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Background: Canine GM1 gangliosidosis is a fatal disease in the Shiba Inu breed, which is one of the most popular traditional breeds in Japan and is maintained as a standard breed in many countries. Therefore, it is important to control and reduce the prevalence of GM1 gangliosidosis for maintaining the quality of this breed and to ensure supply of healthy dogs to prospective breeders and owners. This molecular epidemiological survey was performed to formulate an effective strategy for the control and prevention of this disease., Results: The survey was carried out among 590 clinically unaffected Shiba Inu dogs from the 8 districts of Japan, and a genotyping test was used to determine nation-wide and regional carrier frequencies. The number and native district of affected dogs identified in 16 years from 1997 to June 2013 were also surveyed retrospectively. Of the 590 dogs examined, 6 dogs (1.02%, 6/590) were carriers: 3 dogs (2.27%, 3/132) from the Kinki district and the other 3 dogs from the Hokkaido, Kanto, and Shikoku districts. The retrospective survey revealed 23 affected dogs, among which, 19 dogs (82.6%) were born within the last 7 years. Of the 23 affected dogs, 12 dogs (52.2%) were from the Kinki district. Pedigree analysis demonstrated that all the affected dogs and carriers with the pedigree information have a close blood relationship., Conclusions: Our results showed that the current carrier frequency for GM1 gangliosidosis is on the average 1.02% in Japan and rather high in the Kinki district, which may be related to the high prevalence observed over the past 16 years in this region. This observation suggests that carrier dogs are distributed all over Japan; however, kennels in the Kinki district may face an increased risk of GM1 gangliosidosis. Therefore, for effective control and prevention of this disease, it is necessary to examine as many breeding dogs as possible from all regions of Japan, especially from kennels located in areas with high prevalence and carrier frequency.

Trapped neutrophil syndrome is an autosomal recessive inherited neutropenia in Border collies. The causative mutation is a 4base pair deletion in exon 19 of the canine VPS13B gene. In this study, a real-time PCR assay was developed and a genotyping survey was carried out in Border collies in Japan. The carrier frequency was 11.1%, suggesting that the mutant allele frequency is high enough to warrant measures to control and prevent the disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene. Toy poodles recently were reported as the second breed of dog with SD. The present paper describes the molecular defect of this canine SD as the first identification of a pathogenic mutation in the canine HEXB gene. Genomic and complementary DNA sequences covering exonic regions of the canine HEXB gene, except exon 1, were analysed using DNA and RNA in an affected dog. A homozygous single base pair deletion of guanine in exon 3 was identified at nucleotide position 283 of the putative open reading frame (c.283delG). This mutation has the potential to cause a frameshift resulting in the alteration of valine at amino acid position 59 to a stop codon (p.V59fsX). Genotyping using the mutagenically separated PCR method demonstrated a correlation between phenotype and genotype in dogs with a pedigree related to the disease and that the mutation was rare in a randomly-selected population of toy poodles. These results strongly suggest that the deletion is pathogenic., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To describe a Hokkaido dog, one of the traditional Japanese breeds that was affected by Collie eye anomaly (CEA), and to report the genotype of this dog and the Hokkaido dog allelic frequency of the CEA-associated mutation., Case: A nine-month-old intact female Hokkaido dog without any obvious visual disturbance was diagnosed ophthalmoscopically with CEA. Severe choroidal hypoplasia was observed in the bilateral temporal area adjacent to the optic nerve head, appearing as whitish areas. Therefore, the dog was suspected of possessing the CEA-associated mutation that was previously reported as an intronic 7.8-kilo base deletion in the canine NHEJ1 gene., Procedures: SYBR Green-based real-time PCR with a melting curve analysis, conventional PCR with agarose gel electrophoresis, and direct DNA sequencing were carried out to determine the genotype of the dog. Furthermore, a preliminary genotyping survey was carried out in 17 Hokkaido dogs from three kennels using the real-time PCR method, and the pedigree relationships were analyzed using their pedigree papers., Results: The Hokkaido dog affected by CEA was proven to possess the CEA-associated mutation. Of these 17 Hokkaido dogs, 12 dogs were heterozygous carriers and five dogs were affected by this mutation. The preliminary genotyping survey and pedigree analysis demonstrated that the allelic frequency of the CEA-associated mutation is very high in Hokkaido dogs., Conclusion: These data suggest that the Hokkaido breed is highly susceptible to CEA because of the known CEA-associated mutation much like the Collie-related breeds., (© 2011 American College of Veterinary Ophthalmologists.)

P-glycoprotein, encoded by the MDR1 or ABCB1 gene, is an integral component of the blood-brain barrier as an efflux pump for xenobiotics crucial in limiting drug uptake into the central nervous system. Dogs homozygous for a 4-base pair deletion of the canine MDR1 gene show altered expression or function of P-glycoprotein, resulting in neurotoxicosis after administration of the substrate drugs. In the present study, the usefulness of microchip electrophoresis for genotyping assays detecting this deletion mutation was evaluated. Mutagenically separated polymerase chain reaction (MS-PCR) and real-time PCR assays were newly developed and evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies dogs in Japan to determine the allele frequency in this breed. Microchip electrophoresis showed advantages in detection sensitivity and time saving over other modes of electrophoresis. The MS-PCR assay clearly discriminated all genotypes. Real-time PCR assay was most suitable for a large-scale survey due to its high throughput and rapidity. The genotyping survey demonstrated that the carrier and mutant allele frequencies were 0.49% and 0.25%, respectively, suggesting that the mutant allele frequency in Border Collies is markedly low compared to that in the susceptible dog breeds such as rough and smooth Collies.

Neuronal ceroid lipofuscinosis (NCL) constitutes a group of recessively inherited lysosomal storage diseases that primarily affect neuronal cells. Such diseases share certain clinical and pathologic features in human beings and animals. Neuronal ceroid lipofuscinosis in Border Collie dogs was first detected in Australia in the 1980s, and the pathogenic mutation was shown to be a nonsense mutation (c.619C>T) in exon 4 in canine CLN5 gene. In the present study, novel rapid genotyping assays including polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR primer-induced restriction analysis, mutagenically separated PCR, and real-time PCR with TaqMan minor groove binder probes, were developed. The utility of microchip electrophoresis was also evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies in Japan using these assays to determine the current allele frequency in Japan, providing information to control and prevent this disease in the next stage. All assays developed in the current study are available to discriminate these genotypes, and microchip electrophoresis showed a timesaving advantage over agarose gel electrophoresis. Of all assays, real-time PCR was the most suitable for large-scale examination because of its high throughput. The genotyping survey demonstrated that the carrier frequency was 8.1%. This finding suggested that the mutant allele frequency of NCL in Border Collies is high enough in Japan that measures to control and prevent the disease would be warranted. The genotyping assays developed in the present study could contribute to the prevention of NCL in Border Collies.

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.