Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan

Abstract

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.

Genotyping Assays for the Canine Degenerative Myelopathy-Associated c.118G>A (p.E40K) Mutation of the <i>SOD1</i> Gene Using Conventional and Real-Time PCR Methods: A High Prevalence in the Pembroke Welsh Corgi Breed in Japan

“…DNA templates for the real-time PCR assay were also pre-pared using a disc 1.2 mm in diameter punched from the FTA card as reported previously [6]. Real-time PCR amplification was conducted with the primers and probes listed in Table 1 using the same protocol and reagents described previously [6], except for the annealing temperature (58°C).…”

“…DNA templates were prepared using whole blood spotted onto Flinders Technology Associates filter paper (FTA card; Whatman International Ltd., Piscataway, NJ, U.S.A.). For the PCR-restriction fragment length polymorphism (RFLP) assay, a disc 1.2 mm in diameter punched out of the FTA card was used as a template after quick washing as reported previously [6]. Each PCR test was carried out targeting a sequence around c.118G>A of the canine SOD1 gene with forward and reverse primers shown in Table 1 using a general protocol.…”

Trapped Neutrophil Syndrome in a Border Collie Dog: Clinical, Clinico-Pathologic, and Molecular Findings

“…This is the first case of TNS to be molecularly diagnosed in Japan. To our knowledge, only two reports have described TNS in Border Collies [1,9]. One case report in New Zealand 15 years ago described in some detail the clinical features of two littermates suspected of having inherited neutropenia, but not molecularly diagnosed with TNS [1].…”

“…The LP analysis was conducted with the forward and reverse primers listed in Table 2 using the same protocol and reagents as for the direct DNA sequencing. The analysis of the products was performed using a microchip electrophoresis system (MCE-202 MultiNA Microchip Electrophoresis System, Shimadzu Corp., Kyoto, Japan), as reported previously [8,9].…”

“…Blood samples from four healthy Beagles, stored using FTA cards, were used as a control. A disc punched out of a blood-or saliva-spotted FTA card was used directly as a template for PCR after quick washing as reported previously [8,9].…”