Diagnostic methods for assessing the risk of developing delayed-onset ataxia in dogs
By detecting the PLP1 gene allele in canines, PCR technology was used to diagnose and prevent delayed motor incoordination in toy Chow Chows. This solved the problem of difficulty in diagnosing and preventing the disease in existing technologies, and achieved the effect of early identification and reduction of the risk of disease.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- THE INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH
- Filing Date
- 2022-01-12
- Publication Date
- 2026-04-24
AI Technical Summary
Current technologies are insufficient for the effective diagnosis and prevention of delayed motor incoordination in small breeds such as toy Chow Chows, and there is a lack of targeted treatments.
By detecting the alleles of the protein lipoprotein 1 (PLP1) gene deletion in the genome of canines and using PCR technology for diagnosis, canines carrying the PLP1 gene deletion allele are identified as having a high risk of developing delayed motor incoordination, providing diagnostic tools and methods to avoid breeding animals carrying this genotype.
It enables early diagnosis and prevention of delayed motor incoordination, reduces the risk of disease in offspring, provides targeted treatment options, and reduces the spread of the disease.
Smart Images
Figure 0007850951000003 
Figure 0007850951000004 
Figure 0007850951000005
Abstract
Description
Technical Field
[0001] The present invention relates to a method for diagnosing the onset risk of canine tardive ataxia. More specifically, the present invention relates to a method for diagnosing the onset risk of tardive ataxia, a kit for diagnosing tardive ataxia, a method for breeding dogs with a reduced onset risk of tardive ataxia, and dogs with a low onset risk of tardive ataxia.
Background Art
[0002] Ataxia refers to a pathological condition in which the coordination of various movements related to the intended movement deteriorates and cannot be performed smoothly. Progressive ataxia in humans is said to be difficult to diagnose due to its heterogeneity and the rarity of individual causes. Progressive ataxia often causes diagnostic uncertainty in general neurological examinations and is often diagnosed as idiopathic or remains undiagnosed.
[0003] Most treatment methods for human ataxia only alleviate symptoms and do not address the root cause. However, for some ataxias in which genetic defects have been elucidated, treatment methods targeting the molecular mechanisms related to the causative genes have been developed. For example, miglustat used in the treatment of Niemann-Pick disease type C is an example (see, for example, Non-Patent Document 1). Therefore, it is important to identify the genetic causes of the disease in order to develop treatment methods for ataxia.
[0004] Incidentally, dog breeds lack genetic diversity due to reasons such as the repeated use of specific males in breeding, which contributes to a predisposition to various genetic diseases that are very rare in humans. However, once disease-causing variants are identified, pathological and therapeutic data can be collected from dogs carrying those variants. For example, in Non-Patent Literature 2, after a pathogenic variant causing Duchenne muscular dystrophy in ataxic golden retrievers was identified, studies were conducted on dogs that developed the disease, and the long-term safety and effectiveness of gene therapy were analyzed. Thus, identifying variants that cause genetic diseases in dogs is useful not only for veterinary diagnosis and prevention but also for improving human medicine.
[0005] In small dog breeds such as Toy Poodles, there is a rare but fatal form of delayed-onset ataxia. The symptoms are similar to those of amyotrophic lateral sclerosis (ALS), and the disease is progressive. Although it is not fetal lethal, puppies are born normally, but the disease develops after sexual maturity (after 1 year of age), exhibiting neurological symptoms such as ataxia, and dying a few months later. The cause of this disease was previously unknown. [Prior art documents] [Non-patent literature]
[0006] [Non-Patent Document 1] Patterson MC, et al., Stable or improved neurological manifestations during miglustat therapy in patients from the international disease registry for Niemann-Pick disease type C: an observational cohort study, Orphanet J Rare Dis, 10, 65, 2015. [Non-Patent Document 2] Le Guiner C., et al., Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy, Nat Commun, 8, 16105, 2017. [Overview of the Initiative] [Problems that the invention aims to solve]
[0007] The present invention aims to provide a technology for diagnosing the risk of developing delayed-onset ataxia in dogs. The present invention also aims to provide a diagnostic kit for delayed-onset ataxia in dogs, a method for breeding dogs with a reduced risk of developing delayed-onset ataxia, and dogs with a low risk of developing delayed-onset ataxia. [Means for solving the problem]
[0008] The present invention includes the following embodiments. [1] A method for diagnosing the risk of developing late-onset ataxia, comprising the step of detecting a haplotype lacking the Proteolipid protein 1 (PLP1) gene in the genome of a test dog, wherein the detection of the haplotype indicates that the test dog is at risk of developing late-onset ataxia or that the test dog will produce offspring that are at risk of developing late-onset ataxia. [2] The diagnostic method according to [1], wherein the haplotype lacking the PLP1 gene has the nucleotide sequence described in Sequence ID No. 1. [3] The diagnostic method according to [1] or [2], wherein the dog being examined is a toy poodle (Canis lupus familiaris.) or a hybrid thereof. [4] A diagnostic kit for delayed-onset ataxia, comprising a reagent for detecting haplotypes lacking the PLP1 gene in the genome of a test dog. [5] The kit according to [4], wherein the haplotype lacking the PLP1 gene has the nucleotide sequence described in Sequence ID No. 1. [6] A method for breeding dogs with a reduced risk of developing delayed-onset ataxia, comprising the step of breeding dogs that do not have a haplotype lacking the PLP1 gene. [7] The method according to [6], wherein the haplotype lacking the PLP1 gene has the nucleotide sequence described in Sequence ID No. 1. [8] Dogs that do not have a PLP1 gene deletion haplotype and have a low risk of developing late-onset ataxia. [Effects of the Invention]
[0009] The present invention provides a technology for diagnosing the risk of developing delayed-onset ataxia in dogs. It also provides a diagnostic kit for delayed-onset ataxia in dogs, a method for breeding dogs with a reduced risk of developing delayed-onset ataxia, and dogs with a low risk of developing delayed-onset ataxia. [Brief explanation of the drawing]
[0010] [Figure 1] Figure 1 is a schematic diagram showing the structure of the base sequence of a normal X chromosome and the base sequence of a mutant X chromosome. [Figure 2] Figure 2 shows T1 and T2-weighted magnetic resonance images of the cerebrum (frontal lobe), cerebrum (thalamus), and cerebellum in dogs that developed delayed-onset ataxia, as measured in Experimental Example 1. [Figure 3] Figure 3 shows the pedigree tree of the Toy Poodle created in Experimental Example 2. [Figure 4] Figure 4(a) is a Manhattan plot showing the results of the genome-wide association study in Experimental Example 3. A QQ plot is shown in the upper left. Figure 4(b) is a magnified plot of the X chromosome portion of Figure 4(a). Figure 4(c) shows the haplotypes around associated SNPs in dogs with and without late-onset ataxia, along with information from the NCBI Genome Data Viewer for the corresponding region. [Figure 5]Figure 5 is a diagram showing the result of mapping reads obtained from a male Toy Poodle that developed tardive dyskinesia in Experimental Example 4 to the canine reference genome (CanFam3.1). [Figure 6] Figure 6(a) is a dot plot showing the result of comparing the DNA base sequences in Experimental Example 4. Figure 6(b) is a schematic diagram showing the structure of the mutated genome revealed in Experimental Example 4. [Figure 7] Figure 7 is a diagram showing the result of mapping reads of the whole genome sequence to the canine reference genome (CanFam3.1) modified to have the mutated genome in Experimental Example 4. [Figure 8] Figure 8(a) is a diagram showing the result of analyzing the gene expression level in the canine cerebrum in Experimental Example 5. Also, Figure 8(b) is a diagram showing the result of analyzing the gene expression level in the canine cerebellum in Experimental Example 5. [Method for diagnosing the onset risk of tardive dyskinesia in dogs]
[0011] [イヌの遅発性運動失調症の発症リスクの診断方法] In one embodiment, the present invention includes a step of detecting a haplotype lacking the PLP1 gene on the genome of a test dog, and the detection of the haplotype indicates whether the test dog has a risk of developing tardive dyskinesia or produces offspring having a risk of developing tardive dyskinesia, thereby providing a method for diagnosing the onset risk of tardive dyskinesia.
[0012] As will be described later in the examples, the inventors have clarified that having a haplotype lacking the PLP1 gene is the cause of dogs developing tardive dyskinesia. By the method of this embodiment, the onset risk of tardive dyskinesia in a test dog can be diagnosed. If the test dog does not have a haplotype lacking the PLP1 gene, it can be diagnosed that the test dog is normal and has no risk of developing tardive dyskinesia caused by this haplotype.
[0013] Having a risk of developing tardive dyskinesia means that the test dog has a high likelihood of developing tardive dyskinesia in the future, that the test dog may produce offspring that have a possibility of developing tardive dyskinesia, and so on. Alternatively, it means that the dyskinesia that the test dog has already developed is tardive dyskinesia.
[0014] The genomic sample can be extracted from a biological sample of the test dog. The biological sample is not particularly limited, and examples include oral cells, blood, and the like.
[0015] PLP1 is a gene located on the X chromosome and encodes a transmembrane lipid proteolipid protein that is a major component of myelin. The NCBI Gene ID of the canine PLP1 gene is 481002. Also, the NCBI accession number of the cDNA of the canine PLP1 gene is NM_001013834.2 etc., and the NCBI accession number of the canine PLP1 protein is NP_001013856.1 etc.
[0016] The haplotype lacking the PLP1 gene (hereinafter, may be referred to as the "mutant haplotype") may be a haplotype having a mutation that suppresses the expression of the PLP1 gene, or may be a haplotype having a mutation that causes the deletion of the expression of the PLP1 gene. More specifically, the mutant haplotype may have the nucleotide sequence set forth in SEQ ID NO: 1. The nucleotide sequence set forth in SEQ ID NO: 1 is the nucleotide sequence identified in the examples described later.
[0017] In this specification, a haplotype lacking the PLP1 gene is a haplotype having an allele lacking the PLP1 gene. That is, the diagnostic method of this embodiment includes a step of detecting an allele lacking the PLP1 gene on the genome of a test dog, and the detection of the allele indicates that the test dog is at risk of developing late-onset ataxia or that the test dog will produce offspring that are at risk of developing late-onset ataxia.
[0018] The method for detecting mutant haplotypes (mutant alleles) is not particularly limited, but it can be easily performed by PCR, etc. Specifically, by performing PCR using a primer set for detecting mutant haplotypes (primer set for amplification of mutant sequences) and a primer set for detecting wild-type haplotypes (primer set for amplification of normal sequences), the risk of developing tardive ataxia can be diagnosed.
[0019] Diagnosis using PCR will be explained with reference to Figure 1. Figure 1 is a schematic diagram showing the structure of the base sequence of a normal X chromosome (normal sequence) and the base sequence of a mutant X chromosome having the base sequence described in Sequence ID No. 1 (mutant sequence). The structure of the mutant sequence will be described later in the examples.
[0020] For normal sequence amplification, a primer set that amplifies the PLP1 gene can be used. In Figure 1, a solid arrow indicates an example of a primer set for normal sequence amplification. Examples of primer sets for normal sequence amplification include a set of primers consisting of the nucleotide sequence shown in SEQ ID NO: 2 and a set of primers consisting of the nucleotide sequence shown in SEQ ID NO: 3.
[0021] For mutant sequence amplification, a primer set that amplifies the nucleotide sequence characteristic of the mutant allele can be used. In Figure 1, a dotted arrow indicates an example of a mutant sequence amplification primer set. Examples of mutant sequence amplification primer sets include a set of primers consisting of the nucleotide sequence shown in SEQ ID NO: 4 and a set of primers consisting of the nucleotide sequence shown in SEQ ID NO: 5.
[0022] Based on the PCR results using these primer sets, the risk of developing delayed-onset ataxia can be diagnosed. Table 1 below shows whether or not amplification by PCR occurred in normal dogs and dogs that develop delayed-onset ataxia. In Table 1, "normal dogs" refers to dogs that do not develop delayed-onset ataxia caused by this haplotype, and "diseased dogs" refers to dogs that develop delayed-onset ataxia. Also, "+" means that amplification by PCR was observed, and "-" means that amplification by PCR was not observed. It should be noted that primer sets with similar functions are not limited to the above primer sets, and it is common technical knowledge to those skilled in the art that they can identify usable primer sets.
[0023] The PLP1 gene is located on the X chromosome. Therefore, healthy male dogs have one copy of the wild-type allele. Male dogs with the disease have one copy of the mutant allele. On the other hand, healthy female dogs have two copies of the wild-type allele. Female dogs with the disease may have one copy of the mutant allele and one copy of the wild-type allele, or two copies of the mutant allele.
[0024] [Table 1]
[0025] As shown in Table 1, in healthy male dogs, amplification was observed by PCR using the primer set for normal sequence amplification, but not by PCR using the primer set for mutant sequence amplification. In addition, in diseased male dogs, amplification was not observed by PCR using the primer set for normal sequence amplification, but was observed by PCR using the primer set for mutant sequence amplification.
[0026] Furthermore, in healthy female dogs, amplification was observed by PCR using a primer set for normal sequence amplification, but not by PCR using a primer set for mutant sequence amplification. In addition, in female diseased dogs, if one set of mutant haplotypes and one set of wild-type haplotypes, amplification was observed by PCR using a primer set for normal sequence amplification, and also by PCR using a primer set for mutant sequence amplification. In addition, in female diseased dogs, if two sets of mutant haplotypes, amplification was not observed by PCR using a primer set for normal sequence amplification, but amplification was observed by PCR using a primer set for mutant sequence amplification.
[0027] In diagnostic methods using PCR, a further step of sequencing the PCR amplification product may be performed to confirm that it is the target base sequence.
[0028] In the diagnostic method of this embodiment, the test dog is preferably a Toy Poodle (Canis lupus familiaris) or a hybrid thereof.
[0029] [Diagnostic kit for delayed-onset ataxia in dogs] In one embodiment, the present invention provides a diagnostic kit for delayed-onset ataxia, comprising a reagent for detecting a haplotype lacking the PLP1 gene in the genome of a test dog. The kit of this embodiment allows for the suitable implementation of the diagnostic method for assessing the risk of developing delayed-onset ataxia in dogs described above.
[0030] In the kit of this embodiment, the haplotype lacking the PLP1 gene (mutant haplotype) is the same as described above, and is a haplotype having an allele (mutant allele) lacking the PLP1 gene.
[0031] Using the kit of this embodiment, the risk of developing delayed-onset ataxia in dogs can be diagnosed by detecting mutant alleles in genomic samples extracted from biological samples of test dogs. The biological samples are the same as those described above.
[0032] The mutant haplotype may have the nucleotide sequence described in SEQ ID NO: 1. The nucleotide sequence described in SEQ ID NO: 1 is the nucleotide sequence identified in the examples described later.
[0033] A reagent for detecting haplotypes lacking the PLP1 gene (mutant haplotypes) is the aforementioned primer set for amplifying mutant sequences. The kit of this embodiment may further include a primer set for amplifying normal sequences in addition to the primer set for amplifying mutant sequences. Including the primer set for amplifying normal sequences makes it possible to determine whether or not the test dog has a wild-type allele.
[0034] The kit of this embodiment may further include reagents for extracting genomic DNA from a biological sample, such as a column and buffer, and PCR reaction reagents such as Taq DNA polymerase, dNTPs, and buffer.
[0035] [Methods for breeding dogs with a reduced risk of developing delayed-onset ataxia] In one embodiment, the present invention provides a method for breeding dogs with a reduced risk of developing delayed-onset ataxia, comprising the step of breeding dogs that do not have a haplotype lacking the PLP1 gene.
[0036] The method of this embodiment involves diagnosing the risk of developing delayed-onset ataxia in a test dog using the diagnostic method described above, breeding test dogs (normal dogs) that are diagnosed as having a low risk of developing delayed-onset ataxia, and not breeding test dogs (sick dogs) that are diagnosed as having a risk of developing delayed-onset ataxia.
[0037] The method of this embodiment makes it possible to breed dogs with a reduced risk of developing delayed-onset ataxia. In this way, in the field of companion animals, it is possible to prevent the onset of disease in the next generation through diagnosis by genetic testing.
[0038] In the method of this embodiment, the haplotype lacking the PLP1 gene may have the nucleotide sequence described in SEQ ID NO: 1. The nucleotide sequence described in SEQ ID NO: 1 is the nucleotide sequence identified in the examples described later.
[0039] In the method of this embodiment, the dog is preferably a toy poodle or a hybrid thereof.
[0040] [Dogs with a low risk of developing delayed-onset ataxia] In one embodiment, the present invention provides a dog that does not have a haplotype lacking the PLP1 gene and has a low risk of developing delayed-onset ataxia. In this embodiment, the dog is preferably a toy poodle or a hybrid thereof.
[0041] As described later in the examples, the inventors have revealed that having a haplotype lacking the PLP1 gene is the cause of delayed-onset ataxia in dogs. Therefore, dogs that do not have a haplotype lacking the PLP1 gene are normal and have a low risk of developing delayed-onset ataxia.
[0042] Here, "low risk" means that dogs are less likely to develop late-onset ataxia compared to dogs with a haplotype lacking the PLP1 gene. [Examples]
[0043] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[0044] [Materials and Methods] All experimental protocols using animals and animal-derived samples were approved by the Animal Experiment Committee of Kagoshima University.
[0045] (Dogs, clinical course, and pedigree analysis) The pedigree analysis was conducted using pedigree certificates issued by the Japan Kennel Club for toy poodles that developed delayed-onset ataxia and toy poodles that did not.
[0046] (DNA extraction, genome-wide association study, and haplotype analysis) A genome-wide association study (GWAS) was performed using genomic DNA collected from 65 Toy Poodles. Of these, 27 (15 males, 11 females, and 1 unknown) developed late-onset ataxia, 9 were relatives who did not develop the disease, and 29 (15 males and 14 females) were unrelated Toy Poodles who did not develop the disease.
[0047] Low call rate (<95%), low minor allele frequency (MAF) (<0.01), and deviation from Hardy-Weinberg equilibrium (p<1×10⁻¹⁰). -6 Single nucleotide polymorphisms (SNPs) accompanied by ) were excluded. Autosomal SNPs were analyzed in all dogs, but only male samples were used for X chromosome analysis. Association analysis of case-control studies was performed using a linear mixed model implemented in GEMMA v0.94.1. Kinship matrices were included as random effects, and p-values were determined using likelihood ratio tests. The threshold for genome-wide significance was p = 3.56 × 10⁻⁶. -7 It was set to (0.05 / 140,422).
[0048] Based on the hypothesis that male dogs with delayed-onset ataxia share a risk haplotype containing the causative gene, and that affected female dogs have at least one risk haplotype, we analyzed genotype data of SNPs around specific regions of the X chromosome that showed association in GWAS.
[0049] First, the haplotype shared among all male dogs that developed late-onset ataxia was determined. Next, the haplotype of the female sample was estimated using the shared haplotype. We also checked whether the above haplotype was present in dogs that did not develop late-onset ataxia.
[0050] (Whole genome sequencing of a dog with delayed-onset ataxia) Whole-genome sequencing (WGS) and subsequent bioinformatics analysis were performed on male dogs that developed late-onset ataxia and unaffected male relatives (grandfathers).
[0051] A fragment library was prepared using a commercially available kit (Illumina). Paired-end reads (2 × 150 bp) were acquired with approximately 30x coverage using a HiSeq sequencing system (Illumina).
[0052] Reads were de-adapted using Cutadapt v1.1 and Trimmomatic v0.32, and mapped to a canine reference genome (CanFam3.1) using BWA v0.7.10. PCR duplicates were removed using Picard v1.133. Local realignment and variant calling were performed using GATK v2.3.0. Functional effects of detected mutations were predicted using SnpEff v4.1g and CanFam3.1 annotation. Mapped reads were visualized using IGV v2.8.13.
[0053] (Genome structure analysis) To determine the structure of the large deletion detected by WGS, Sanger sequencing was performed using PCR products from male dogs with delayed-onset ataxia. PCR and Sanger sequencing were performed using standard methods. Table 2 below shows the nucleotide sequences of the primers used for PCR and Sanger sequencing.
[0054] [Table 2]
[0055] Using Genome Workbench v3.6.0, DNA sequences obtained by Sanger sequencing were compared with CanFam3.1 using BLASTn, and the results were visualized by dot plotting.
[0056] (RNA extraction and RNA-seq analysis) Total RNA was extracted from the cerebrums of three dogs—a male and a female Toy Poodle with delayed-onset ataxia, and a male Beagle as a control—using Direct-zol RNA MiniPrep (Zymo Research). Total RNA from the cerebellum was also extracted from the same dogs. RNA concentration and integrity were measured using the Agilent 2100 RNA 6000 Nano Kit (Agilent Technologies).
[0057] Next, the whole RNA library was prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina). Subsequently, it was sequenced using HiSeq (Illumina) to obtain 100 bp paired-end reads.
[0058] Read trimming was performed using cutadapt v3.2 and PRINSEQ v0.20.4, mapping was performed using HISAT2 v2.2.1, and the transcript was quantified using StringTie v2.1.4.
[0059] The reads were mapped to the same canine reference genome, CanFam3.1, used in WGS. The number of reads matching expressed genes was standardized to transcripts per kilobase million (TPM). A pseudocount of 0.5 was added to the expression level values to calculate log2-fold change.
[0060] (Carrier screening of 2,170 dogs of 211 breeds) To estimate whether the structural variations identified in this experiment exist in other regional breeds and populations, we used publicly available SNP data to detect haplotypes shared by dogs with late-onset ataxia. We detected the presence of haplotypes in genotyping data of 404 SNPs from 2,170 male dogs of 211 breeds.
[0061] [Experimental Example 1] (Clinical laboratory tests) Figure 2 shows T1 and T2-weighted magnetic resonance images of the frontal lobe, thalamus, and cerebellum in cross-sections of dogs with delayed-onset ataxia. The results showed ventricular dilation and clarification of sulci in dogs with delayed-onset ataxia.
[0062] [Experimental Example 2] (phylogenetic analysis) A pedigree tree was created based on the pedigree information of 21 Toy Poodles that developed delayed-onset ataxia. Figure 3 shows the created pedigree tree. In Figure 3, "Affected" indicates that the dog developed delayed-onset ataxia, "Unaffected" indicates that the dog did not develop delayed-onset ataxia, and "Unknown" indicates that it is unknown whether or not the dog developed delayed-onset ataxia. Of the 21 dogs, 10 were born to female dogs that developed delayed-onset ataxia, and 9 had at least one littermate that developed delayed-onset ataxia.
[0063] The presence of parents with delayed-onset ataxia suggested a dominant inheritance pattern, but the onset in adult dogs raised the possibility of other inheritance patterns. All dogs that developed delayed-onset ataxia shared a common male ancestor born in the United States. This familial pattern suggested the presence of a strong genetic component for the disease.
[0064] [Experimental Example 3] (Genome-wide association studies and haplotype analysis) We performed a genome-wide association study (GWAS) using toy poodles that developed late-onset ataxia and toy poodles that did not develop the disease, and identified SNPs associated with the disease.
[0065] In the QC process, we observed low call rates (<95%), low minor allele frequencies (MAF) (<0.01), and deviations from the Hardy-Weinberg equilibrium (p<1×10⁻¹⁰). -6 This process eliminated 33,240 SNPs (30,533 on autosomes and 2,707 on the X chromosome). Ultimately, 137,469 SNPs from 25 dogs that developed late-onset ataxia and 38 dogs that did not were used for autosomal analysis. Additionally, 2,953 SNPs from 14 male dogs that developed late-onset ataxia and 21 male dogs that did not were used for X chromosome analysis.
[0066] Figure 4(a) is a Manhattan plot showing the GWAS results. A QQ plot is shown in the upper left. Figure 4(b) is a magnified Manhattan plot of the X chromosome portion of Figure 4(a). In Figures 4(a) and (b), the dotted lines indicate the p-value corrected for Bonferroni at 0.05. Figure 4(c) shows the haplotypes around associated SNPs in dogs with and without late-onset ataxia, along with screenshots of the corresponding regions from the NCBI Genome Data Viewer.
[0067] As a result, of the 140,422 SNPs, five SNPs located on the X chromosome were significantly associated with the development of late-onset ataxia (p<3.56×10⁻¹⁰). -7Furthermore, no SNPs located on autosomes were found to be associated with the development of late-onset ataxia. Additionally, SNPs significantly associated with the development of late-onset ataxia were located between 75.2 Mb and 90.6 Mb on the X chromosome.
[0068] By using the genotype of SNPs around relevant regions on the X chromosome, a haplotype shared by all male dogs that developed late-onset ataxia was detected. The shared haplotype ranged from 74.8 Mb (BICF2G63014737) to 91.5 Mb (BICF2G63010158). None of the male dogs that did not develop late-onset ataxia possessed this haplotype.
[0069] Next, we analyzed corresponding SNPs in female dogs that developed late-onset ataxia using haplotype information from male dogs that developed late-onset ataxia. Of the 11 female dogs that developed late-onset ataxia, 7 had the same haplotype as the male dogs that developed late-onset ataxia.
[0070] In all female dogs that developed delayed-onset ataxia, with the exception of one, a 3' short haplotype was identified. The short haplotype ranged from 74.8 Mb (BICF2G63014737) to 88.0 Mb (BICF2G63011054). The chromosomal region of the SNP adjacent to the haplotype (74.7 Mb to 88.0 Mb) contained 132 genes. Since this haplotype is located on the X chromosome, females may have up to two copies, but female dogs that developed delayed-onset ataxia had only one copy of these haplotypes, and none had two copies. Of the 17 female toy poodles that did not develop delayed-onset ataxia, one had one copy of the haplotype.
[0071] [Experimental Example 4] (Detection of structural variations) We obtained 691,000,000 150 bp paired-end reads from dogs that developed late-onset ataxia and 731,000,000 150 bp paired-end reads from dogs that did not develop the disease, and performed whole-genome sequencing (WGS) with an average coverage of 32.8 times.
[0072] Figure 5 shows the results of mapping leads obtained from male Toy Poodles with delayed-onset ataxia (affected dogs) to the canine reference genome (CanFam3.1). As a control, the results of mapping leads obtained from male Toy Poodles with delayed-onset ataxia (healthy dogs) to the canine reference genome (CanFam3.1) are shown.
[0073] As a result, it was revealed that the haplotype of dogs that developed late-onset ataxia had a deletion of approximately 50 kbp located at positions 77,175,958 to 77,226,911 on the X chromosome. The deleted region contained the complete PLP1 gene. This genomic mutation was not observed in related dogs that did not develop late-onset ataxia.
[0074] Next, primers for Sanger sequencing of the mutated genome were designed based on the DNA sequence predicted from the WGS reads, and Sanger sequencing was performed. Subsequently, the Sanger-sequenced DNA sequence was compared with that of CanFam3.1 using BLASTn.
[0075] Figure 6(a) is a dot plot showing the results of comparing the DNA base sequences. It was revealed that the mutated genome contains deletions of 49 kbp and 1.7 kbp, a duplication of 971 bp originating from the 5' upstream side, and an insertion of 69 bp originating from the complementary strand. Figure 6(b) is a schematic diagram showing the structure of the mutated genome.
[0076] Next, WGS reads from male dogs that developed and did not develop late-onset ataxia were mapped to a canine reference genome (CanFam3.1) that had been artificially modified to contain the mutated genome, and the sequence of the mutated genome was examined to determine whether it was correct.
[0077] Figure 7 shows the results of mapping the reads. In Figure 7, the dashed lines indicate regions where structural mutations exist. As a result, fragmented reads were observed in male dogs that did not develop late-onset ataxia (healthy dogs), whereas no fragmented reads were observed in male dogs that developed late-onset ataxia (affected dogs). This result indicates that the mutated genome sequence is correct. Furthermore, as shown in Figure 7, it was revealed that dogs that developed late-onset ataxia have a small number of nucleotides that are not present in the reference sequence.
[0078] [Experimental Example 5] (RNA-seq analysis) RNA-seq analysis revealed that the PLP1 gene exhibited the expected expression pattern and did not affect the expression of other genes. Because the structural mutation included a complete PLP1 gene, PLP1 gene expression was not observed in the brains of male dogs with delayed-onset ataxia, and it was predicted that in the brains of female dogs with delayed-onset ataxia, the expression level of the PLP1 gene would be half that of male dogs without delayed-onset ataxia due to X chromosome inactivation.
[0079] To detect changes in gene expression in dogs with late-onset ataxia, the cerebral and cerebellar transcriptomes of toy poodles with late-onset ataxia were compared to those of beagles without the condition. 116,700,000 100bp paired-end reads were obtained from the cerebral and cerebellar tissues of three dogs.
[0080] Figure 8(a) shows the results of gene expression analysis in the cerebrum. Figure 8(b) shows the results of gene expression analysis in the cerebellum. Gene expression levels were standardized to transcripts per kilobase million (TPM) values.
[0081] In Figures 8(a) and (b), the size of the plots corresponds to the mean expression level (indicated as "A" in the legend). "R" represents the TPM value of dogs that developed late-onset ataxia, and "G" represents the TPM value of dogs that did not develop late-onset ataxia.
[0082] Analysis of gene expression in the cerebrum revealed that the expression levels of two genes were more than 32 times higher compared to male dogs without late-onset ataxia, and the expression levels of three genes were more than 32 times lower compared to male dogs without late-onset ataxia.
[0083] Of these genes, only the PLP1 gene showed a significant decrease in the cerebellum of dogs that developed late-onset ataxia. Compared to dogs that did not develop late-onset ataxia, the expression level of the PLP1 gene in the cerebrum of female dogs with late-onset ataxia was 0.5 times, and the expression level in the cerebellum was 0.2 times. None of the other genes showed similar expression patterns between the cerebrum and cerebellum, or between male and female dogs with late-onset ataxia.
[0084] These results strongly suggest that genomic changes identified in dogs with late-onset ataxia lead to a deletion in PLP1 gene expression, which is the cause of this disease.
[0085] [Experimental Example 6] (Carrier screening of 2,170 dogs of 211 breeds) We estimated whether the structural variants identified in this experiment exist in other populations. Using publicly available SNP data obtained from the same Illumina array, we detected haplotypes shared by dogs that developed late-onset ataxia. SNP data from 2,170 male dogs of 211 breeds were examined. As a result, it was revealed that one toy poodle had the same haplotype. Other dog breeds did not have this haplotype. [Industrial applicability]
[0086] The present invention provides a technology for diagnosing the risk of developing delayed-onset ataxia. It also provides a diagnostic kit for delayed-onset ataxia, a method for breeding dogs with a reduced risk of developing delayed-onset ataxia, and dogs with a low risk of developing delayed-onset ataxia.
Claims
1. The procedure includes a step of detecting a haplotype lacking the Protein 1 (PLP1) gene in the genome of a test dog, The detection of the aforementioned haplotype indicates that the test dog is at risk of developing late-onset ataxia, or that the test dog will produce offspring that are at risk of developing late-onset ataxia. The haplotype lacking the PLP1 gene has the nucleotide sequence described in Sequence ID No. 1, The aforementioned test dog is a Toy Poodle or a hybrid thereof. Diagnostic methods for assessing the risk of developing delayed-onset ataxia.
2. This product contains a reagent for detecting haplotypes lacking the PLP1 gene in the genome of a test dog. The haplotype lacking the PLP1 gene has the nucleotide sequence described in Sequence ID No. 1, The aforementioned test dog is a Toy Poodle or a hybrid thereof. The reagent for detecting the PLP1 gene-deleting haplotype includes a primer set that amplifies the base sequence described in SEQ ID NO: 1 but does not amplify the wild-type PLP1 allele. Diagnostic kit for delayed-onset ataxia.
3. The kit for diagnosing delayed-onset ataxia according to claim 2, wherein the primer set that amplifies the base sequence described in SEQ ID NO: 1 and does not amplify the wild-type PLP1 allele comprises a primer consisting of the base sequence shown in SEQ ID NO: 4 and a primer consisting of the base sequence shown in SEQ ID NO:
5.
4. A method for breeding dogs with a reduced risk of developing delayed-onset ataxia, A step of diagnosing the risk of developing delayed-onset ataxia in a test dog using the diagnostic method described in claim 1, The process includes the step of breeding the test dogs that have been diagnosed as not having a haplotype lacking the PLP1 gene, A method wherein the test dog is a toy poodle or a hybrid thereof.