Abstract
BackgroundThe 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.ResultFirst, 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.ConclusionIn 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.
Highlights
The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s
Onset at a young age is a distinctive feature, initial GI lesions can arise at a wide range of ages, even after 10 years in JRTs with germline APC variant [6]. These findings indicate the necessity of genetic testing for definitive diagnosis of hereditary GI polyposis
We showed that the polymerase chain reaction (PCR)-RFLP and TaqMan duplex real-time PCR assays were capable of accurately determining the germline APC variant status of carrier and non-carrier dogs without any false-positive or falsenegative errors
Summary
The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. We demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state This disease is an autosomal dominant hereditary disorder. We demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene, c.[462_ 463delinsTT] (OMIA ID: 001916–9615), in the heterozygous state This is an autosomal dominant hereditary disorder [6]. The solitary and multiple polyps were found in either one or both the stomach and colorectum, with a predilection for the gastric antrum and rectum, in the affected dogs, and most of the GI lesions were histopathologically diagnosed as adenomas or adenocarcinomas [6] This cancer-prone disease could possibly be a canine counterpart of attenuated familial adenomatous polyposis (FAP) in humans [7]
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