# Patent

## Title
Method for diagnosing and predicting cerebellar ataxia

## Abstract

The present invention relates to an in vitro method for diagnosing and/or predicting hereditary cerebellar ataxia in a dog, and/or identifying a dog which is healthy carrier of hereditary cerebellar ataxia, comprising determining the presence or absence of an homozygous or heterozygous genetic variation in the arylsulfatase G gene sequence in a biological sample from said dog, as compared with the arylsulfatase G gene sequence of a healthy non-carrier dog, wherein the presence of said homozygous genetic variation indicates that said dog is or will be affected by hereditary cerebellar ataxia, and the presence of said heterozygous genetic variation indicates that said dog is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Provisional Application No. 61/077,656, filed Jul. 2, 2008 (which is hereby incorporated by reference).


FIELD OF THE INVENTION
The present invention relates to methods for diagnosing and predicting cerebellar ataxia.
BACKGROUND OF THE INVENTION
Neuronal ceroid lipofuscinoses (NCLs), a category of hereditary cerebellar ataxia, cerebellar abiotrophy or cerebellar cortical degeneration, is a group of monogenic inherited neurodegenerative storage diseases, found in men and mice, that are characterized by psychomotor retardation, blindness and premature death. In this clinically and pathologically heterogenous group of diseases, progressive loss of neuronal populations occurs within the cerebellum, specifically the Purkinje neurons and the granular cell layer. Loss of neuronal populations from other areas of the central nervous system also may occur, depending on the particular disease. This neuronal degeneration results in the insidious development of ataxia and a number of other neurological signs (depending on the disease) that progress at varying rates until the patient is incapacitated.
Similar hereditary NCLs have been reported in several domestic animals species including cattle (Houweling et al. (2006) Biochim. Biophys. Acta 1762:890-897), goat (Fiske and Storts (1988) Vet. Pathol. 25:171-173), sheep (Tammen et al. (2006) Biochim. Biophys. Acta 1762:898-905; Frugier et al. (2008) Neurobiol. Dis. 29:306-315), cat (Weissembock and Rossel (1997) J. Comp. Pathol. 117:17-24; Bildfell et al. (1995) Vet. Pathol. 32:485-488; Nakayama et al. (1993) J. Vet. Med. Sci. 55:829-831; Green and Little (1974) Can. J. Comp. Med. 38:207-212) and certain dog breeds.
The first American Staffordshire Terriers (AST) displaying the clinical and histopathological features of an adult-onset cerebellar cortical degeneration or cerebellar ataxia were diagnosed in 2002 and 2003 (Thibaud et al. European Society of Veterinary Neurology 15th Annual Symposium, Philadelphia Pa., September 2002; Hazli{hacek over (c)}ek et al. (2003) Schweiz Arch. Tierheilkd 145:369-375). Affected dogs showed first clinical signs from 18 months to 9 years of age with the majority of dogs presented to veterinarians between 4 and 6 years of age. They showed first stumbling, truncal sway and ataxia exacerbated by lifting the head up and negotiating stairs, progressing to an obvious ataxia characterized by dysmetria, nystagmus, coarse intention tremor and falling with transient opisthotonus. Most dogs survived for 2 to 4 years before they were euthanized while they became totally unable to walk without falling repeatedly. Histophathologic findings included loss of Purkinje cells and thinning of the molecular and granular layers.
In 2004, Siso and collaborators showed that a cerebellar cortical degeneration seen in five ASTs and eight American Pit Bull Terriers (APBT) bred in the US could be classified as a neuronal ceroid lipofuscinosis (Siso et al. (2004) Acta Neuropathol. 108:386-392). Indeed, they performed light microscopic, immunocytochemical and ultrastructural investigations and found that the neuronal degeneration not only affected Purkinje cells of the cerebellum but that diffuse nerve cell loss and gliosis were observed in the thalamic nuclei. They reported an accumulation of yellow-brown granules within neurones. These granules exhibited yellow autofluorescence under ultraviolet light; they were positive following periodic-acid-Shiff (PAS) and Sudan black staining thus indicating that the neuronal degeneration in these five ASTs was associated with neuronal ceroid lipofuscinosis
The same year, Olby and collaborators determined that the more consistent mode of inheritance of the disease in the established pedigree of AST from the US was autosomal recessive (Olby et al. (2004) J. Vet. Intern. Med. 18:201-208) but the implicated gene was not identified.
Dogs displaying symptoms of the disease are therefore homozygous for the causal mutation, and healthy carrier dogs clinically undetectable and being heterozygous for the causal mutation also exist. No predicting examination is currently available to determine the status of a dog towards NCL. Indeed, only the confrontation of the breed and the age of the dog, the presence of characteristic symptoms and an MRI exam of the brain enable for diagnosing NCL in an AST.
The appearance of this disease and the apparent increase in its prevalence are of great concern for the American Staffordshire Terrier and the American Pit Bull Terrier breeds because it is an incapacitating disease for which no cure is known. Moreover, the late onset of signs results in affected dogs being bred before they develop ataxia, potentially causing wide dissemination of the disease within the breed.
Accordingly, there is a need for genetic methods to identify healthy carrier and affected dogs before breeding, in order to stop the spreading of the disease. Determination of the causal mutation of the disease is necessary to design such a method.
In humans, NCLs have been attributed to mutations in six genes, namely PPT1(palmitoyl-protein thioesterase 1), TPP1 (tripeptidyl peptidase 1), CLN3 (ceroid lipofuscinosis, neuronal 3), CLN5, CLN6 and CLN8 (Siintola et al. (2006) Biochim. Biophys. Acta 1762:857-864). Mutations in three additional genes are involved in NCLs in animals. In White Swedish Landrace sheep, a mutation in the cathepsin D (CTSD) gene causes autosomal recessive congenital ovine NCL (Tyynela et al. (2000) EMBO J. 19:2786-2792). Furthermore, null mutations in Clcn3 (chloride channel 3), Ppt2 (palrnitoyl-protein thioesterase 2) and Ctsf (cathepsin F) are responsible for various forms of NCLs in mice (Yoshikawa et al. (2002) Genes Cells. 7:597-605; Gupta et al. (2001) Proc. Natl. Acad. Sci. USA 98:1.3566-13571; Tang et al. (2006) Mol. Cell. Biol. 26:2309-2316). Canine NCLs have been diagnosed in at least 18 breeds, and previous studies have found a CLN8 missense mutation associated with NCL in English Setters (Katz et al.
(2005) Biochem. Biophys. Res. Commun. 327:541-547), a CLN5 nonsense mutation associated with NCL in Border Collies (Melville et al. (2005) Genomics 86:287-294), a CTSD missense mutation associated with NCL in American Bulldogs (Awano et al. (2006) Mol/ Genet. Metab. 89:254-260), and a TPP1 single nucleotide deletion at CLN2 associated with NCL in Dachshunds (Awano et al. (2006) Mol. Genet. Metab. 89:254260). No common mutation was therefore identified according to the breed.
Method of Diagnosing and/or Predicting Hereditary Cerebellar Ataxia
The present invention arises from the finding, by the inventors, that ASTs suffering from hereditary cerebellar ataxia displayed an adenosine (A) at nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene, on both alleles of the gene, whereas healthy animals displayed either a guanine (G) at nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene on both alleles of the gene, or a guanine on one allele and an adenosine on the other allele at nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene. In other words, ASTs suffering from hereditary cerebellar ataxia were homozygous A/A at nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene, whereas healthy animals were either homozygous G/G or heterozygous GIA at nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene.
Thus, the present invention relates to an in vitro method for diagnosing and/or predicting hereditary cerebellar ataxia in a dog, comprising determining the presence or absence of an homozygous genetic variation in the arylsulfatase G gene sequence in a biological sample from said dog, as compared with the arylsulfatase G gene sequence of a healthy non-carrier dog, wherein the presence of said homozygous genetic variation indicates that said dog is or will be affected by hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.
The present invention also relates to an in vitro method for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, comprising determining the presence or absence of an heterozygous genetic variation in the arylsulfatase G gene sequence in a biological sample from said dog, as compared with the arylsulfatase G gene sequence of a healthy non-carrier dog, wherein the presence of said heterozygous genetic variation indicates that said dog is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.
As used herein the arylsulfatase G (ARSG) gene refers to the dog arylsulfatase G gene (including the 5′ regulatory region, the promoter, the introns, the exons and the 3′ regulatory region) and to fragments thereof. The arylsulfatase G gene is located on chromosome 9 (CFA09), and is shown in Genbank accession number 480460. As known from one skilled in the art, a gene includes both transcribed and untranscribed regions. The transcribed region may include introns, which are spliced out of the mRNA, and 5′- and 3′untranslated (UTR) sequences along with the protein coding sequences (exons). Accordingly, as used herein, the genomic sequence of the ARSG gene contains 5′- and 3′UTR sequences, introns and exons. Typically, the dog genomic sequence of the ARSG gene is composed of 11 exons and 10 introns. The genomic sequence of the ARSG gene is represented herein by SEQ ID NO: 2. As used herein, the American Staffordshire Terrier cDNA sequence of the ARSG gene consists of the coding sequence of the gene lacking introns. Typically, the cDNA sequence of the ARSG gene is composed of the 11 exons of the genomic sequence. It is herein represented by SEQ ID NO: 1.
In the context of the present invention, the terms “genetic variation” and “polymorphism” are used indifferently and contemplate single nucleotide substitutions, insertions and deletions of nucleotides, repetitive sequences (such as microsatellites), and the total or partial absence of genes (e.g. null mutations). More preferably, a genetic variation according to the invention is a single nucleotide polymorphism (SNP). The term “single nucleotide polymorphism” in the context of the present invention includes single base nucleotide substitutions and short deletion and insertion polymorphisms. Preferably, a SNP according to the invention is an adenosine allele of the ARSG gene.
In order to make the reading easier, the presence of an adenosine (A) at nucleotide position 296 of the cDNA sequence of the ARSG gene, will be called “adenosine allele”, and the presence of a guanine (G) at nucleotide position 296 of the cDNA sequence of the ARSG gene, will be called “guanine allele”.
As defined above, the cDNA sequence of the ARSG gene consists of the exons of the genomic sequence. Accordingly, a determined nucleotide position of the cDNA sequence corresponds to a determined position of the genomic sequence. In the context of the invention, the nucleotide position 296 of the ARSG gene cDNA sequence of SEQ ID NO: 1 corresponds to the nucleotide position 22139 of the ARSG gene genomic sequence of SEQ ID NO: 2. As known from one skilled in the art, introns of a gene may display numerous polymorphisms between subjects. Accordingly, a determined nucleotide position of the cDNA sequence of a gene may not correspond exactly to the same nucleotide position of the genomic sequence of the gene from a subject to another. Moreover, as the 5′-UTR sequence of the ARSG gene has not been clearly identified, the nucleotide position of the genomic sequence corresponding to the nucleotide position 296 of the cDNA sequence may slightly vary. Such a correspondence is nevertheless easily determined by one skilled in the art.
In a particular embodiment, said genetic variation is determined on the transcript or the antisense strand of the ARSG gene. As known from one skilled in the art, the sequence of the antisense strand of a gene is complementary to the sequence of the coding strand. This coding strand is transcribed in RNA, which may be spliced to form mRNA. The sequence of said mRNA is complementary to the sequence corresponding to the juxtaposition of the exons sequences contained in the coding strand. Accordingly, in the context of the invention, an adenosine allele as defined above corresponds to the presence of an uridine (U) at nucleotide position 296 of the mRNA sequence of the ARSG gene or a thymidine (T) at nucleotide position 22139 of the antisense strand sequence of the ARSG gene, and a guanine allele as defined above corresponds to the presence of a cytosine (C) at nucleotide position 296 of the mRNA sequence of the ARSG gene or at nucleotide position 22139 of the antisense strand sequence of the ARSG gene.
In the context of the invention, a genetic variation may be homozygous or heterozygous. A homozygous genetic variation means that the same genetic variation is present on both alleles of the gene carrying said genetic variation. A heterozygous genetic variation means on the contrary that said genetic variation is only present on one allele of the gene carrying said genetic variation.
As used herein, the term “American Staffordshire Terrier” (or “Amstaff”) refers to a dog belonging to group 3 section 3 of the Federation Cynologique Internationale classification, standard n° :286, published on December 1st, 1997. American Staffordshire Terriers display typically the following characteristics. They are 43 to 48 cm tall. They are muscular, stocky and not long-legged. Their coat is short, close, stiff to the touch and can be of any colour. Their head has a medium length with a broad skull, a distinct stop and strong jaws. Their eyes are dark and round. Their ears are set high, cropped or uncropped, and held rose. Their tail is short in comparison to size and low set.
As used herein, the term “American Pit Bull Terrier” refers to a dog belonging to the terrier group according to the United Kennel Club classification. It is typically a medium-sized dog, solidly built, with a short coat and smooth, well-defined muscle structure. American Pit Bull Terriers display typically the following characteristics. They are muscular and stocky. Their coat is short, stiff to the touch and may be black and brown-red, brown-yellow, or black and grey or white-black. Their head has a medium length with round uniformly coloured eyes, and high set, cropped or uncropped ears. Their tail is short compared to the body length. The American Pit Bull Terrier should be both powerful and agile, the actual weight and height being less important than the correct proportion of weight to height.
As used herein, the term “Pit Bull type” refers to dogs without pedigree having at least one American Staffordshire Terrier or at least one American Pit Bull Terrier in their parents or grand-parents.
According to the invention, dogs are of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type. More preferably, they are of a breed selected among American Staffordshire Terrier and American Pit Bull Terrier. Most preferably, they are American Staffordshire Terrier.
The terms “hereditary cerebellar ataxia”, “hereditary cerebellar cortical degeneration”, “neuronal ceroid lipofuscinose” and “cerebellar abiotrophy” are used herein indifferently and refer to any inherited progressive neuropathy. Preferably, said disease is a cerebellar disease which is characterized by a neuronal cell loss or by the accumulation of autofluorescent cytoplasmic granules of lipopigments in cells. Hereditary cerebellar ataxia may affect any subject which is a mammal, in particular dogs or human beings, more particularly dogs.
As used herein, the term “diagnosing” includes determining, monitoring, confirming, subclassifying and predicting of the relevant disease, disorder, complication, or risk.
As used herein, the term “predicting” refers to making a finding that a dog has a significantly enhanced probability of developing a hereditary cerebellar ataxia.
In the context of the invention, the term “healthy non-carrier” refers to a subject that is not and will not be affected by hereditary cerebellar ataxia and that will never transmit the disease to its progeny. Typically, according to the invention, a subject which is healthy non-carrier of hereditary cerebellar ataxia is homozygous G/G at nucleotide position 296 of the cDNA sequence of the ARSG gene.
In the context of the invention, the term “healthy carrier” refers to a subject that can transmit the disease to its progeny but which does not develop the disease. Typically, according to the invention, a subject which is healthy carrier of hereditary cerebellar ataxia only carries a heterozygous genetic variation in the arylsulfatase G gene. As hereditary cerebellar ataxia is an autosomal recessive disease, said subject will not develop the disease. However, it has one chance on two to transmit the allele displaying the genetic variation to its progeny. Accordingly, if the progeny also receives an allele displaying the genetic variation from the other parent, which is healthy carrier of hereditary cerebellar ataxia or is or will be affected by hereditary cerebellar ataxia, said progeny will be affected by hereditary cerebellar ataxia.
As used herein, the term “biological sample” means a substance of biological origin. In particular the biological sample comprises nucleic acids from the subject to be diagnosed. Examples of biological samples include, but are not limited to, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like; organs such as kidney, liver, heart, lung, and the like, saliva and mouth epithelial cells.
Numerous methods allowing determining the presence of a genetic variation in a biological sample are well known from the one skilled in the art. These methods include, without being limited, hybridization methods with DNA probes specific of said genetic variation, such as comparative genomic hybridization (CGH), matrix-CGH, array-CGH, oligonucleotide arrays, representational oligonucleotide microarray (ROMA), high-throughput technologies for SNP genotyping, for example Affymetrix SNP chips, and amplification methods such as quantitative PCR and real-time PCR.
Preferably, the presence or absence of said genetic variation is determined by polymerase chain reaction (PCR) and pyrosequencing, by sequencing or by specific amplification of said genetic variation. Such methods are well known to one skilled in the art. In particular, pyrosequencing is a method of DNA sequencing based on the “sequencing by synthesis” principle, wherein a single strand of DNA is sequenced by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. An example of pyrosequencing method is described in Ahmadian et al. (2000) Anal. Biochem. 280:103-110. More preferably, the presence or absence of said genetic variation is determined by quantitative PCR or by realtime PCR.
Nucleic Acids, Probes, Primers and Uses Thereof
The present invention also relates to an isolated nucleic acid comprising a sequence at least 80% identical to the sequence selected in the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, a fragment of said sequence, or a complementary sequence thereof, wherein said nucleic acid comprises an adenosine at nucleotide position 296 of SEQ ID NO: 1 or an adenosine at nucleotide position 22139 of SEQ ID NO: 2.
Said isolated nucleic acid may comprise or consist of a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence selected in the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, to a fragment of said sequence, or a complementary sequence thereof wherein said nucleic acid comprises an adenosine at nucleotide position 296 of SEQ ID NO: 1 or an adenosine at nucleotide position 22139 of SEQ ID NO: 2.
The percentage of sequence identity is calculated by comparing the sequence of said nucleic acid optimally aligned with the reference sequence, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions of the reference sequence, and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment may be performed by Needleman-Wunsch global alignment (Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453)
“Isolated nucleic acid” refers herein to both RNA and DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acids can have any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of nucleic acids include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant nucleic acids, and branched nucleic acids. A nucleic acid may contain unconventional or modified nucleotides. Isolated nucleic acids according to the invention may be purified or recombinant.
The fragments may be of any length, e.g. at least 10, 15, 25, 50, 100, 500 or 1000 nucleotides long.
The invention also provides for the use of said nucleic acid for diagnosing and/or predicting hereditary cerebellar ataxia in a dog of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, and/or for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.
The present invention also relates to a method for diagnosing and/or predicting hereditary cerebellar ataxia in a dog of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, and/or for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, wherein a nucleic acid as defined above is used.
The present invention also relates to an isolated probe comprising a nucleic acid as defined above.
As used herein, a “probe” refers to an oligonucleotide capable of binding in a base-specific manner to a complementary strand of nucleic acid. Isolated probes according to the invention may be purified or recombinant. They may be labelled with a detectable moiety, i.e. a moiety capable of generating a detectable signal, such as a radioactive, calorimetric, fluorescent, chemiluminescent or electrochemiluminescent signal. Numerous such detectable moieties are known in the art. By way of example, the moiety may be a radioactive compound or a detectable enzyme (e.g., horseradish peroxidase (HRP)).
In a preferred embodiment, the probe according to the invention comprises or is constituted of from about 10 to about 1000 nucleotides. Preferably, it is a fragment comprising a contiguous span of at least 12 nucleotides of SEQ ID NO: 1. Most preferably, it is a fragment consisting of a contiguous span of at least 12 nucleotides of SEQ ID NO: 1.
The present invention further relates to an isolated primer comprising a fragment of the sequence selected in the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
As used herein, the term “primer” refers to an oligonucleotide which is capable of annealing to a target sequence and serving as a point of initiation of DNA synthesis under conditions suitable for amplification of the primer extension product which is complementary to said target sequence. The primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The length of the primer depends on several factors, including temperature and sequence of the primer, but must be long enough to initiate the synthesis of amplification products. Preferably the primer is from 10 to 35 nucleotides in length. A primer can further contain additional features which allow for detection, immobilization, or manipulation of the amplified product. The primer may furthermore comprise covalently-bound fluorescent dyes, which confer specific fluorescence properties to the hybrid consisting of the primer and the target-sequence or non covalently-bound fluorescent dyes which can interact with the double-stranded DNA/RNA to change the fluorescence properties. Fluorescent dyes which can be used are for example SYBR-green or ethidium bromide.
In a preferred embodiment, the 3′ end of the primer according to the invention is located within 800 nucleotides, more preferably within 500 nucleotides, still preferably within 100 nucleotides upstream of nucleotide position 296 of SEQ ID NO: 1 or of nucleotide position 22139 of SEQ ID NO: 2.
In a particular embodiment, the primer according to the invention comprises a contiguous span of at least 12 nucleotides of SEQ ID NO: 1 or of SEQ ID NO: 2.
Preferably, the 3′ end of the primer according to the invention is located at nucleotide position 296 of SEQ ID NO: 1 or at nucleotide position 22139 of SEQ ID NO: 2, or one nucleotide upstream of nucleotide position 296 of SEQ ID NO: 1, or one nucleotide upstream of nucleotide position 22139 of SEQ ID NO:2. More preferably, the 3′ end of the primer according to the invention is located at nucleotide position 296 of SEQ ID NO: 1 or one nucleotide upstream of nucleotide position 296 of SEQ ID NO: 1.
Another object of the present invention relates to a pair of primers comprising a first and a second primer each comprising a fragment of the sequence SEQ ID NO: 1 or SEQ ID NO: 2, wherein

    
    
        a) said first primer hybridizes to a first DNA strand of said arylsulfatase G gene;
        b) said second primer hybridizes to the strand complementary to said first DNA strand of arylsulfatase G gene; and
        c) the 3′ ends of said first and second primers are located within 500 nucleotides of nucleotide position 296 of SEQ ID NO: 1 or of nucleotide position 22139 of SEQ ID NO: 2.
    
    


In the context of the invention, the terms “hybridize” or “hybridization,” as is known to those skilled in the art, refer to the binding of a nucleic acid molecule to a particular nucleotide sequence under suitable conditions, namely under stringent conditions.
The term “stringent conditions” or “high stringency conditions” as used herein corresponds to conditions that are suitable to produce binding pairs between nucleic acids having a determined level of complementarity, while being unsuitable to the formation of binding pairs between nucleic acids displaying a complementarity inferior to said determined level. Stringent conditions are the combination of both hybridization and wash conditions and are sequence dependent. These conditions may be modified according to methods known from those skilled in the art (Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, high stringency conditions are selected to be about 5° C. lower than the thermal melting point (Tm), preferably at a temperature close to the Tm of perfectly base-paired duplexes (Andersen, Nucleic acid Hybridization, Springer, 1999, p. 54). Hybridization procedures are well known in the art and are described for example in Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,Seidman, J. G., Smith, J. A., Struhl, K. eds. (1998) Current protocols in molecular biology. V. B. Chanda, series ed. New York: John Wiley & Sons.
High stringency conditions typically involve hybridizing at about 50° C. to about 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at about 60° C. to about 68° C.
In a preferred embodiment, the first primer of the pair of primers as defined above comprises a contiguous span of at least 12 nucleotides of SEQ ID NO: 1; and the second primer of the pair of primers as defined above comprises a contiguous span of at least 12 nucleotides of a sequence complementary to SEQ ID NO: 1.
More preferably, the 3′ end of the first primer of the pair of primers as defined above is located at nucleotide position 296 of SEQ ID NO: 1 or at nucleotide position 22139 of SEQ ID NO: 2 or one nucleotide upstream of nucleotide position 296 of SEQ ID NO:1 or one nucleotide upstream of nucleotide position 22139 of SEQ ID NO: 2, and the 3′ end of the second primer of the pair of primers as defined above is located at nucleotide position 296 of a sequence complementary to SEQ ID NO: 1, or at nucleotide position 22139 of a sequence complementary to SEQ ID NO: 2, or one nucleotide upstream of nucleotide position 296 of a sequence complementary to SEQ ID NO: 1, or one nucleotide upstream of nucleotide position 22139 of a sequence complementary to SEQ ID NO: 2.
Most preferably, the 3′ end of the first primer of the pair of primers as defined above is located at nucleotide position 296 of SEQ ID NO: 1 or one nucleotide upstream of nucleotide position 296 of SEQ ID NO: 1 and the 3′ end of the second primer of the pair of primers as defined above is located at nucleotide position 296 of a sequence complementary to SEQ ID NO: 1 or one nucleotide upstream of nucleotide position 296 of a sequence complementary to SEQ ID NO: 1.
The nucleic acids, the probes, the primers and the pairs of primers according to the invention can be used to determine the presence or absence of an homozygous genetic variation in the arylsulfatase G gene sequence in a biological sample from a dog to be diagnosed, wherein the presence of said homozygous genetic variation indicates that said dog is or will be affected by hereditary cerebellar ataxia. Preferably, the nucleic acids, the probes, the primers and the pairs of primers according to the invention are used to determine the presence or not of an adenosine allele at the nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene or at the nucleotide position 22139 of the genomic sequence of the arylsulfatase G gene, more preferably of an adenosine allele at the nucleotide position 296 of SEQ ID NO: 1 or at the nucleotide position 22139 of SEQ ID NO: 2.
It also relates to the use of respectively a nucleic acid, a probe, a primer and a pair of primers as defined above for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.
Namely, the nucleic acids, the probes, the primers and the pairs of primers according to the invention can be used to determine the presence or absence of heterozygous genetic variation in the arylsulfatase G gene sequence in a biological sample from a dog to be diagnosed, wherein the presence of said heterozygous genetic variation indicates that said dog is healthy carrier of hereditary cerebellar ataxia. Preferably, the nucleic acids, the probes, the primers and the pairs of primers according to the invention are used to determine the presence or not of an adenosine allele at the nucleotide position 296 of the cDNA sequence of the arylsulfatase G gene or at the nucleotide position 22139 of the genomic sequence of the arylsulfatase G gene, more preferably of an adenosine allele at the nucleotide position 296 of SEQ ID NO: 1 or at the nucleotide position 22139 of SEQ ID NO: 2.
Arrays and Kits
The present invention also relates to an array for diagnosing and/or predicting hereditary cerebellar ataxia in a dog of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type and/or for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, wherein said array comprises probes as defined above.
In a particular embodiment, the probes as defined above are assembled on a same solid support, preferably a standardized support. Its size can vary according to the apparatuses used to detect the presence or absence of a genetic variation as defined above.
Advantageously, the combination of probes according to the invention is in form of a DNA matrix, comprising a support on which probes likely to hybridize to target sequences are deposed, preferably in a standardized way. The size of such supports varies according to the preparation and detection methods used. Such small supports are also referred to array.
As used herein, the term “array” refers to a set of genes, fragment of genes, oligonucleotides deposited on a support (glass slide, nylon membrane . . . ) with a high density. Numerous scientific publications about the preparation and the use of arrays are available.
The present invention further relates to a kit for diagnosing and/or predicting hereditary cerebellar ataxia in a dog of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, and/or for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, said dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, wherein said kit comprises primers or a pair of primers as defined above.
Genotyping
The present invention also relates to the use of a nucleic acid as defined above, for genotyping a dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, wherein said dog suffers of hereditary cerebellar ataxia.
The present invention further relates to a method for genotyping a dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, wherein said dog suffers of hereditary cerebellar ataxia, wherein a nucleic acid as defined above is used.
As used herein, the term “genotyping” a dog involves determining at least one specific allele or specific nucleotide carried by a dog at at least one gene of interest.
In particular, in the context of the invention, said specific nucleotide is at the nucleotide position 296 of the arylsulfatase G gene cDNA sequence of SEQ ID NO: 1 or at the nucleotide position 22139 of the arylsulfatase G gene genomic sequence of SEQ ID NO: 2 and said gene of interest is arylsulfatase G gene.
Any well-known method of genotyping may be used in the frame of the present invention. Such methods include methods such as e.g. conventional dot blot analyzes, single strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, heteroduplex analysis and mismatch cleavage detection. Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat. No. 4,656,127. Oligonucleotide microarrays or solid-phase capturable dideoxynucleotides and mass spectrometry may also be used. Preferred methods involve directly determining the identity of the nucleotide present at a specific nucleotide position by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay.
The present inventors identified the mutation in the arylsulfatase G gene defined above, associated with hereditary cerebellar ataxia, by sequencing a representative number of dogs suffering of hereditary cerebellar ataxia. Nevertheless, it cannot be excluded that other mutations in the arylsulfatase G gene exist that are associated with said disease. Accordingly, the present invention also relates to a method of identifying mutations in the arylsulfatase G gene associated with hereditary cerebellar ataxia, by genotyping a dog being of a breed selected in the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type, wherein said dog suffers of hereditary cerebellar ataxia.
The invention will be further illustrated by the following figures and examples.



FIGURES
FIG. 1 shows the genome-wide association mapping of the NCL of the AST. Results of the whole genome scan performed with the MSS2 (minimal screening set 2) panel and 38 healthy and 39 affected French dogs are displayed. A single locus with strong significance was identified on CFA09. The Bonferroni corrected —log(pvalue) were calculated with STRAT software and reported on the y axis.
FIG. 2 shows the completed genome-wide association mapping of the NCL of the French AST. It displays the results of the whole genome scan with the MSS2 panel completed with 14 new SNPs and microsatellites markers located on CFA09. The Bonferroni corrected—log(pvalue) were calculated with STRAT software and reported on the y axis.
FIG. 3 shows the fine mapping of the NCL of the French AST. Significance of association was calculated with the STRAT software for markers within the CFA09 critical region (Bonferroni corrected pvalues; significance when—log(pvalue)≧3). The candidate region spans 19.3 Mb between markers GALK1 and REN206J15 and can be divided into two large blocks of markers. Block 1 spans 10 Mb between markers GALK1 and FH3596 while block 2 spans 9.3 Mb between markers FH3596 and REN206J15.
FIG. 4 shows the two blocks of markers defined by the two-point linkage disequilibrium (LD) analysis between 16 markers from CFA09 in French ASTs. Linkage disequilibrium (in LD score×100) between CFA09 markers is indicated in each square. A square with no value indicates a linkage of 100% between two markers: The position of each marker is indicated above its name. Analyses were performed with HAPLOVIEW software. The two blocks of markers defined by the fine mapping (FIG. 3) could be restricted to two haplotypic regions spanning 3.3 Mb for block 1 and 690 kb for block 2.
FIGS. 5 and 6 show the strong genotype-phenotype correlation of NCL to Block 1 and Block 2 haplotype. The disease AST allele is shown in black and the alternative allele in white. Frequency, indicated right to each haplotype, was calculated with HAPLOVIEW using the genotypes of the 77 French ASTs.
FIG. 7 shows histograms displaying the arylsulfatase activity (in nmol/h/mg of protein) measured after 1 (1h) or 2 hours (2h) of incubation with the substrate in homozygous affected (A/A) and healthy control (G/G) French ASTs. The number of dogs analyzed is given within each histogram and vertical bars represent the standard error of the mean, Mann-Whitney-Wilcoxon pvalues are indicated above the columns.



EXAMPLE
This example describes the identification of the genetic variation responsible for hereditary cerebellar ataxia or hereditary cerebellar cortical degeneration in ASTs.
Materials and methods
Dogs
A total of 104 affected dogs were included in this study. Seventy affected dogs were diagnosed by European or American board-certified Veterinary neurologists at the Alfort School or Lyon School of Veterinary Medicine, France and at the NC College of Veterinary Medicine, Raleigh, N.C., USA. For each dog, clinical history was collected and a complete clinical and neurological evaluation was performed. Routine hematology and serum biochemistry parameters were assessed. With owner consent, the dogs were anesthetized and the cerebrospinal fluid was sampled from the cerebello-medullary citern and routine analysis was performed. Brain examination was carried out using magnetic resonance imaging. Clinical feedbacks were continuously collected. Affected dogs diagnosed by veterinarians in private practice underwent complete physical and neurologic examinations, blood cell count and serum biochemistry. Healthy ASTs were recruited as controls. These healthy dogs were thirty eight≧3-year old French ASTs who followed the complete procedure and failed to exhibit any of the symptoms seen in affected dogs at the time of the initial genome scan. For all these dogs, clinical feedbacks from owners and veterinarians were continuously obtained and allowed the update our clinical database. None of them became affected.
Brain and cerebellum histology
Following their owner's request, 14 dogs aged 4-8 years were euthanized after they had reached the irreversible terminal stage of the disease. The whole brains with cerebellum were removed and weighed. The brains were cut in halves, soaked in 10% buffered formalin for 7 days and embedded in paraffin. Five μm-thick representative sections from midbrain, olfactory bulb, pons, cerebellum, (vermis), frontal lobe, caudate nucleus, basal ganglia, thalamus and occipital lobe were processed and stained with hematoxylin and eosin, Luxol fast blue and Periodic Acid-Schiff Reagent (PAS).
Determination of the mode of inheritance
Pedigrees of affected and healthy dogs were recovered; the number of affected siblings per litter was obtained from their owners. The observed and expected numbers of affected and healthy dogs in the litters were compared using a χ2 test.
MSS2 and SNP genotyping
DNA was isolated from ethylenediaminetetraacetic acid stabilized blood samples of dogs. PCR amplification of each of the 327 canine microsatellite markers of the Minimal Screening Set 2 (MSS2) (Clark et al. (2004) Genomics 84:550-554) was carried out separately on 30 ng of genomic DNA using a classical PCR protocol with AmpliTaqGOLD® DNA polymerase (Applied Biosystems). One μl of each fluorescent PCR product from each chromosome-specific panel was loaded onto a 3130 XL genetic analyser (Applied Biosystems) and resolved with an internal size standard (GeneScan 500 LIZ, PE Biosystems). Results were analysed using GeneMapper software v3.7 (Applied Biosystems).
SNP genotypes were determined using a pyrosequencing method adapted from Ahmandian et al. (2000) Anal Biochem 280:103-110, on a Biotage PSQTM 96 pyrosequencer. PCR primers for the ABCA5-SNP were 5′-biotinyl-TTCCATCCCTTTCACAGTCTTT-3′ (SEQ ID NO: 3) with 5′ACGATGGTTTTCAAATCTTACCT-3′ (SEQ ID NO: 4); and, the sequencing primer was 5′-GTTTTCAAATCTTACCTTCT-3′ (SEQ ID NO: 5). PCR primers for the ARSG-SNP were 5′-biotinyl-CTCCTGGCCTGGCTTTCTGT-3′ (SEQ ID NO: 6) with 5′ATCCCCGTGACGTAGCCG-3′ (SEQ ID NO: 7); and, the sequencing primer was 5′TTGTGCGTGACTCCG-3′ (SEQ ID NO: 8). PCR primers for the CA4-SNP were 5′-CTCTTCTTTCGGGTGGACCT-3′ (SEQ ID NO: 9) with 5′-biotynyl-CAGCAGACAGTAGGGAAACTGAT-3′ (SEQ ID NO: 10); and, the sequencing primer was 5′-GGAAGTGGTTCTTTGC-3′ (SEQ ID NO: 11).
Association study
For genome-wide mapping, the inventors performed a case control association analysis. In a first step, genotypes at MSS2 loci were analysed using STRAT software. The p-values were corrected for the number of comparisons according to Bonferroni procedures. In a second step, fine mapping of the CFA09 critical region was performed using PLINK software. Individuals were tested for both a phenotype and a genotype at each locus. It was assumed that both genotype and phenotype were binary, denoting the alleles by A and non-A. A-class allele was defined as the disease associated allele (most frequent allele in the affected dog cohort). Multiallelic loci were accommodated by focusing on the A allele and grouping the remaining as non-A allele. The p-values were corrected for the number of comparisons according to Bonferroni procedures. Linkage disequilibrium analysis was performed using PLINK software and, finally, the disease associated haplotypes were identified using Haploview software (Purcell et al. (2007) Am. J. Hum. Genet. 81:559-575).
RT-PCR
A collection of organ samples (in particular from brain, muscle (biceps femoris), gut, liver, lung, oesophagus, kidney, pancreas, aorta, diaphragm and ovary) were collected and frozen. They were obtained from two dogs who were euthanized for medical reasons, namely a 7 year-old affected AST and a 4 year-old Labrador Retriever suffering from a non-neurological incurable disease. Also, biopsies of a biceps femoris and a lymph node from a living 5 year-old healthy AST were obtained under anesthesia. Poly(A)+ RNA were extracted using the Ambion Poly(A)Purist MAG kit (Ambion). One hundred ng of Poly(A)+mRNA were reverse-transcribed using the SuperScript III RT kit (Invitrogen). The cDNA were then amplified using the Q-Bio Taq DNA Polymerase (Qbiogen) and the primers listed in Table 1. PCR products were analysed by electrophoresis on a 1.5% agarose gel.







TABLE 1







Primers used for RT-PCR expression profiles of candidate genes














Annealing




RT-PCR oligonucleotides
Localiza-
temperature
Product


Gene
(forward and reverse)
tion
(° C.)
size (bp)














AMZ2
5′-CAAACAGTACGGCACTCTGAA-
exon 1
56
1116



3′ (SEQ ID NO: 12)






5′-
exon 6





GTGCAAGTGTTTATTTCAATAACTA






TG-3′ (SEQ ID NO: 13)





 


SLC16A6
5′-TTTGTTCCAGAGCCAATGTTT-
exon 1
56
1166



3′ (SEQ ID NO: 14)






5′-GAAGGCTCGGCTGTAAATCTT-
exon 6





3′ (SEQ ID NO: 15)





 


ARSG
5′-GTTCCTGGGGGTGACTTTCT-3′
exon 8
58
400



(SEQ ID NO: 16)






5′-ATCTGCCTGTGGGGAAATC-3′
exon 11





(SEQ ID NO: 17)





 


WIPII
5′-GCTTCTCCTTCAACCAGGACT-
exon 1
62
593



3′ (SEQ ID NO: 18)






5′-TACAGAGAACACCCGGATGAC-
exon 7





3′ (SEQ ID NO: 19)





 


FAM20A
5′-CAAAGAGCAGCTCAACCTCAC-
exon 1
60
687



3′ (SEQ ID NO: 20)






5′-TCTTTTCCTGCCAGCGAGTA-3′
exon 6





(SEQ ID NO: 21)





 


ABCA8
5′-GTCAACAGACCTGGGCATTA-3′
exon 1
58
395



(SEQ ID NO: 22)






5′-
exon 3





CGTGAACTTCAAATGATATGAGAA






TG-3′ (SEQ ID NO: 23)





 


ABCA9
5′-GCACATAAGTGTCGGTCAGC-3′
exon 1
58
378



(SEQ ID NO: 24)






5′-
exon 3





AAGAGGACTTTCACTGCATCTACA-






3′ (SEQ ID NO: 25)





 


ABCA6
5′-AAACTCAAGCACTTCTGTGCAA-
exon 1
58
360



3′ (SEQ ID NO: 26)






5′-
exon 3





TGTCATGAAAGATGATTCCAATG-3′






(SEQ ID NO: 27)





 


ABCA5
5′-
exon 5
60
501



GGAGTTTTTAAAGATAATGGGACT






TCA-3′ (SEQ ID NO: 28)






5′-
exon 8





TAGACGGCCAAGAGGACATAGAA-






3′ (SEQ ID NO: 29)





 


KCNJI2
5′-CTCTCCTGGCTGTTCTTTGG-3′
exon 1
60
386



(SEQ ID NO: 30)






5′-AAGTGGCTCTTCCGAAGGTT-3′
exon 1





(SEQ ID NO: 31)









Statistical analysis

Chi-square test (χ2) and non parametric Mann-Whitney-Wilcoxon test for small distributions were performed with StatView F-4.1 software (Abacus Concepts).
Enzymatic assays
ARSG activity towards p-nitrocatechol sulphate was assayed in leucocytes according to Frese et al. (2008) J. Biol. Chem. 283:11388-11395.
Results
Clinical and histopathological characterization of the disease
American Staffordshire Terriers (ASTs) suffering from locomotor ataxia are weekly seen in specialized pet medical centers. A cohort of 54 males and 50 females exhibiting locomotion disablements were analyzed in France (n=66) and the USA (n=38). Seventy dogs were directly evaluated by board-certified neurologists from a French School of Veterinary Medicine or the College of Veterinary Medicine, North Carolina State University and 14 who had been initially diagnosed by their regular veterinarian were confirmed by one of the inventors. They were mainly from France and the USA, with the exception of 3 dogs living in Belgium and one in Germany. The 66 identified affected French ASTs included 36 males and 30 females. Fifty-two were evaluated by a board-certified neurologist or an internist at Alfort; one was evaluated at the School of Veterinary Medicine in Lyon (France). Fourteen dogs were evaluated by their regular veterinarian and details of the findings were evaluated by the inventors. One of these 14 dogs was directly related (sibling) to an individual with confirmed disease. For 5 dogs, no pedigree information was available. Four dogs were not registered as ASTs in the pedigrees from the French Canine Association (Société Centrale Canine).
To test affected dogs against cerebellar cortical degeneration it was proposed to their owners a magnetic resonance imaging (MRI). Among the 54 dogs for which owners agreed to MRI examination, 53 exhibited significant cerebellar atrophy or enlarged cerebellar sulci. Interestingly, these features were not observed in one 4 year-old dog examined in the very early steps of the observed locomotor problems. Owners noticed the first neurologic signs manifested by their dogs when aged between 18 months and 8 years (between 21 months and 7 years for the French ASTs group). The majority of affected ASTs (70% [72% in the French ASTs group]) begun to show ataxia between 3 and 5 years of age. The first clinical signs of the disease included stumbling when negotiating stairs, walking uphill or downhill or turning corners. Later, the affected dogs developed ataxia with hypermetria and coarse intention tremor. They had difficulty in initiating movements and fall when shaking the head. When rolled onto their backs, dogs showed vertical, horizontal or rotary nystragmus. Mentation and behaviour remained normal. The affected ASTs were euthanized between 3 months and more than 9 years after they have been diagnosed. One affected AST, born in January 1999 was still alive in June 2008.
Post-mortem examination was performed on three 6, 7 and 8 year-old affected males and two 4 and 6 year-old affected females. Representative sections from several brain areas were prepared and stained with HE, Luxol Fast Blue and PAS. Luxol fast blue was used for staining of myelin and myelinated axons. PAS staining was used to identify carbohydrates like cerebrosides or glycogen in brain sections. At necropsy, affected ASTs had cerebellum atrophy and inconsistent unilateral enlargement of lateral ventricles. The most prominent histological finding seen in each of them was a marked loss of Purkinje cells in all areas of the cerebellum. Dystrophic neurones were observed in the center of the granular layer. Massive accumulation of lipofuscin pigments in neurones and macrophages of the cerebellum and cerebral cortex was observed in each of them. Remaining Purkinje cells had a massive accumulation of pigments. Such an accumulation was also noticed in neurones of the hypoglossal nucleus and motor ocular nerves, in pyramidal cells of hippocampus, and in occipital cortex and pyramidal cells of horn of Ammon. The most prominent accumulation was observed in thalamic neurones of posterior nuclei and pulvinar. Storage material appeared blue on HE-stained sections, pink on PAS-stained sections and blue-green on Luxol-Fast-Blue-stained sections. It was made of multiple granules that swelled and distorted the cells and displaced the nuclei and Nissl substance to the periphery of the cells. Storage material was observed in the cytoplasmic arms of axons and in macrophages located where Purkinje cells had disappeared. White matter appeared histologically normal in each necropsied dogs. The inventors concluded from these histopathological features that the locomotor ataxia frequently seen in AST is highly reminiscent of a storage disease called “Neuronal Ceroid Lipofuscinose” or “Neuronal Ceroid Lipofuscinosis” (NCL).
Inheritance
Pedigrees were recovered for 84 dogs. Dogs of both sexes were affected. The ratio of affected males (36) to females (30) was 1.2 to 1. In 2 litters, at least 2 littermates were affected. In 3 litters, at least 3 littermates were affected. Lastly, in 2 litters, 4 littermates were affected. In 4 families, one parent and at least one offspring were affected. The pedigrees of 73 dogs for which the inventors had full information concerning the status of the littermates of the propositus, were used to assess the mode of inheritance. Data are presented in Table 2.







TABLE 2







Numbers of affected and healthy dogs in AST litters


Minus one affected dog present per litter










Affected
Healthy











Parents
Observed
Expected
Observed
Expected














2 healthy parents
7
4.5
11
13.5








Chi Square test
χ2 = 1.18 no statistical difference











1 affected parent
12
13
14
13








Chi Square test
χ2 = 0.154 no statistical difference







No affected dogs in litters








1 affected parent
27/27 healthy dogs in a total of 4 litters









Assuming an autosomal recessive mode of inheritance, no statistical difference was seen between the expected and the observed numbers of affected and healthy dogs for the two types of litters available (two healthy parents and one affected parent). Two litters of 8 healthy dogs each, 1 litter of 6 healthy dogs and 1 litter of 5 healthy dogs were born from couples of dogs including one affected parent. These data are in complete agreement with those of Olby et al. (2004) J. Vet. Intern. Med. 18:201-208, who analysed 37 relative dogs. A posteriori analysis of the complete panel of affected dogs (France, n=66 and US, n=35) showed that the clinical phenotype had a 22.8% penetrance at 3 years, 78.2% at 5 years and 100% at 8 years of age. In particular, penetrance was complete at 7 years of age in French ASTs and reduced to 78% and 24% at 5 and 3 years of age respectively, while in US bred ASTs penetrance was complete at 9 years of age (Olby et al., 2004). Expressivity of the disease was very variable, dogs been euthanized, while they became totally unable to walk without falling repeatedly, between a few month to more than 9 years after they have been diagnosed. One affected dog, diagnosed at 2 years of age, was 9 years of age and still alive in June 2008. But most ataxic ASTs survived for 2 to 4 years before they were euthanized.
The inventors therefore conducted mapping analyses by postulating an autosomal recessive mode of inheritance with complete penetrance in dogs aged more than 9 years and reduced penetrance in dogs less than 9 years of age and with variable expressivity. Finally, dogs from both sexes were affected and their relative number was not significantly different (males: n=54; females: n=50; χ2 =0.15), thus showing an identical penetrance in both genders.
Mapping of the morbidity locus on CFA09 by association study
A total of 77 French ASTs were recruited for a whole genome study: 39 (22 males, 17 females) exhibited clear signs of NCL while 38 (22 males, 16 females) were healthy. All healthy dogs were older than 3 years when recruited. The 77 ASTs were not related excepted for 2 affected siblings, 2 affected half-siblings, an affected mother and its healthy daughter and a healthy mother and its 3 healthy pups. The inventors therefore chose to perform a case-control study. Two hundred forty-seven autosomal microsatellites markers were selected from the MSS2 panel (Clark et al. (2004) Genomics 84:550-554) on two criteria: (i) their polymorphism in various breeds of dogs and (ii) the amplification accuracy under standard PCR conditions. The 77 ASTs were genotyped for the 247 markers. Two hundred thirty-one markers showed correct PCR amplification and were polymorphic in the case patients and control subjects. The coverage of the genome was 9.85 Mb. To determine if any of the markers were associated with the clinical signs of NCL, the inventors performed a case-control test using the program STRAT. This analysis revealed strong association between the clinical signs of the disease and C09.173 marker on canine chromosome 9 (CFA09) (χ2 test, Bonferroni corrected p-value =4.10×10−13) (FIG. 1). The inventors concluded that NCL locus was located on CFA09.
Limiting the critical interval by fine mapping and haplotype analysis
Because of the linkage of NCL locus with C09.173 marker, the inventors selected 17 additional CFA09 markers, either microsatellites or SNPs. Several of these markers were from the NCBI dog database, while others were identified in the inventors' laboratory (Table 3).







TABLE 3







Additional CFA09 markers tested for the whole genome scan


Microsatellites forward primers were 5′end labeled with  


6-carboxyfluorescein (6-FAM). Annealing temperature


 was a 61-51° C. touch down.














Position





Product
 from
Polymor-



Primers
size
centromere
phism in


Marker name
(forward and reverse)
 (bp)
(Mb)
AST dogs














ACP1
5′-6-FAM-
86
14.63
yes


(RENacp1)
ACCCCCTGTGCACCTCATCA






CTTA-3′ (SEQ ID NO: 32)






5′-






AGGTCACTGTCTGTACACGT






AGTG-3′ (SEQ ID NO: 33)





 


G4935
5′-6-FAM-
242
16.12
yes


(RENg4935)
ACATCAGGTGAAGAGCTTG






C-3′ (SEQ ID NO: 34)






5′-






GAAGTTGGCTGGGGAAGG-






3′ (SEQ ID NO: 35)





 


RGS93
5′-6-FAM-
151
17.94
yes


(RENrgs9.3)
AGTTAGACTGCCTTCTGATG






AAGTG-3′ (SEQ ID NO: 36)






5′-6-FAM-






TGTCTATCGATTCTTCCCAA






CTAAC-3′ (SEQ ID NO: 37)





 


RGS92
5′-6-FAM-
171
17.96
yes


(RENrgs9.2)
GCACGACTCCAGGAATATA






GTAGAA-3' (SEQ ID NO: 38)






5′-






TGGGACTTAAACGCTAAATT






GTATG-3′ (SEQ ID NO: 39)





 


Ren198p23
5′-6-FAM-
132
18.09
yes



TTGTACATTATCTGTTCTAC






CTCGG-3′ (SEQ ID NO: 40)






5′-






TCTTCAGCAGGCCTTTTCTC-






3′ (SEQ ID NO: 41)





 


FH3596
5′-6-FAM-
285
18.11
yes



ACATCAGGTGAAGAGCTTG






C-3′ (SEQ ID NO: 42)






5′-






GAAGTTGGCTGGGGAAGG-






3′ (SEQ ID NO: 43)





 


Mic1
5′-6-FAM-
191
18.18
yes


(DTR9Alf1)
TTCCAGGGGCACTTTCTACT






T-3′ (SEQ ID NO: 44)






5′-






TCTCCCTCTGCCTATGTCTC






A-3′ (SEQ ID NO: 45)





 


Mic2
5′-6-FAM-
235
18.23
yes


(DDR9A1f2)
GGGCGTTGAACAGATCAAA






TA-3′ (SEQ ID NO: 46)






5′-






TCATCTCCACACCAGGAGA






CT-3′ (SEQ ID NO: 47)





 


Mic3
5′-6-FAM-
173
18.24
yes


(DDR9Alf3)
GGGCTCTGAGTCTGGTCTTT






T-3′ (SEQ ID NO: 48)






5′-






ACGTATGTGCGTATCCCGTA






T-3′ (SEQ ID NO: 49)





 


ABCA5-SNP
5′-biotinyl-
60
18.84
yes



TTCCATCCCTTTCACAGTCT






TT-3′ (SEQ ID NO: 50)






5′-






ACGATGGTTTTCAAATCTTA






CCT-3′ (SEQ ID NO: 51)





 


KCNJ2
5′-6-FAM-
193
19.60
no



TATCTGGCGAATGAGATCCT






CT-3′ (SEQ ID NO: 52)






5′-






CGTTTTCGTAGCAAAAGGA






GTT-3' (SEQ ID NO: 53)





 


FH1014
5′-6-FAM-
245
22.87
no



AGGCTATTAACCCCTGATCG-






3′ (SEQ ID NO: 54)






5′-






CGATGCCTTACTTAAACAAA






CC-3′ (SEQ ID NO: 55)





 


Ren144119
5′-6-FAM-
216
24.83
yes



TGTCATCCTGCATCCAATGT-






3′ (SEQ ID NO: 56)






5′-






CAATTTACTTTTGGGCGTCA-






3′ (SEQ ID NO: 57)





 


Zubeca 3
5′-6-FAM-
201
26.16
no



TCAGGCCTTTGATGATTTCA-






3′ (SEQ ID N: 58)






5′-






CAGGGCTGGCATTTATGTAA






G-3′ (SEQ ID NO: 59)





 


Ren206j15
5′-6-FAM-
227
27.45
yes



CCCCCAACAATCAAATGTTT






A-3′ (SEQ ID NO: 60)






5′-






AATGCAGCTATATGGGCCA






C-3′ (SEQ ID NO: 61)





 


FH4059
5′-6-FAM-
396
30.54
yes



GGATCTGTGTTTCTTCGTTA






GC-3′ (SEQ ID NO: 62)






5′-






TTGATTAAAGAGCAGCTTA






GCC-3′ (SEQ ID NO: 63)





 


CA4-SNP
5′-
79
39.65
yes



CTCTTCTTTCGGGTGGACCT-






3′ (SEQ ID NO: 64)






5′-biotynyl-






CAGCAGACAGTAGGGAAAC






TGAT-3′ (SEQ ID NO: 65)









These markers span a 25 Mb chromosomal region around C09.173. Fourteen of them were polymorphic in the studied dogs. The additional markers allowed to confirm the mapping of NCL locus onto canine chromosome 9 (FIG. 2). Interestingly, the strongest association was obtained for ABCA5-SNP, a synonymous SNP located within exon 36 of the ABCA5 gene (GeneBank ID: FM211813; χ2 test, Bonferroni corrected p-value=1.10×10−15). Furthermore, these markers narrowed the critical interval to a 19.30 Mb chromosomal segment extending from GALK1 (χ2 test, Bonferroni corrected p-value=3.40) to REN206J15 (χ2 test, Bonferroni corrected p-value=56.85) which could be divided into two regions of homozygosity (10 Mb and 9.3 Mb) interrupted by a region of 1 Mb. Block 1 covered the sequence between GALK1 and FH3596 (χ2 test, Bonferroni corrected p-value=0.026) and block 2 covered the sequence between FH3595 and REN206J15 (FIG. 3).
Using the HAPLOVIEW program, the inventors showed that these two blocks were in linkage disequilibrium on CFA09 and haplotype analysis revealed a 4.21 Mb region (14.63-18.84 Mb) that included two distinct narrowed blocks with perfect genotype-phenotype correlation (FIG. 4). Each of these two narrowed blocks included 5 markers (first narrowed block of 3.33 Mb [RENacpl—RGS92]; second narrowed block of 690 kb [DTR9A1f1—ABCA5-SNP]). The haplotype of the first narrowed block was present in 76.9% of affected AST and in 20.9% of healthy controls while the haplotype of the second narrowed block was present in 95.3% of affected AST and in 16.2% of healthy controls (FIG. 5, FIG. 6). From these data, the inventors concluded that the NCL causing gene location was located in the narrowed block 2 and more particularly in the vicinity of the ABCA5-SNP position.
Candidate gene selection
To search for candidate genes that could account for NCL in dogs, the inventors took advantage of the available annotation of the Dog genome. The 690 kb region contained 8 genes and 3 non annotated transcriptional units and the inventors included 5 additional genes from the flanking ends of the region. From the centromeric to the telomeric part of the region of interest, the 16 candidate genes were AA/22 (archaelysin family metallopeptidase 2), SLCA16A6 (solute carrier family 16, member 6), ARSG (arysulfatase G), WIP11 (WD repeat domain, phosphoinositide interacting 1), PRKARIA (protein kinase, cAMP-dependent, regulatory, type I, alpha), FAM20A (family with sequence similarity 20, member A), LOC610988, LOC610995, ABCA8 (ATP-binding cassette, sub family A, member 8), U6, ABCA9 (ATP-binding cassette, sub family A, member 9), ABCA6 (ATP-binding cassette, sub family A, member 6), ABCA5 (ATP-binding cassette, sub family A, member 5), MAP2K6 (mitogen-activated protein kinase kinase 6), KCNJ16 (potassium inwardly-rectifying channel, subfamily J, member 16) and KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2). Each candidate gene was considered for its expression levels in mouse and human tissues, its molecular functions, its participation to biological processes and pathway, and lastly its expression in dog tissues. Their molecular function in biological processes and pathways were obtained from the Panther classification system and GeneCards database (Table 4). From the Gene Expression Atlas database (SymAtlas alias, Genomics Institute of the Novartis Research Foundation), all annotated genes but KCNJ16 seem ubiquitously expressed in human and mouse tissues and using a panel of ̃10 organs collected from a 4 year-old female Labrador Retriever (brain, skeletal muscle, heart, gut, liver, lung, esophagus, kidney, pancreas, skin and ovary), the inventors confirmed that the 13 annotated genes are also ubiquitously expressed in canine tissues, with the exceptions of ABCA8 and ABCA9 that could not be detected in kidneys (Table 4).







TABLE 4







Candidate genes in the critical region
















Expression in



Position



Human and
Expression


in Mb
Gene
Name
Function
Mouse
in Dog





18.12
AMZ2
Archaelysin family
Metallopeptidase
Ubiquitous
Ubiquitous




metallopeptidase 2





18.14
SLC16A6
Solute carrier family
Monocarboxylic acid
Ubiquitous
Ubiquitous




16, member 6
transporter




18.18
ARSG
Arylsulfatase G
Sulphatase activity.
Ubiquitous
Ubiquitous





Lysosomal enzyme




18.24
WIPII
WD repeat domain,
WD40 repeat protein of 
Ubiquitous
Ubiquitous




phosphoinositide
49 kDa interacting with
high expression





interacting 1
phosphoinositides
in heart



18.31
PRKAR1A
Protein kinase,
Protein kinase. Tumor-
Ubiquitous
Ubiquitous




cAMP-dependent,
suppressor gene. When






regulatory, type I,
mutated in humans






alpha (tissue- specific
PRKAR1A is






extinguisher 1)
responsible for the







Carney complex.




18.33
FAM20A
Family with sequence
Murine FAM20A is a
Ubiquitous
Ubiquitous




similarity 20, member
secreted protein






A
expressed in







hematopoietic cells




18.43
LOC610988
Similar to 60S
Hypothetical ribosomal
Unknown
Not tested




ribosomal protein
protein






L23a





18.44
LOC610995
None
Unknown
Unknown
Not tested


18.55
ABCA8
ATP-binding cassette,
ABC transporter
Ubiquitous,
Ubiquitous




sub-family A

high expression
except




(ABC1), member 8

in olfactory
kidney






bulb (Human)







and liver







(Mouse).



18.57
U6
U6 spliceosomal
Hypothetical
Unknown
Not tested




RNA
spliceosomal RNA




18.64
ABCA9
ATP-binding cassette,
ABC transporter
Ubiquitous
Ubiquitous




sub-family A


except




(ABC1), member 9


kidney


18.72
ABCA6
ATP-binding cassette,
ABC transporter
Ubiquitous
Ubiquitous




sub-family A







(ABC1), member 6





18.85
ABCA5
ATP-binding cassette,
ABC transporter
Ubiquitous
Ubiquitous




sub-family A







(ABC1), member 5





18.97
MAP2K6
Mitogen-activated
Protein kinase.
Ubiquitous
Ubiquitous




protein kinase kinase
Activates p38 MAP






6
kinase.




19.57
KCNJ16
Potassium inwardly-
Ion channel
Thyroid, kidney
Ubiquitous




rectifying channel,

and pancreas





subfamily J, member







16





19.60
KCNJ2
Potassium inwardly-
Ion channel
Ubiquitous,
Ubiquitous




rectifying channel,

high expression





subfamily J, member

in whole blood





2

in Human









Semi-quantitative expression levels were also assessed by the inventors in a panel of tissues from a 7 year-old affected AST and no difference could be observed for any of them (data not shown). Using genomic DNA from 4 healthy and 4 affected AST, the inventors therefore decided to batch sequence and compare in the 2 groups of dogs the coding and intron-exon boundaries sequences of those genes primarily suspected to play a role in neuronal homeostasis.
They found no difference for KCNJ2, MAP2K6 and SLC16A6. One synonymous SNP was detected in the single exon of KCNJ16 [Genebank ID: FM211814] and was not associated with the disease. In addition to the previously described synonymous SNP in exon 36 of the ABCA5 (ABCA5-SNP) highly associated with the NCL locus, 2 non synonymous SNPs were detected in exons 14 and 17 [Genebank ID: FM211419 and FM211812] and were not linked to the disease. Finally, a single non-synonymous (G/A) SNP was detected in the exon 2 of the ARSG gene and in this panel of 8 dogs, specifically segregated with NCL. The inventors therefore tested the strength of its association with NCL using a larger group of dogs.
ARSG polymorphism in affected dogs
A large panel of dogs was thus genotyped for the SNP identified in the ARSG gene. First, 71 French healthy control ASTs and the 66 French and 38 US affected dogs allowed to confirm that the allele segregation of the SNP was compatible with the autosomal recessive inheritance of NCL. Indeed, 100% of affected dogs were AIA, all healthy ASTs were G/G or G/A, and only one clinically healthy dog was AIA (Table 5). Importantly, this dog shares a 2.77 Mb haplotype [RENg4935 - ABCA5-SNP] with affected ASTs. He was born in September 2000 and because the owner did not give his consent, could not be MRI-assessed. His clinical status is continuously investigated by the inventors. The panel also included 525 non-NCL dogs from 9 putatively AST- related breeds, 132 dogs from additional 45 breeds and 3 French mongrels. None of the 1320 genotyped chromosomes contained the A allele which seemed therefore specific of the AST breed and more precisely associated with the NCL predisposition (Table 5).







TABLE 5





Genotypes for the ARSG-SNP







a-Genotype frequencies for the ARSG-SNP


in the healthy and affected ASTs groups. 









Clinical status 




of dogs
Healthy
Affected
















Genotype 
G/G
G/A
A/A
G/G
G/A
A/A


at the








ARSG-SNP 








locus








French 
53.5%
45.1%
1.4%
0
0
100%


American
(n = 38)
(n = 32)
(n = 1)


(n = 66)


Staffordshire 








Terrier








US American
ND
ND
ND
0
0
100


Staffordshire 





(n = 38)


Terrier










b-Panel of Bulls and Terriers dogs potentially 


related to the AST breed and AST non


related dogs with a confirmed G/G genotype. 











Number 


 
Breed
of dogs






Staffordshire 
22



Bull Terrier




American Bulldog
18



Bull Terrier
139



Bull Terrier Toy
33



Bullmastiff
23



Mastiff
31



English Bulldog
82



Dogo Argentino
18



German Boxer
159



American Cocker 
2



Spaniel




Australian Cattle 
3



Dog




Barzoi
5



Beagle
5



Beauceron
2



Belgian Shepherd 
6



Dog




Bermese Mountain 
3



Dog




Bichon
1



Bordeaux Dogge
1



Brittany Spaniel
2



Chow-Chow
2



Collie
1



Coton de Tulear
2



Dalmatien
5



Das chund
5



Drahthaar
1



English Cocker 
2



Spaniel




English Setter
2



Fox Terrier
4



French Bulldog
4



German Shepherd 
7



dog




German Short-haired 
2



Pointing Dog




Golden Retriever
7



Great Dane
2



Husky
1



Irish Setter
2



Jack Russell Terrier
4



Labrador Retriever
8



Leonberger
5



Lhassa Apso
1



Malamute
1



Newfoundland
1



Poodle
5



Pyrenean Mountain 
3



Dog




Rottweiler
5



Saluki
1



Samoyede
2



Schnauzer
1



Shar Pei
6



Shih Tzu
2



Tatra Shepherd Dog
1



Tibet Dogge
1



Tibetan Terrier
2



Whippet
1



Yorkshire Terrier
3



Mongrel
3





The number of dogs is indicated into brackets.


ND: not determined.


Exhaustive list of Bulls and Terriers dogs potentially related to the AST breed (bolded) and AST non related dogs which have been genotyped for the ARSG SNP. With no exception, these dogs were all G/G.






The ARSG SNP is located at position 296 of the open reading frame and causes the substitution of an histidine for the arginine 99 (Arg99His) in the protein [Genebank ID: FM246885].
To evaluate the functional importance of the Arg99 substitution, the inventors searched whether this residue had been under a positive selective pressure by aligning the sequence of the canine ARSG protein with known metazoan orthologs and with biochemically characterized human ARS family members, namely ARSA, ARSB, ARSC (alias name is STS steroid sulfatase), ARSD, ARSE and ARSF. Global alignments attested that canine and human ARSG are highly conserved (86%), as are canine and murine proteins (79%). In addition, the inventors observed a 100% conservation between the canine ARSG and all the human ARS members for the 10 critical residues known to operate in the catalytic activity of these proteins. Refering to positions in human ARSA (Ghosh (2007) Cell. Mol. Life Sci. 64:2013-2022; Ghosh (2005) Methods Enzymol. 400:273-293), these residues are Asp29, Asp30, HFGS69 (hydroxyformylglycine sulphate 69, the post-translationally modified cysteine 69), Arg73, Lys123, His125, His229, Asp281, G1n282 and Lys303. Although Arg99 (Arg84 refering to human ARSA) was not previously identified as a key component of the catalytic activity, the inventors observed that it is included in a domain of 10 residues displaying a high alignment score between human ARS and ARSG from Dog, Human, Mouse, Chicken, Zebrafish, Fugu, and two species of worms (Caenorhabditis elegans and Ciona intestinalis). Indeed, 5 of these 10 residues, including Arg99, are conserved. Altogether, these data highly suggested that Arg99 is a critical residue for ARSG activity and that its substitution may impair long-term neuronal survival in AST dogs.
Reduced arylsulfatase activity in affected dogs
In many instances, missense mutations lead to a rapid degradation of the encoded enzyme in the endoplasmic reticulum (Ellgaard et al. (1999) Science 286:1882-1888). Before this hypothesis could be directly tested using an antibody and biochemical cellular tests under characterization and because the inventors found the canine ARSG gene ubiquitously expressed, they decided to evaluate the impact of the Arg99His substitution on leucocytes arylsulfatase activity using a method adapted from Frese et al. (Frese et al.
(2008) J. Biol. Chem. 283:11388-11395). In addition, unwanted compensatory activity by redundant ARS from the family was avoided by the use of pH conditions for STS, ARSF and ARSI (Puca et al. (1997) Genomics 42:192-199;, Oshikawa et al. (2009) Mol. Vis. 15:482-494) and specific inhibitors for ARSE (warfarin (Franco et al. (1995) Cell 81:1525)), ARSB (chloride ions (Bostick et al. (1978) Clin. Chem. 24:1305-1316)). Leucocyte's arylsulfatase activity from 5 A/A homozygous affected ASTs was compared with leucocytes activity of 5 homozygous G/G healthy ASTs, taken as the 100% reference activity level. The inventors observed that after one hour of incubation, the arylsulfatase activity of leucocytes from affected dogs was significantly reduced to 24.7% of the normal activity (Mann-Whitney-Wilcoxon p-values=0.009; FIG. 7) and that after 2 hours of incubation, the activity remained reduced to 28.5% (Mann-Whitney-Wilcoxon p-values=0.025; FIG. 7). Therefore, leucocyte arylsulfatase activity from affected dogs carrying an Arg99His substitution in ARSG could not reach a normal level, strongly suggesting that the GIA mutation detected in the exon 2 of the ARSG gene of American Staffordshire terriers is the disease-causing mutation.
Accordingly, the inventors have demonstrated that the presence of an homozygous A allele in the arylsulfatase G gene of ASTs instead of a G allele is associated with hereditary cerebellar ataxia or hereditary cerebellar cortical degeneration, and that the determination of this allele would allow to diagnose and/or predict hereditary cerebellar ataxia or hereditary cerebellar cortical degeneration in dogs.

## Claims

1. An in vitro method for diagnosing and/or predicting hereditary cerebellar ataxia in a dog, comprising :
obtaining a biological sample from the dog,
determining in the biological sample the presence of a homozygous adenosine allele at the nucleotide position 296 of the arylsulfatase G gene cDNA sequence of SEQ ID NO: 1, and
diagnosing and/or predicting the dog is or will be affected by hereditary cerebellar ataxia when the homozygous adenosine allele is detected,
wherein said dog is selected from the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.

2. A method according to claim 1, wherein the presence of the homozygous adenosine allele is determined by polymerase chain reaction (PCR) and pyrosequencing, by sequencing or by specific amplification of said allele.

3. An in vitro method for identifying a dog which is healthy carrier of hereditary cerebellar ataxia, comprising:
obtaining a biological sample from the dog,
determining in the biological sample the presence of a heterozygous adenosine allele at the nucleotide position 296 of the arylsulfatase G gene cDNA sequence of SEQ ID NO: 1, and
identifying the dog as a healthy carrier of hereditary cerebellar ataxia when the heterozygous adenosine allele is detected,
wherein said dog is selected from the group consisting of American Staffordshire Terrier, American Pit Bull Terrier and Pit Bull type.

4. A method according to claim 3, wherein the presence of the heterozygous adenosine allele is determined by polymerase chain reaction (PCR) and pyrosequencing, by sequencing or by specific amplification of said allele.