 kb in Golden Retriever and Labrador Retriever, 2.4 Mb in Akita, and 3-3.2 Mb in Bernese Mountain Dog and Pekingese - see

http ://www.ncbi . nlm . nih .gov/entrez/query.fcgi?cmd = Retrieve&db=pubmed&dop t=Abstract&l ist_uids= 15545498&query_hl =4) . Thus genetic variations which are proximal to the β-ΙΙΙ spectrin gene (SPTBN2) and in linkage disequilibrium with the hereinbefore mentioned deletion mutation are also within the scope of the invention .

A region which is described as 'proximal' or 'sufficiently close' to a polymorphic marker may be within about 3000kb, 2000kb or lOOOkb of the β-ΙΙΙ spectrin gene (SPTBN2), preferably within about 500kb away, and more preferably within about lOOkb, more preferably within 50 kb, more preferably within 10 kb of the β-ΙΙΙ spectrin gene (SPTBN2) . For these other genetic variations (e.g . SN P or microsatellite polymorphisms), the method will generally involve determining the identity of a nucleotide or nucleotides at the position of said polymorphism . In one embodiment, assessment of the SN Ps at the positions described above will establish whether or not the individual is heterozygous or homozygous for the allele at these sites.

Materials

The invention further provides oligonucleotides for use in probing or amplification reactions, which may be fragments of the β-ΙΙΙ spectrin gene (SPTBN2) . Nucleic acid for use in the methods of the present invention, such as an oligonucleotide probe and/or pair of amplification primers, may be provided in isolated form and may be part of a kit, e.g . in a suitable container such as a vial in which the contents are protected from the external environment. The kit may include instructions for use of the nucleic acid, e.g . in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample. A kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleotides, buffer solution etc. The nucleic acid may be labelled. A kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g . a swab for removing cells from the buccal cavity or a syringe for removing a blood sample (such components generally being sterile) .

The various embodiments of the invention described above may also apply to the following : a diagnostic means for determining the cerebellar cortical degeneration status of a canine mammal; a diagnostic kit comprising such a diagnostic means; and the use, in the manufacture of means for assessing the cerebellar cortical degeneration status of a canine mammal of sequences (e.g ., PCR primers) to amplify a region of the β-ΙΙΙ spectrin gene (SPTBN2) as described herein .

Therapy

According to a further aspect the invention there is provided a method of treating cerebellar cortical degeneration in a canine mammal, which method comprises assessing the cerebellar cortical degeneration status of a canine mammal by use of a method as defined herein and if the canine mammal is identified as affected by cerebellar cortical degeneration, treating said canine mammal to prevent or reduce the onset of cerebellar cortical degeneration .

Gene replacement therapy

As noted above the present inventors have identified a mutation in exon 29 of the β-ΙΙΙ spectrin gene (SPTBN2) in the DNA which changes the reading frame of the DNA, in turn introducing a 'premature stop codon' which causes the protein to be prematurely terminated.

Thus, according to a further aspect of the invention, there is provided a method of treating cerebellar cortical degeneration in a canine mammal, which method comprises increasing the level of non-mutant, wild-type β-ΙΙΙ spectrin gene (SPTBN2) expression and/or β-ΙΙΙ spectrin gene (SPTBN2) product activity in the canine mammal.

Normal (i.e. non-mutant) β-ΙΙΙ spectrin gene (SPTBN2) nucleic acid sequences described above can, for example, be utilized for the treatment of cerebellar cortical degeneration. Such treatment can be administered, for example, in the form of gene replacement therapy. Specifically, one or more copies of a normal β-ΙΙΙ spectrin gene (SPTBN2) or a portion of the β-ΙΙΙ spectrin gene (SPTBN2) that directs the production of a β-ΙΙΙ spectrin gene (SPTBN2) product exhibiting normal β-ΙΙΙ spectrin gene (SPTBN2) function, may be inserted into the appropriate cells within a canine mammal in need of the same, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.

Because the β-ΙΙΙ spectrin gene (SPTBN2) is expressed inter alia in the nervous system and the highest levels of expression are found in Purkinje cell soma and dendrites, such gene replacement therapy techniques should be capable of delivering β-ΙΙΙ spectrin gene (SPTBN2) sequences to this cell type. Also included are methods using liposomes either in vivo, ex vivo or in vitro wherein β-ΙΙΙ spectrin gene (SPTBN2) DNA is delivered to the cytoplasm and nucleus of target cells. In another embodiment, techniques for delivery involve direct administration of such β-ΙΙΙ spectrin gene (SPTBN2) sequences to the site of the cells in which the β-ΙΙΙ spectrin gene (SPTBN2) sequences are to be expressed. Additional methods that may be utilized to increase the overall level of β-ΙΙΙ spectrin gene (SPTBN2) expression and/or β-ΙΙΙ spectrin gene (SPTBN2) product activity include the introduction of appropriate β-ΙΙΙ spectrin gene (S TB/V2)-expressing cells, preferably autologous cells, into the canine mammal at positions and in numbers that are sufficient to ameliorate the symptoms of cerebellar cortical degeneration . Such cells may be either recombinant or non-recombinant. The expression of the β-ΙΙΙ spectrin gene (SPTBN2) sequences is controlled by the appropriate gene regulatory sequences to allow such expression in the necessary cell types. Such gene regulatory sequences are well known to the skilled artisan . Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g ., Anderson, U.S. Pat. No. 5,399,349.

When the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system .

Thus, for example, the invention provides a method of gene therapy one or more copies of a nucleic acid sequence as described herein (e.g. non-mutant β-ΙΙΙ spectrin gene (SPTBN2) or an active variant thereof) may be inserted into the appropriate cells within the canine mammal, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Example gene therapy vectors for use in the method of this invention include retroviral or episomal vectors expressing particular desired genes under the control of the promoter and/or the supplemental control sequences disclosed herein (see, e.g ., Axel, et al ., U .S. Pat. No. 4,399,216, and Pastan, et al ., U .S . Pat. No. 5, 166,059, both incorporated herein by reference) . Delivery systems as contemplated herein include both viral and liposomal delivery systems (see, e.g., Davis, et al ., U .S. Pat. No. 4,920,209, incorporated herein by reference). Such gene therapy vectors may incorporate targeting signals to the appropriate membrane or organ . Alternatively, or additionally cell or organelle specific promoters may be used. The invention also provides such vectors and DNA molecules for use in a method of treatment of cerebellar cortical degeneration in a canine mammal. The invention further provides use of such DNA molecules in the preparation of a medicament, for example for the treatment of a canine mammal .

Assessment of Genetic Variation

Methods for detecting or assessing genetic variations are reviewed by Schafer and Hawkins, (Nature Biotechnology (1998)16, 33-39, and references referred to therein) and include: allele specific oligonucleotide probing, amplification using PCR, denaturing gradient gel electrophoresis, RNase cleavage, chemical cleavage of mismatch, T4 endonuclease VII cleavage, multiphoton detection, cleavase fragment length polymorphism, E. coli mismatch repair enzymes, denaturing high performance liquid chromatography, (MALDI-TOF) mass spectrometry, analysing the melting characteristics for double stranded DNA fragments as described by Akey et al (2001) Biotechniques 30; 358-367. These references, inasmuch as they may be used in the performance of the present invention by those skilled in the art, are specifically incorporated herein by reference.

The assessment of the genetic variation may be carried out on a DNA microchip, if appropriate. One example of such a microchip-system may involve the synthesis of microarrays of oligonucleotides on a glass support. Fluorescently - labelled PCR products may then be hybridised to the oligonucleotide array and sequence specific hybridisation may be detected by scanning confocal microscopy and analysed automatically (see Marshall & Hodgson (1998) Nature Biotechnology 16 : 27-31 , for a review). Some preferred examples of such methods will now be discussed in more detail .

Use of nucleic acid probes

The method of detecting or assessing the genetic variation may comprise determining the binding of an oligonucleotide probe to the nucleic acid sample. Thus, in one embodiment, the detection step is performed by determining the binding of oligonucleotide probes to the nucleic acid sample, wherein the probes comprise all or part of the wild-type or mutant β-ΙΙΙ spectrin gene (SPTBN2) . The probe may comprise a nucleic acid sequence which binds specifically to a particular allele of a polymorphism and does not bind specifically to other alleles of the polymorphism . Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single- stranded DNA. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined .

Binding of a probe to target nucleic acid (e.g . DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled.

Polymorphisms may be detected by contacting the sample with one or more labelled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof under conditions favorable for the specific annealing of these reagents to their complementary sequences within the relevant gene.

As is understood by those skilled in the art, a 'complement' or 'complementary' or 'reverse complement' sequence (the terms are equivalent) is one which is the same length as a reference sequence, but is 100% complementary thereto whereby by each nucleotide is base paired to its counterpart running in anti- parallel fashion i .e. G to C, and A to T or U . In one embodiment, the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed from the nucleic acid :gene hybrid. The presence of nucleic acids that have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtitre plate or polystyrene beads. In this case, after incubation, non- annealed, labeled nucleic acid reagents are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal gene sequence in order to determine whether a gene mutation is present.

Approaches which rely on hybridisation between a probe and test nucleic acid and subsequent detection of a mismatch may be employed. Under appropriate conditions (temperature, pH etc.), an oligonucleotide probe will hybridise with a sequence which is not entirely complementary. The degree of base-pairing between the two molecules will be sufficient for them to anneal despite a mismatch. Various approaches are well known in the art for detecting the presence of a mis-match between two annealing nucleic acid molecules. For instance, RN'ase A cleaves at the site of a mis-match. Cleavage can be detected by electrophoresing test nucleic acid to which the relevant probe or probe has annealed and looking for smaller molecules (i.e. molecules with higher electrophoretic mobility) than the full length probe/test hybrid. Other approaches rely on the use of enzymes such as resolvases or endonucleases.

Thus, an oligonucleotide probe that has the sequence of a region of the normal gene (either sense or anti-sense strand) in which polymorphisms associated with the trait of interest are known to occur may be annealed to test nucleic acid and the presence or absence of a mis-match determined. Detection of the presence of a mis-match may indicate the presence in the test nucleic acid of a mutation associated with the trait. On the other hand, an oligonucleotide probe that has the sequence of a region of the gene including a mutation associated with disease resistance may be annealed to test nucleic acid and the presence or absence of a mis-match determined. The presence of a mismatch may indicate that the nucleic acid in the test sample has the normal sequence, or a different mutant or allele sequence. In either case, a battery of probes to different regions of the gene may be employed. As discussed above, suitable probes may comprise all or part of the β-ΙΙΙ spectrin gene (SPTBN2) sequence (or reverse complement thereof), or all or part of a mutant form of the sequence (or reverse complement thereof ). The mutant form may contain one or more of the genetic variations described herein. Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.

Suitable selective hybridisation conditions for oligonucleotides of 17 to 30 bases include hybridization overnight at 42°C in 6X SSC and washing in 6X SSC at a series of increasing temperatures from 42°C to 65°C. One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989) : T_m = 81.5°C + 16.6Log [Na⁺] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex.

Other suitable conditions and protocols are described in Molecular Cloning : a Laboratory Manual : 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press and Current Protocols in Molecular Biology, Ausubel et al . eds., John Wiley & Sons, 1992.

Amplification-based methods The hybridisation of such a probe may be part of a PCR or other amplification procedure. Accordingly, in one embodiment the detection step is performed by amplifying all or part of the β-ΙΙΙ spectrin gene (SPTBN2) . The assessment of the genetic variation in the amplification product may then be carried out by any suitable method, e.g., as described herein . An example of such a method is a combination of PCR and low stringency hybridisation with a suitable probe. Unless stated otherwise, the methods of assessing the genetic variation described herein may be performed on a genomic DNA sample, or on an amplification product thereof.

Where the method involves PCR, or other amplification procedure, any suitable β-ΙΙΙ spectrin gene (SPTBN2) PCR primers flanking the marker of interest may be used .

In one embodiment, the amplified region which the primers flank is less than 500 nucleotides, such as less than 300 nucleotides, in particular 50 to 300 (e.g. 268) nucleotides in length . In one embodiment, the detection step is performed by amplifying all or part of exon 29 of the β-ΙΙΙ spectrin gene (SPTBN2), such as between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (CanFam 2.0) .

In a further embodiment, the detection step is performed by use of primers which flank or include part of the region defined between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (CanFam 2.0) .

An oligonucleotide for use in nucleic acid amplification may be about 30 or fewer nucleotides. Generally specific primers are upwards of 14 nucleotides in length, but are suitably 15-25 inclusive, more preferably 18-20. Those skilled in the art are well versed in the design of primers for use processes such as PCR. Various techniques for synthesizing oligonucleotide primers are well known in the art, including phosphotriester and phosphodiester synthesis methods. Suitable polymerase chain reaction (PCR) methods are reviewed, for instance, in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant. Biol ., 51 : 263, (1987), Ehrlich (ed), PCR technology, Stockton Press, NY, 1989, and Ehrlich et al, Science, 252 : 1643-1650, (1991)). PCR comprises steps of denaturation of template nucleic acid (if double- stranded), annealing of primer to target, and polymerisation. An amplification method may be a method other than PCR. Such methods include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction, rolling circle amplification and ligation activated transcription. For convenience, and because it is generally preferred, the term PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.

In one embodiment, "Amplified Fragment Length Polymorphism" (AFLP) may be carried out using primers devised on the basis of the sequences disclosed herein. Analysis of the products can be carried out using e.g. by gel electrophoresis, capillary electrophoresis.

In one embodiment, described in the Examples hereinafter, the region of DNA that contains the mutation is amplified using PCR and the length of the resulting fragment of DNA is measured.

Examples of results from the genotyping assay are shown below. Sequencing

The genetic variation may be assessed or confirmed by nucleotide sequencing of a nucleic acid sample to determine the presence of the genetic variation. The identity may be determined by comparison of the nucleotide sequence obtained with the native, non-mutant, wild-type sequence. Nucleotide sequence analysis may be performed on a genomic DNA sample, or amplified part thereof, or RNA sample as appropriate, using methods which are standard in the art.

Where an amplified part of the genomic DNA sample is used, the genomic DNA sample may be subjected to a PCR amplification reaction using a pair of suitable primers. In this way the region containing a particular polymorphism or polymorphisms may be selectively amplified (PCR methods and primers are discussed in more detail above). The nucleotide sequence of the amplification product may then be determined by standard techniques.

Other techniques which may be used are single base extension techniques and pyrosequencing.

Mobility based methods

The assessment of the genetic variation may be performed by single strand conformation polymorphism analysis (SSCP). In this technique, PCR products from the region to be tested are heat denatured and rapidly cooled to avoid the reassociation of complementary strands. The single strands then form sequence dependent conformations that influence gel mobility. The different mobilities can then be analysed by gel electrophoresis.

Assessment may be by heteroduplex analysis. In this analysis, the DNA sequence to be tested is amplified, denatured and renatured to itself or to known wild-type DNA. Heteroduplexes between different alleles contain DNA "bubbles" at mismatched basepairs that can affect mobility through a gel. Therefore, the mobility on a gel indicates the presence of sequence alterations. Restriction site based methods

Where an SNP creates or abolishes a restriction site, the assessment may be made using RFLP analysis. In this analysis, the DNA is mixed with the relevant restriction enzyme (i.e., the enzyme whose restriction site is created or abolished). The resultant DNA is resolved by gel electrophoresis to distinguish between DNA samples having the restriction site, which will be cut at that site, and DNA without that restriction site, which will not be cut.

Where the SNP does not create or abolish a restriction site the SNP may be assessed in the following way. A mutant PCR primer may be designed which introduces a mutation into the amplification product, such that a restriction site is created when one of the polymorphic variants is present but not when another polymorphic variant is present. After PCR amplification using this primer (and another suitable primer), the amplification product is admixed with the relevant restriction enzyme and the resultant DNA analysed by gel electrophoresis to test for digestion.

The following studies illustrate the invention : Methods

Clinical examinations and sample collection

Three affected, full-sibling dogs from two different litters were clinically evaluated. Two dogs belonged to a first litter of seven puppies and the third affected dog belonged to a second litter of seven puppies. The affected dog from the second litter was examined at the Animal Health Trust (AHT) and the medical records and video footages of the two affected puppies from the first litter were obtained from the breeder and reviewed. The affected puppy from the second litter underwent post-mortem examination and cerebellar tissue was preserved in RNAIater (Life Technologies) for RNA extraction. Paraffin-embedded sections of cerebellum of one of the two affected puppies belonging to the first litter were available for histopathological studies. The affected puppy and its six siblings from the second litter, the dam and the sire underwent complete physical and neurological examination. All DNA samples used for SPTBN2 mutation frequency analysis in the Beagle and other breeds had been recruited for independent investigations, and were collected from pet dogs by either buccal swabbing or from residual blood drawn as part of a veterinary procedure.

Pathologic Study A full post-mortem was performed and the brain preserved in 10 % buffered formalin for 10 days prior to processing. Upon fixation the cerebellum was transected longitudinally along the midline. The left 'hemisphere' was serial transected along the longitudinal axis (rostral-dorsal); the right 'hemisphere' was serial sectioned along the horizontal axis (medial-lateral). Cerebral serial transverse sections were taken.

Tissues were processed routinely on the Shandon Excelsior ES. Paraffin- embedded tissues were sectioned at 4-6 μηη . Slides were stained on the Shandon Linistainer with Mayer's hematoxylin and 1 % alcoholic eosin. Previous slides, and their associated paraffin-embedded blocks, from the full-sibling clinically affected female puppy were retrieved for comparison and review.

Nucleic acid preparation

Genomic DNA was extracted from whole blood samples preserved in EDTA using the Nucleon BACC2 kit (Tepnel Life Science), from buccal swabs using the QiaAmp Midi kit (Qiagen), and from formalin fixed paraffin embedded (FFPE) tissue using the Nucleospin FFPE DNA kit (Macherey Nagel). RNA was extracted using the Qiagen RNeasy Midi kit (Qiagen), including the on column DNase treatment stage. mRNA was isolated from 4.9

total RNA using Sera-Mag oligo(dt) particles (Thermo Fisher) and Sera-Mag mRNA isolation buffer kit (Thermo Fisher). mRNA-seq

Libraries were prepared using the N EBNext® mRNA Sample Prep Master Mix Set 1, consisting of RNA fragmentation, first strand cDNA synthesis, second strand synthesis, end repair, dA tailing, and PCR amplification modules. Fragmented RNA was purified using the Qiagen RNeasy mini kit (Qiagen). Reverse transcription of RNA fragments was performed using Superscript II Reverse Transcriptase (Life Technologies). Clean-up after the second strand synthesis, end repair, dA tailing, and PCR amplification modules was performed using the QIAquick PCR purification mini kit (Qiagen). The adaptor ligated library was size selected by band excision after agarose gel electrophoresis, and purified using the QIAquick gel extraction kit (Qiagen) before PCR amplification, using primers for Illumina paired-end multiplexed sequencing. The final mRNA-seq library was quantified by qPCR using the Kapa library quantification kit (Kapa Biosystems). Paired-end sequencing (51 bp reads) was carried out on a partial lane of an Illumina HiSeq2000, producing a 1.39 Gb dataset. Reads were aligned to the canine reference genome (CanFam 2.0) using BWA [21] . Quality scores were recalculated using GATK [22] . Aligned reads were viewed using The Integrative Genomics Viewer (IGV) [23] .

Polymerase chain reaction, genotyping and capillary sequencing

Polymerase chain reaction (PCR) for capillary sequencing was performed in 6 μΙ reactions consisting of 0.2 mM dNTPs (N EB), lx PCR buffer (Qiagen), 0.83 μΜ forward primer (5'-TACTGGACACCACGGACAAGT-3' (SEQ ID NO : 1)), 0.83 μΜ reverse primer (GGCAGAGACGTGAGTTAGCAC; (SEQ ID NO : 3)), 0.5 units HotStarTaq Plus DNA polymerase (Qiagen) and ultrapure water, with an expected product size of 578 bp. Cycling parameters for PCR were 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds, and completed with a final elongation stage of 72°C for 10 minutes. Reactions used for fragment analysis on ABI3130xl genetics analysers included 0.12 μΜ R110 labelled dUTP and used an alternative reverse primer (5'- G G C CTCTATCTCTG C CTTG AT- 3 ' (SEQ ID NO : 2)), for an expected product size of 268 bp. Genotyping data was analysed using Genemapper software (Applied Biosystems). 578 bp PCR products were Sanger sequenced using Big Dye v3.1 (Applied Biosystems) for capillary electrophoresis on an ABI3130xl genetic analyser. Sequencing data were analysed using Gap4 (Staden package) [24]. All primers were designed using Primer3 [25] and manufactured by IDT.

Quantitative PCR (qPCR) assays were carried out on an Illumina Eco machine in 10 μΙ reactions containing 5 μΙ Kapa Probe Fast qPCR mastermix (Kapa Biosystems), 1 x IDT PrimeTime qPCR assay mix and 2 μΙ cDNA (primer sequences listed in Table 1) :

Table 1: Primers Used for qPCR assays of the SPTBN2 gene

The probe was 5'6-FAM and 3' Iowa black labelled, with internal ZEN labelling Primer „

Assay name ,. Sequence

Type

SPTBN2 Forward TGGATGGTGAAGAGCAGAAC (SEQ ID NO : 4)

SPTBN2 Reverse TTCTCAGTGAACTTGGGTGGCTTCTC (SEQ ID NO : 5)

SPTBN2 Probe TCCTTCAATTCCTATCGCACG (SEQ ID NO : 6)

Reaction efficiencies were calculated using a seven point 2 x serial dilution to create a standard curve. The efficiency for the SPTBN2 assay was estimated at 99.3 %, with a standard curve r² value of > 0.995. Assays for ACTB and TBP genes were used as controls.

Immunoblotting

Canine cerebellum samples (~30 mg) were homogenised in 1 ml ice cold RIPA lysis buffer (Sigma-Aldrich), containing one complete protease inhibitor cocktail tablet per 10 ml (Roche). Protein concentrations were measured using a Qubit fluorometer (Invitrogen). Protein samples were separated by denaturing 6 % SDS-PAGE (National Diagnostics). Separated proteins were transferred to a nitrocellulose membrane, which was blocked for 16 hours with 5 % non-fat dried milk in phosphate-buffered saline/0.1% Tween 20 (PBS-T). Blocked nitrocellulose membranes were incubated for one hour in 1 : 200 goat anti- SPTBN2 (Santa Cruz Biotechnology) or 1 : 1000 mouse ant\-ACTB (Camlab) primary antibody in blocking buffer. After washing in PBS-T, blots were incubated in 1 : 10,000 HRP-conjugated donkey anti-goat or 1 : 1000 HRP- conjugated goat anti-mouse secondary antibody in blocking buffer. Immunoreactive proteins were detected using HRP-conjugate substrate kit for enhanced chemiluminescence.

Results

Clinical investigations

A four week-old male beagle puppy presented to the AHT with ten-day history of severe cerebellar ataxia. The dog was the only affected one from a litter of seven puppies. The breeder noticed that the affected puppy was not able to ambulate normally from the onset of walking and the clinical signs had remained stable since then. The puppy was otherwise eating and drinking well and there were no signs of systemic illness in the littermates, in the dam (also during gestation) or in the sire. Physical examination did not reveal any gross abnormalities apart from the neurological signs. Neurological examination revealed severe cerebellar ataxia, with tendency to lean and fall towards both sides, resulting in inability to walk without assistance. Proprioceptive positioning was normal while hopping reactions were abnormal with delayed onset of protraction and exaggerated response, once initiated. Spinal reflexes were normal in all four limbs. Cranial nerve examination revealed an absent menace response bilaterally with normal vision. Occasionally when the head was positioned in extension spontaneous rotatory nystagmus was observed. A lesion involving mainly the cerebellum and spinocerebellar tracts was suspected. The main differential diagnoses included degenerative central nervous system disease, such as neonatal onset of cerebellar cortical degeneration and less likely inflammatory/infectious central nervous system disease, metabolic disease and neoplasia. Haematology and comprehensive biochemistry did not reveal any significant abnormalities for a four week old puppy. Brainstem auditory evoked responses identified clear waves I to V. Based on the severity of the clinical signs, normal haematology and comprehensive biochemistry, a degenerative condition was considered the most likely underlying cause and the breeder elected euthanasia. Post-mortem examination was performed an hour after euthanasia, and failed to reveal gross pathology. The brain in toto weighed 42 g, whilst the cerebellum weighed 5 g (12 %, normal 10-12 %) [2] . Narrowing of folia was not noted.

Physical and neurological examination of the dam, sire and six clinically unaffected littermates were performed at the AHT and did not reveal any abnormalities. The only previous litter from the same dam and sire consisted of seven puppies, of which two (one female and one male) had clinical signs consistent with NCCD and the remainder were clinically normal based on clinical history and video footages provided by the breeder when the puppies were eight weeks old. Both clinically affected puppies were euthanized at eight weeks of age and paraffin- embedded sections of cerebellum from the female were available for histopathological examination.

Histopathological examination results

Histopathologically, the lesions were confined to the cerebellum. Examination of serial cerebellar sections of the four week old puppy identified mild loss of Purkinje cells, with corresponding increased numbers of astrocytes. Moderate numbers of Purkinje cells were shrunken with angular cell margins, hypereosinophilic cytoplasm, and condensed nuclei (Figure 1A). Occasional associated swollen dendritic processes were identified. Spheroids were rarely seen. Mild spongiosis was present at the granular cell layer - Purkinje cell interface Bieslcowsky fiber stain was performed and demonstrated the subacute loss of Purkinje cells, also called "empty baskets" (Figure 2). Spatial characterisation after calbindin-immunohistochemistry and haematoxylin counterstain was also performed (Figure 3).

Examination of slides made from the paraffin embedded tissues of the eight week old female pup from the previous litter identified marked Purkinje cell loss with rare remaining, often abnormal, Purkinje cells (Figure I B). Variably swollen or shrunken, hypereosinophilic Purkinje cells were identified within the remaining population. Correlating astrocytosis replaced previously lost Purkinje cells. Swollen dendritic processes and small numbers of spheroids were present. There was moderate to marked thinning of the subjacent granular layer. Cerebellar nuclei in both puppies were normal. Neuronal storage products were not identified in either puppy.

Genetic investigations

Of a total of 14 dogs from the same sire and dam mating, 3 dogs (2 male and 1 female) were affected by NCCD, which is consistent with an autosomal recessive mode of inheritance.

A review of the scientific literature and the Online Mendelian Inheritance in Man (OMIM) database indicated 41 human ataxia loci have been identified for which 28 causal genes have been characterised. Twenty seven of the genes causing human ataxia have orthologous canine genes, and these genes were considered as candidates. The mRNA-seq experiment using cerebellum tissue from a single NCCD case produced a 1.39 Gb dataset, which has been submitted to the NBCI Sequence Reads Archive (Accession number SRA051411). The dataset consisted of 13.64 million reads, with 97.1 % of the reads mapping to the dog genome. The dataset was sufficient for complete exonic coverage of 24 of the 27 candidate genes when aligned to the CanFam 2.0 genome build. No polymorphisms were identified in 11 of the genes. Three genes contained polymorphisms in non-coding regions only. Heterozygous SN Ps were identified in four genes, excluding association with NCCD. Two genes contained synonymous SN Ps. Non-synonymous changes were identified in ITPR1 (chr20 : 15,780,361G>C;p. E2491Q), BEAN1 (chr5 :85,782,181C>T;p. R247Q), and ADCK3 (chr7 :41,059,467A>G;p.S328P). For ITPR1 and ADCK3 the non- reference residue is highly conserved amongst vertebrate species and therefore could be ruled out as causal. Alignment across vertebrate species at the site of the BEAN1 polymorphism indicates that both glutamic acid and glutamine residues occur naturally, enabling that variant to also be excluded. An 8 bp deletion was detected in exon 29 of SPTBN2, chrl8 : 53,691,704_53,691,711del, and is shown in Figure 4A. The deletion was confirmed by Sanger sequencing (Figure 4B). The frameshift is predicted to result in a run of 27 aberrant amino acids, followed by premature termination with a 410 amino acid truncation p. G1952i nsRDRGQGRPLLLMHRHGAGAACQEPLCS*. The mutation is located in the 16^th of 17 spectrin repeat domains located in β-ΙΙΙ spectrin (Figure 5). Genotyping experiments were performed to establish whether the 8 bp SPTBN2 deletion could be potentially causal . The sire and dam of the affected dogs were both heterozygous for the 8 bp deletion, and out of the ten clinically unaffected siblings tested, seven were heterozygous and three were homozygous for the wild-type allele. DNA extracted from FFPE tissue of a previous NCCD case from the same sire and dam mating genotyped homozygous for the deletion. Seven other clinically unaffected half-siblings, with the same sire as the affected dogs, were either heterozygous or wild-type homozygous. An extended pedigree is shown in Figure 6. An additional 145 Beagles, which were collected for an unrelated project and clinically normal with respect to NCCD, were also genotyped. Eight dogs were heterozygous for the deletion, and the remaining 137 dogs were homozygous wild-type, in full concordance with the mutation being causal. In addition 513 dogs from 37 other breeds were also genotyped; all were homozygous for the wild-type allele.

Limited qPCR experiments to assess the expression levels of SPTBN2 in affected and normal cerebellum tissues suggested a 68 fold reduction in SPTBN2 transcript levels in the affected Beagle (data shown in Table 2). Table 2: Relative Expression Analysis Data

Expression levels of SPTBN2 were measured relative to ACTB and TBP using qPCR. Fold change was calculated based on changes in threshold cycle (Ct) measurements within (ACt) and between (ΔΔΟ:) the case and control.

Using Western blot analysis with primary antibodies targeting the N-terminal region of β-ΙΙΙ spectrin, no full length or truncated β-ΙΙΙ spectrin could be detected in cerebellum tissue of the NCCD case, suggesting expression of the protein may have been abolished (Figure 7).

Discussion

In this study we have described NCCD in three Beagle dogs and report the use of genome-wide mRNA sequencing of a single case to identify an 8 bp deletion in the β-ΙΙΙ spectrin gene, SPTBN2, that segregates consistently with the disorder. Initial clinical presentation and progression of the three affected Beagle puppies were highly suggestive of NCCD, with histopathological studies confirming Purkinje cell degeneration and necrosis in different stages according to disease progression in the two puppies that underwent necropsy. Histologically both the four-week old puppy and eight-week old puppy revealed Purkinje cell degeneration and loss. Lesions present in the eight-week old puppy demonstrated disease progression expected with the prolonged survival time, with increased proportions of lost Purkinje cells, and the presence of corresponding degeneration of the granular cell layer. Changes in both puppies were limited to Purkinje cell death and secondary changes associated with the loss of Purkinje cells (ie astrocytosis, granular cell degeneration, axonal swelling). Regional variation, as previously described by Yasuba et al, was not identified [10], but all the affected Beagles in that study were euthanized at 14 weeks of age, and the regional variation could just be related with the progression of the disease.

The identification of an 8 bp deletion in SPTBN2, a gene associated with spinocerebellar type 5 (SCA5) in humans [26], that fully segregates with the disease provides a strong candidate variant for NCCD in the Beagle.

The 8 bp deletion in the dog is located at a tandem repeat sequence, suggesting homologous recombination as the deletion mechanism. The position of a SNP (c.5580T>C) 18 bp downstream of the deleted sequence removes a possible termination site for the mutant protein and extends the sequence of potential aberrant amino acids from 6 to 27. Although expression analysis was limited, due to the availability of only one case and one control, results are suggestive of a 68 x reduction in the relative levels of SPTBN2 in the NCCD case cerebellum, which may be due to nonsense mediated decay. Even though SPTBN2 expression is greatly reduced in NCCD affected cerebellum tissue, sufficient read depth from the mRNA-seq expression was still achieved, because of the high levels of SPTBN2 expression normally seen in cerebellum tissue. Further to a reduction in mRNA levels, no full length or truncated β-ΙΙΙ spectrin was detectable in NCCD affected cerebellum tissue by Western blot analysis. This may indicate that the 8 bp deletion results in a full knock-out of SPTBN2. A full gene knock-out eliminates the possibility of a dominant negative effect that could be caused by a truncated form of the β-ΙΙΙ spectrin protein, which is in agreement with heterozygous dogs showing no clinical signs. Although NCCD is likely to be heterogeneous in different canine breeds, screening for the SPTBN2 deletion in non-beagle cases has not been investigated to confirm this. It is possible that the mutation could exist at very low frequencies in other breed populations, especially those closely related to the beagle, but extensive screening of large numbers of individuals would be required to fully investigate this possibility.

Conclusion

This study demonstrated that a spontaneous canine model of spinocerebellar ataxia type 5 is caused by a mutation in SPTBN2, the gene encoding β-ΙΙΙ spectrin, which offers an invaluable opportunity for further understanding of the disease pathogenesis and treatment. The Beagle is a naturally occurring, animal model for SCA5 and may help to increase understanding of disease progression in humans. This study also shows that mRNA-seq sequencing is a feasible method to identify mutations associated with rare diseases when a suitable tissue resource is available. The genetic assessment of the invention therefore provides the significant advantage of identifying carriers and to eradicate the disease from the breed.

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Claims

1. β-ΙΙΙ spectrin gene (SPTBN2) for use as a biomarker for the in vitro diagnosis of cerebellar cortical degeneration in a canine mammal.

2. An in vitro method of assessing the cerebellar cortical degeneration status in a canine mammal, the method comprising the step of detecting genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2).

3. The method as defined in claim 2, wherein the canine mammal is a dog which is a breed selected from the list consisting of: Beagle, Rhodesian Ridgeback, Samoyed and Irish Setter.

4. The method as defined in claim 3, wherein the canine mammal is a Beagle.

5. The method as defined in any of claims 2 to 4, which comprises the steps of:

(i) providing a sample of nucleic acid from the canine mammal;

(iii) correlating the result from (ii) with the cerebellar cortical degeneration status of the canine mammal.

6. The method as defined in claim 5, wherein the nucleic acid is genomic DNA.

7. The method as defined in any one of claims 2 to 6, wherein the cerebellar cortical degeneration is neonatal cerebellar cortical degeneration (NCCD).

8. The method as defined in any one of claims 2 to 7, wherein the genetic variation is within exon 29 of the β-ΙΙΙ spectrin gene (SPTBN2).

9. The method as defined in any one of claims 2 to 8, wherein the genetic variation comprises a deletion mutation within the β-ΙΙΙ spectrin gene (SPTBN2) .

10. The method as defined in claim 9, wherein the deletion mutation comprises an 8 bp deletion between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (CanFam 2.0) .

11. The method as defined in any one of claims 2 to 10, which additionally comprises the step of establishing whether or not the canine mammal is heterozygous or homozygous for the genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2) .

12. The method as defined in claim 11, wherein if the canine mammal is homozygous for the genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2), it is diagnosed as a canine mammal suffering from cerebellar cortical degeneration .

13. The method as defined in claim 11, wherein if the canine mammal is heterozygous for the genetic variation within the β-ΙΙΙ spectrin gene (SPTBN2), it is selected as being suitable for breeding with a canine mammal of the same breed which is homozygous for the wild-type β-ΙΙΙ spectrin gene (SPTBN2) .

14. The method as defined in claim 11, wherein if the canine mammal is homozygous for the wild-type β-ΙΙΙ spectrin gene (SPTBN2), it is selected as being suitable for breeding with a canine mammal of the same breed which is homozygous or heterozygous for the wild-type β-ΙΙΙ spectrin gene (SPTBN2) .

15. The method as defined in any one of claims 2 to 14, wherein the sample is assessed for one or more other markers which are linked or associated with canine disorders.

16. The method as defined in any one of claims 2 to 15, wherein the detection step is performed by determining the binding of oligonucleotide probes to the nucleic acid sample, wherein the probes comprise all or part of the wild-type or mutant β-ΙΙΙ spectrin gene (SPTBN2) .

17. The method as defined in any one of claims 2 to 16, wherein the detection step is performed by amplifying all or part of the β-ΙΙΙ spectrin gene

(SPTBN2) .

18. The method as defined in claim 17, wherein the amplified region is less than 500 nucleotides in length, such as less than 300 nucleotides in length, in particular 50 to 300 nucleotides in length .

19. The method as defined in claim 17 or claim 18, wherein the detection step is performed by amplifying all or part of exon 29 of the β-ΙΙΙ spectrin gene (SPTBN2), such as between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (CanFam 2.0) .

20. The method as defined in any of claims 17 to 19, wherein the detection step is performed by use of primers which flank or include part of the region defined between nucleotides 53,691,704 and 53,691,711 on chromosome 18 (CanFam 2.0).

21. The method as defined in claim 20, wherein the primers are :

Forward : 5'-TACTGGACACCACGGACAAGT -3' (SEQ I D NO : 1); and

Reverse : 5'-GGCCTCTATCTCTGCCTTGAT -3' (SEQ ID NO : 2) .

22. A primer pair for use in a method of assessing the cerebellar cortical degeneration status in a canine mammal, wherein said primers are capable of amplifying all or part of the β-ΙΙΙ spectrin gene (SPTBN2), wherein the amplified region is less than 500 nucleotides in length, such as less than 300 nucleotides in length, and wherein the primers are as defined in claim 21.

23. A kit for use in a method of assessing the cerebellar cortical degeneration status in a canine mammal, wherein said kit comprises : (a) a primer pair, wherein said primers are capable of amplifying all or part of the β-ΙΙΙ spectrin gene (SPTBN2), wherein the amplified region is less than 500 nucleotides in length, such as less than 300 nucleotides in length, and wherein the primers are as defined in claim 21 ; and

(b) means for providing a test sample from the canine mammal .

24. A method of treating cerebellar cortical degeneration in a canine mammal, which method comprises assessing the cerebellar cortical degeneration status of a canine mammal by use of a method as defined in any of claims 2 to 21 and if the canine mammal is identified as affected by cerebellar cortical degeneration, treating said canine mammal to prevent or reduce the onset of cerebellar cortical degeneration .

25. A method of treating cerebellar cortical degeneration in a canine mammal, which method comprises increasing the level of non-mutant, wild-type β-ΙΙΙ spectrin gene (SPTBN2) expression and/or β-ΙΙΙ spectrin gene (SPTBN2) product activity in the canine mammal .