Kyoko Yoshizaki1, Akihiro Hirata2, Hiroyuki Matsushita1, Naohito Nishii3, Mifumi Kawabe4, Takashi Mori5,6, Hiroki Sakai1,6. 1. Laboratory of Veterinary Pathology, Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. 2. Laboratory of Veterinary Pathology, Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. akatsuki@gifu-u.ac.jp. 3. Laboratory of Veterinary Internal Medicine, Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. 4. Laboratory of Veterinary Clinical Radiology, Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. 5. Laboratory of Veterinary Clinical Oncology, Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. 6. Center for Highly Advanced Integration of Nano and Life Sciences, Gifu University (G-CHAIN), 1-1 Yanagido, Gifu, 501-1193, Japan.
Abstract
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.
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.
Entities:
Keywords:
Diagnosis; Genetic testing; Hereditary gastrointestinal polyposis; Jack Russell terrier
Authors: Keijiro Mizukami; Hye-Sook Chang; Akira Yabuki; Takuji Kawamichi; Natsuko Kawahara; Daisuke Hayashi; Mohammad A Hossain; Mohammad M Rahman; Mohammad M Uddin; Osamu Yamato Journal: J Vet Diagn Invest Date: 2011-11 Impact factor: 1.279