Real-Time PCR Genotyping Assay for GM2 Gangliosidosis Variant 0 in Toy Poodles and the Mutant Allele Frequency in Japan

Rahman, Mohammad Mahbubur; Yabuki, Akira; Kohyama, Moeko; Mitani, Sawane; Mizukami, Keijiro; Uddin, Mohammad M.; Chang, Hye-Sook; Kushida, Kazuya; Kishimoto, Miori; Yamabe, Remi; Yamato, Osamu

doi:10.1292/jvms.13-0443

Cited by 8 publications

(6 citation statements)

References 15 publications

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“…In Japan, molecular epidemiological surveys have been carried out for several inherited canine diseases to determine the associated mutant allele frequencies and thereby evaluate the necessity of prevention measures [27][28][29][30][31]. Among these, lethal diseases characterized by progressive neurological dysfunction include GM1 gangliosidosis in Shiba Inus (mutant allele frequency = 0.00509) [27] and Mame Shibas (0.00246) [28], neuronal ceroid lipofuscinosis (NCL) in Chihuahuas (0.00645) [30], Sandhoff disease in Toy Poodles (0.00101) [29], and NCL in Border Collies (0.0405) [31].…”

Section: Discussionmentioning

confidence: 99%

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate

Maki

Islam

Itoh

et al. 2022

Animals

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Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy–Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs’ owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.

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Section: Discussionmentioning

confidence: 99%

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate

Maki

Islam

Itoh

et al. 2022

Animals

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“…These animals included a 14-month-old Shiba Inu with GM1 gangliosidosis diagnosed in 2009, an 11-month-old domestic shorthair cat with GM1 gangliosidosis diagnosed in 2004, a 20-month-old Toy Poodle with GM2 gangliosidosis diagnosed in 2006, and a 20-month-old domestic shorthair cat with GM2 gangliosidosis diagnosed in 2010. The diagnosis of these animals was established using genetic and/or biochemical tests reported previously [ 11 , 43 – 45 ]. Stored paraffin-embedded cerebral cortex samples of a dog and a cat without any brain disease were also used as controls.…”

Section: Methodsmentioning

confidence: 99%

In situ detection of GM1 and GM2 gangliosides using immunohistochemical and immunofluorescent techniques for auxiliary diagnosis of canine and feline gangliosidoses

et al. 2016

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BackgroundGM1 and GM2 gangliosidoses are progressive neurodegenerative lysosomal storage diseases resulting from the excessive accumulation of GM1 and GM2 gangliosides in the lysosomes, respectively. The diagnosis of gangliosidosis is carried out based on comprehensive findings using various types of specimens for histological, ultrastructural, biochemical and genetic analyses. Therefore, the partial absence or lack of specimens might have resulted in many undiagnosed cases. The aim of the present study was to establish immunohistochemical and immunofluorescent techniques for the auxiliary diagnosis of canine and feline gangliosidoses, using paraffin-embedded brain specimens stored for a long period.ResultsUsing hematoxylin and eosin staining, cytoplasmic accumulation of pale to eosinophilic granular materials in swollen neurons was observed in animals previously diagnosed with GM1 or GM2 gangliosidosis. The immunohistochemical and immunofluorescent techniques developed in this study clearly demonstrated the accumulated material to be either GM1 or GM2 ganglioside.ConclusionsImmunohistochemical and immunofluorescent techniques using stored paraffin-embedded brain specimens are useful for the retrospective diagnosis of GM1 and GM2 gangliosidoses in dogs and cats.

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“…Once the disease-causing variant is identified, other PCR-based genetic testing methods such as PCR-restriction fragment length polymorphism (PCR-RFLP) and TaqMan real-time PCR assays can be designed, enabling rapid detection of the target variant in any standard laboratory. These assays are available for the detection of single-base changes, such as point mutations and single nucleotide polymorphisms (SNPs), and used as genotyping assays for canine hereditary diseases [12][13][14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

et al. 2021

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Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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Real-Time PCR Genotyping Assay for GM2 Gangliosidosis Variant 0 in Toy Poodles and the Mutant Allele Frequency in Japan

Cited by 8 publications

References 15 publications

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate

In situ detection of GM1 and GM2 gangliosides using immunohistochemical and immunofluorescent techniques for auxiliary diagnosis of canine and feline gangliosidoses

PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

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