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 mutations, c.[462A > T; 463A > T] . Importantly, false-positive and false-negative errors were never detected in both PCR-RFLP and TaqMan real-time PCR assays, demonstrating the high specificity and sensitivity of the established assays. While PCR-RFLP and TaqMan PCR assays are capable of detecting even a single-base difference, the target APC mutation is a two-base substitution , providing substantial advantage to enhance the specificities of the established assays.
Although the PCR-RFLP assay is one of the most common methods for genotyping, a lack of suitable restriction enzymes hinders its application in some cases. In this study, we successfully developed the PCR-RFLP assay for hereditary GI polyposis by taking advantage of the causative germline APC mutations creating a new restriction enzyme site for MseI. In the PCR-RFLP assay, MseI digestion yielded diagnostic fragments of 51 and 58 bp from the mutant APC allele and differentiated carrier and non-carrier dogs. In this assay, the PCR amplicon contained two restriction sites of MseI besides the mutation site; therefore, the 156-bp band disappeared after the digestion regardless of the genotypes, contributing to the prevention of false negative errors by incomplete digestion. Furthermore, the entire process of this assay can be completed within half a day using an ordinary thermal cycler and electrophoresis apparatus, enabling rapid diagnosis in any standard laboratory. In addition, by using the buccal swab as test materials, determination of the genotypes is possible in a less invasive manner. Moreover, when dogs are suspected to have a hereditary disease on histopathological examination of their GI lesions, it is possible to determine their APC genotype from FFPE samples without the need for additional blood or swab samples. This approach is also applicable to retrospective studies using archival FFPE samples.
TaqMan duplex real-time PCR assay requires dedicated equipment capable of measuring multiple fluorescences in real time, but this assay enables high-throughput genotyping. Therefore, it is suitable for large-scale molecular epidemiological studies. Another advantage of this assay is determination of genotypes in a single PCR without any additional post-amplification manipulations, thus minimizing the risk of human errors.
The genetic tests developed in this study would provide significant benefits in small animal practice. Clinical veterinarians need to differentiate between hereditary and sporadic GI cancers in dogs for the following reasons. First, JRTs affected with the hereditary disease are much more likely than sporadic cases to have multiple cancers . Second, in the case of the hereditary GI polyposis, the affected dogs can be expected to have a longer survival time than sporadic cases although they have an increased lifelong risk of disease recurrence . The genetic testing enables the differential diagnosis between hereditary and sporadic GI cancers. In addition, JRTs with chronic GI symptoms such as vomiting and bloody stool can be candidates for genetic testing . Considering that endoscopic examination requires general anesthesia in dogs, genetic testing could be an option to predict the occurrence of GI lesions in JRTs. The positive test results strongly support the need for further examinations such as endoscopy. Furthermore, genetic testing would be useful for the assessment of future risk for hereditary GI polyps. Many commercial laboratories have recently offered genetic tests for canine and feline hereditary diseases , available to pet owners and veterinarians for assessment of lifelong risk. Hereditary GI polyposis is an adult-onset disease, and initial GI lesions can arise at variable ages, reportedly between 2 to over 10 years . Therefore, knowing the lifelong risk is necessary for early detection and treatment.
Genetic testing is indispensable for preventing the spread of hereditary diseases in dogs [2, 9, 10]. In adult-onset diseases, there is a risk of the mutation carriers being unintentionally used for breeding before the disease onset. If a popular sire, such as a dog show champion, is a mutation carrier, causative mutations can spread rapidly within the breed due to their extensive use for breeding . Our previous retrospective study revealed that the incidence of GI polyposis in JRTs increased since the late 2000s, and that all the affected dogs were born during the first decade of the 2000s . The spread of hereditary GI polyposis among JRTs in Japan might be due to the popular side effect during this period. Proper genetic screening of sires and dams before breeding would prevent the transmission of germline APC mutations to future generations. This approach would substantially reduce the future incidence and eventually eradicate this hereditary disease in the future. Therefore, genetic screening should preferably become a routine for animal breeding .
The genotyping assays established in this study would certainly facilitate large-scale epidemiological studies. There are no reports of JRTs with hereditary GI polyposis in other countries, except Japan; thus, the germline APC mutations are possibly prevalent only among JRTs in Japan. However, when examining the pedigree certificates of the JRTs affected by hereditary GI polyposis, some ancestor dogs were introduced from Australia (unpublished data), and thus the possibility of the presence of JRTs with the germline APC mutation in other countries cannot be completely denied. Furthermore, the prevalence of germline APC mutations in JRTs in Japan remains to be investigated. Further studies are needed to clarify the prevalence of germline APC mutations in JRTs in Japan and other countries. Moreover, in future epidemiological studies of healthy JRTs, the carrier dogs could be identified before disease onset, indicating the usefulness of genetic testing for future risk assessment.