Molecular Epidemiological Study of Germline APC Variant Associated with Hereditary Gastrointestinal Polyposis in Dogs: Current Prevalence in Jack Russell Terriers in Japan and Breed Distribution

doi:10.21203/rs.3.rs-1557638/v1

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Molecular Epidemiological Study of Germline APC Variant Associated with Hereditary Gastrointestinal Polyposis in Dogs: Current Prevalence in Jack Russell Terriers in Japan and Breed Distribution

https://doi.org/10.21203/rs.3.rs-1557638/v1

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Background: Cases of gastrointestinal (GI) neoplastic polyps in Jack Russell Terriers (JRTs) have increased in Japan since the late 2000s. We recently demonstrated that JRTs with GI polyps heterozygously harbor an identical germline variant in the adenomatous polyposis coli (APC) gene, c.[462_463delinsTT]; therefore, this is an autosomal dominant hereditary disease. We conducted a molecular epidemiological study to explore the current prevalence of germline APC variants in JRTs in Japan and the breed distribution of this disease.

Results: Peripheral blood samples from 792 JRTs were collected at 93 veterinary hospitals in Japan in 2020. Using an established polymerase chain reaction-restriction fragment length polymorphism assay, the germline APC variant was detected in 15 JRTs, with an overall prevalence of 1.89%. The prevalence was not significantly different for sex, age, and coat type criteria. Notably, the variant carriers had a current or previous history of GI neoplastic polyps, providing further evidence of the association of the germline APC variant with GI polyposis. Pedigree analysis of carrier dogs revealed that the germline APC variant was no longer confined to a few specific bloodlines but was widely spread among JRTs in Japan. Furthermore, some ancestors of the carriers were from Australia or New Zealand, suggesting the possible presence of carriers in countries other than Japan. Next, we retrospectively investigated the germline APC variant status of dogs with GI epithelial tumors using genomic DNA samples extracted from archived pathological specimens, as well as those stored in a canine genome bank. In total, 67 purebred dogs of 24 breeds, including four JRTs, and four mixed-breed dogs were examined. While three variant carriers were found in JRTs, the germline APC variant was not detected in any of the other breeds.

Conclusion: The current prevalence of the germline APC variant was approximately 2% in JRTs in Japan and the prevalence remained roughly flat during the last 15 years. In addition, hereditary GI polyposis associated with the variant was virtually specific to JRTs.

hereditary gastrointestinal polyposis

Jack Russell Terrier

germline APC variant

epidemiology

prevalence

breed distribution

gastric cancer

colorectal cancer

In dogs, breed predisposition often provides the first clue to the discovery of a novel hereditary disorder [1]. Although gastrointestinal (GI) epithelial tumors are rare in dogs [2, 3], the number of cases of Jack Russell Terriers (JRTs) with GI neoplastic polyps has recently increased in Japan [4–6] despite a lack of a corresponding increase in JRT population [7]. In a recent study, we demonstrated that JRTs with GI polyps harbor an identical germline variant in the adenomatous polyposis coli (APC) gene, c.[462_463delinsTT] (GenBank ID: LC598892.1) in a heterozygous state; thus, this is an autosomal dominant hereditary disorder (OMIA ID 001916–9615) [6]. JRTs affected by hereditary GI polyposis develop solitary or multiple neoplastic polyps in the GI tract, and most lesions are histopathologically diagnosed as adenomas or adenocarcinomas [6]. These polyps occur primarily in the stomach and large intestine, with a predilection for the gastric antrum and rectum, and multiple lesions can develop simultaneously at both locations [6]. Early onset and frequent recurrence of GI polyps are also features of hereditary GI polyposis [6]. Based on similarities in the responsible genes and disease phenotypes, this disease can be considered a canine counterpart of familial adenomatous polyposis in humans [8].

When compared with dogs affected with sporadic GI cancers, JRTs with hereditary GI polyposis are expected to have longer survival times, with reported 1- and 2-year survival rates of 100%; but at the same time, they have an increased lifelong risk of recurrence [6]. Therefore, it is critical for veterinary clinicians to distinguish cases of hereditary GI polyposis from those of sporadic GI cancer to provide appropriate treatment and accurately predict prognosis. Once a disease-causing variant is identified, it is possible to develop specific genetic testing for the target variant. In a recent study, we established polymerase chain reaction (PCR)-based genotyping assays capable of detecting the germline APC variant [9]. These assays can facilitate not only clinical diagnosis, but also large-scale epidemiological studies of this novel hereditary disease.

Our previous retrospective analysis revealed that cases of JRTs with hereditary GI polyposis began to increase in the late 2000s, and most affected JRTs were born in the first decade of the 2000s [9], suggesting that the germline APC variant could have spread among JRTs in Japan during this period. However, the extent to which germline APC variants spread in JRTs remains unexplored. A large-scale epidemiological study would determine current prevalence and fluctuation of the APC variant among JRTs during the last one or two decades.

According to well-established online databases [10–13], there are currently hundreds of hereditary diseases with known causative gene variants in dogs. While many of these diseases occur at a greater frequency in a single breed or several related breeds, some disorders are common to multiple breeds [1, 13]. Recently, Donner et al. investigated 18,000 purebred dogs among 330 different breeds using a DNA panel screening test, testing for 152 known genetic variants, and clearly demonstrated that several known disease-associated variants were more widespread across different breeds than previously recognized [14]. However, this study validated the fact that several disease-associated variants still show complete or virtually complete breed-specificity [14]. Therefore, it is necessary to determine whether this novel hereditary disease is specific to JRTs.

In the present study, using the established genotyping assays [9], we explored the current prevalence of the germline APC variant associated with hereditary GI polyposis in JRTs in Japan and investigated the breed distribution of this disease by retrospectively examining archived samples of dogs with GI epithelial tumors.

Epidemiological survey of JRTs

Current prevalence of germline APC variant associated with hereditary GI polyposis in JRTs

The peripheral blood of 792 JRTs collected at 93 veterinary clinics in Japan in 2020 was analyzed. Concentrations of the extracted genomic DNA ranged widely from 2.2 to 489.0 ng/µL. An established PCR-restriction fragment length polymorphism (PCR-RFLP) assay successfully determined the APC genotypes of all examined JRTs, and 15 JRTs were found to be carriers of the germline APC variant (Fig. 1A). The presence of the APC variant, c.[462_463delinsTT], was validated by PCR-direct sequencing for all 15 JRTs (Fig. 1B). The overall prevalence of germline APC variants in JRTs was 1.89%. There was no significant difference in prevalence depending on sex and coat type (Table 1). When divided into 5-year age groups, the prevalence of the APC variant was around 2% in all age groups without significant fluctuations.

Medical history and current symptoms of APC variant carriers

Interviews with veterinarians and dog owners revealed information on the current and previous medical histories and clinical symptoms of the detected APC variant carriers. As expected, of ten carriers with available information, five developed GI cancers before or after the present epidemiological survey (Table 2). Case no. JRT216 had a previous history of repeated occurrence of rectal adenocarcinoma at the age of 4 and 6 years. Despite the lack of histopathological examination, the dog had recurrence of rectal polyp at age 7. As for case no. JRT152, the 8-year-old female dog was presented to a veterinary clinic with blood-tingled vomitus during the present epidemiological study, and polyps were endoscopically detected in the cardia and antrum of the stomach (Fig. 1C). Based on histopathological examination of the biopsy specimen, both gastric polyps were diagnosed as adenocarcinomas (Fig. 1D, E). In case no. JRT203, the detection of the germline APC variant in the present study led to the early detection of gastric cancer. The dog was prone to vomiting, and therefore in response to the positive genetic test result, she underwent endoscopic examination at the age of 6 years. An antrum polyp was detected and histopathologically diagnosed as an adenocarcinoma. Furthermore, two dogs developed GI cancers after the survey. The first case was of JRT317; the dog had no GI symptoms at the time of blood sampling. Six months later, bloody stool was observed, and rectal adenocarcinoma was surgically resected when the dog was 3 years of age. The second dog, case no. JRT342, had a history of intermittent vomiting for many years; when the dog was 11 years of age, endoscopic biopsy revealed adenocarcinoma in the antrum of the stomach. Three months later, abdominal surgery was performed, but tumor resection was abandoned due to adhesion to adjacent organs, including the pancreas. During abdominal surgery, the dog underwent Billroth II gastrojejunostomy to relieve the gastric outlet obstruction but died 2 months later.

Our previous study showed that vomiting and bloody stool are the most frequent symptoms in JRTs after the onset of hereditary GI polyposis [6]. Despite the absence of a clinical examination for GI cancers, GI symptoms were present in four carriers (Table 2), of which JRT056 showed serious GI symptoms before death from an uncertain cause at the age of 10 years. Among the detected variant carriers with the available information, only JRT221 remained asymptomatic until the age of 3.5 years.

Pedigree analysis of the APC variant carriers

Pedigree certificates of nine carriers were available with the cooperation of the dog owners, and they contained the information of three generations in the ancestry of each dog. The blood relationships of the carriers were analyzed along with five additional previous cases (Supplemental Table 1) [6].

Three groups with blood relationships were identified among the carriers. The first group consisted of paternal half-sisters, JRT203 and JRT317, and their mothers were also non-littermate full-sisters (Fig. 2A). Either the father or one of the maternal grandparents had introduced the germline APC variant. In the second group, JRT216 was a maternal half-brother of JRT-P04, and JRT196 was also a descendant of their common mother dog (Fig. 2B). The last group contained four JRTs that descended from a common breeding pair, and the mother of JRT221 was a full sister of JRT-P05 (Fig. 2C). It should also be noted that the remaining two carriers, JRT152 and JRT447, had no relationship with any other carriers in the past three generations, indicating that the germline APC variant was no longer confined to a few specific bloodlines and was widely spread among JRTs in Japan.

The pedigree analysis also revealed that many JRTs from Australia and New Zealand were present among the ancestors of the APC variant carriers, mainly in the great-grandparents’ generation (Table 3).

Breed distribution of hereditary GI polyposis in dogs

To investigate breed distribution of hereditary GI polyposis, germline APC variant status of dogs with GI epithelial tumors was retrospectively examined using samples obtained from the pathology archive and canine genome bank.

Analysis of archived pathological samples

Using formalin-fixed paraffin-embedded (FFPE) specimens, 32 cases were examined, including 28 purebred dogs of 14 different breeds and four mixed-breed dogs (Table 4). JRTs were excluded because they had already been evaluated in our previous study [6]. Characteristics of the examined cases and information on the tumors are summarized in Supplemental Table 2. Except for a single case of gastric tumor, all tumors were located in the small and large intestines (n=13 and 18, respectively). While all tumors in the stomach and small intestine were adenocarcinomas, tumors in the large intestine were diagnosed as adenomas (n=13) or adenocarcinomas (n=5).

APC variant status was first analyzed using the PCR-RFLP assay, but clear fragment patterns were not observed for some samples, possibly due to the lower quality of the extracted DNA (Fig. 3A). Subsequently, all samples were also analyzed by PCR-direct sequencing, and the APC variant was not detected in any case (Fig. 3B).

Analysis of canine genome bank samples

Additional DNA samples were obtained from the canine genome bank at Azabu University, which contained 39 purebred dogs of 19 different breeds including four JRTs and a mixed-breed dog (Table 4). Characteristics of the individual dogs and information on GI cancers are provided in Supplemental Table 3. The cases were affected by adenocarcinomas of the stomach (n=10) or adenomas and adenocarcinomas of the intestine (n=6 and 23, respectively). The registered histopathological diagnoses of tumors in JRTs were gastric adenocarcinoma (n=2) and intestinal adenocarcinoma (n=2).

The PCR-RFLP assays revealed that while three JRTs were carriers of the germline APC variant, the remaining 36 dogs were non-carriers (Fig. 3C). A JRT with rectal adenocarcinoma did not harbor the germline APC variant and was determined to be a sporadic case. The presence or absence of APC variants in all samples was validated using PCR-direct sequencing (data not shown).

Furthermore, the genome bank samples were analyzed by TaqMan duplex real-time PCR assay to validate the practical usefulness of the previously established assay [9]. By taking advantage of high-throughput genotyping, all genome bank samples were analyzed at once. Real-time amplification plots of 6-carboxyfluorescein (FAM) and 2-chloro-7’phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC) fluorescence intensities indicated the genotypes of each case (Fig. 3D), which was consistent with the results of PCR-RFLP and PCR-direct sequencing analyses. In addition, when allelic discrimination analysis was conducted based on the VIC or FAM signal intensity ratio at the endpoints of PCR amplification, the examined samples were divided into two clusters, as expected (Fig. 3E). Although one anomalous sample was excluded from the wild-type cluster, its genotype was readily determined by real-time amplification plot, as with other samples (data not shown).

In the present study, we successfully obtained a sufficient number of blood samples from JRTs without additional invasive procedures by collecting them during the annual test for filarial infection. The overall prevalence of the germline APC variant was determined to be 1.89% during the screening of 792 JRTs in Japan. To appropriately evaluate this value, it is necessary to consider the difference in the mode of inheritance: a majority of canine hereditary diseases are inherited in a recessive manner [1, 13, 15]; however, this is a dominant disease. The disease onset risk might be comparable to that of recessive disease, with a heterozygous carrier prevalence of more than 20% [16]. The prevalence of the germline APC variant in JRTs did not significantly change depending on birth year, suggesting that there was no substantial fluctuation in the prevalence during the last 15 years. Once disease-causing gene variants are identified in dogs, it is possible to substantially reduce the prevalence by preventing transmission of the deleterious variant to future generations. It has been reported that several disease variants have been significantly reduced in frequency or eradicated in the general dog population [1, 14]. As hereditary GI polyposis is an adult-onset disease, there is a risk that germline APC variant carriers will be unintentionally used for breeding before disease onset. Therefore, breeding programs incorporating genetic screening for germline APC variants are indispensable for disease control. In the present study, pedigree analysis of the detected carrier dogs revealed that the germline APC variant was no longer confined to closely related bloodlines and is likely to spread widely in JRTs, indicating the need to examine all sires and dams without exception.

The present study provides further evidence of the association between germline APC variants and GI polyposis in JRTs. As expected, among the APC variant carriers with available medical history, half of the JRTs had current and/or previous history of adenocarcinomas of the stomach or large intestine. In addition, consistent with our previous study [6], early onset and recurrent cases were present in the carrier dogs. Notably, the detection of the germline APC variant in the present epidemiological study led to the early detection of gastric cancer in a JRT. Considering that genetic testing is less invasive, it would be a better diagnostic option for JRTs with refractory GI symptoms in a clinical setting. Positive results would prompt further examination, such as GI endoscopy. Moreover, in the present study, a follow-up survey of the variant carriers revealed that genetic testing preceded the onset of GI cancer in two cases. This result demonstrates the usefulness of genetic testing for predicting the lifelong risk of GI cancer in JRTs. Considering that initial GI lesions can arise at variable ages, reportedly between 2 and above 10 years, in JRTs with the germline APC variant [6], future risk assessment is important for the early detection of onset. In contrast, there were also APC variant carriers without a history of GI cancer, although some dogs showed GI symptoms suggestive of cancer development [6]. However, some carriers, including an asymptomatic dog, did not reach the reported average age of GI polyposis onset of 7.7 years [6]. Further follow-up surveys of carriers are required to determine whether the APC variant exhibits complete penetrance in JRTs.

Even within the same dog breed, the prevalence of certain hereditary diseases can differ among geographically separated subpopulations [17–19]. While JRT is a popular dog breed in other countries as well, there has been no report of JRTs with hereditary GI polyposis in any country other than Japan. In contrast, pedigree analysis revealed that many JRTs were introduced from Australia and New Zealand in the past three generations of the detected APC variant carriers. Taken together, the germline APC variant has become regionally more prevalent among JRTs in Japan, and it is likely that variant carriers remain hidden in other countries. Owing to the lower prevalence of the germline APC variant in JRTs, the disease may not have attracted much attention in other countries. Therefore, it is necessary for veterinary clinicians and pathologists worldwide to raise awareness of this novel hereditary disease.

In the present study, despite the relatively limited number of examined cases, the germline APC variant was not detected in any dogs with GI cancer in any breeds other than JRTs, suggesting that the variant is considered virtually breed-specific. In dogs, although many causative variants are breed-specific, several variants, such as those underlying degenerative myelopathy or hyperuricosuria, are common to multiple breeds [1, 13, 14, 16, 20]. Such variants are thought to be ancient in origin and are therefore potentially widespread across breeds. The absence of the germline APC variant in breeds other than JRTs implies that the variant arose de novo in a JRT after the establishment of this dog breed.

In veterinary research, it is often difficult to collect sufficient high-quality genomic DNA samples from animals with rare disorders. In the present study, we overcame the low incidence of GI epithelial tumors in dogs [2, 3] by utilizing DNA samples stored in the canine genome bank recently established in Japan. As shown in a previous study [21], canine biobanks will assist in the discovery of novel hereditary disorders by validating the association between identified germline variants and specific diseases. In fact, JRTs with germline APC variants have been found in dogs with GI cancers in the canine genome bank.

Another purpose of the present study was to verify the utility and detection limits of PCR-based genotyping assays established in our previous study [9]. While concentrations of blood-derived genomic DNA samples of JRTs were extremely variable ranging from 2.2 to 489.0 ng/µL possibly due to difference in condition and period of freezing preservation at each hospital, the PCR-RFLP assay successfully determined the APC genotypes of all the examined JRTs, demonstrating the practical usefulness of this assay. In fact, this assay has been put into practical use in Japan and is offered by a commercial laboratory as DNA testing for hereditary GI polyposis. In contrast, the retrospective analysis revealed that it would be difficult to always make a precise diagnosis from FFPE samples using only the PCR-RFLP assay because of the difficulty in obtaining high-quality genomic DNA from FFPE samples. In such cases, as shown in the present study, the combined use of PCR-direct sequencing can compensate for the defect.

In the present study, an epidemiological survey revealed that the current prevalence of the germline APC variant associated with hereditary GI polyposis in JRTs in Japan was 1.89%, and the prevalence remained roughly flat during the last 15 years. Proper genetic screening of breeding animals will prevent transmission of the germline APC variant to future generations, which will substantially reduce the prevalence and eventually eradicate this hereditary disease. In addition, the germline APC variant was not detected in any dogs with GI epithelial tumors of other breeds; therefore, hereditary GI polyposis was considered to be virtually specific to JRTs.

Sample collection and preparation

JRTs

Peripheral blood was collected from 792 JRTs at 93 veterinary clinics in Japan between March and June 2020. In Japan, during spring and early summer, dogs undergo annual blood tests for filarial infection before starting anti-filarial agents. When blood was collected from JRTs for this purpose, an additional 500 µL was withdrawn for the present study with the consent of dog owners. All JRTs who visited the veterinary clinics within the study period, irrespective of their current and previous medical history, were included in the present study. Blood samples were anticoagulated with EDTA and stored at -20°C until DNA extraction. Genomic DNA was extracted from the blood samples using a DNeasy Blood & Tissue Kit (QIAGEN, Venlo, Netherlands). The concentration and purity (A260/A280 ratio) of the extracted DNA were measured using a NanoDrop^TM Lite spectrophotometer (Thermo Fisher Scientific).

Dogs with GI tumors of multiple breeds

FFPE samples

Archival FFPE specimens, which were diagnosed as adenocarcinomas of the GI tract in dogs between 2014 and 2018 at the Laboratory of Veterinary Pathology, Gifu University, were used (Table 4). Genomic DNA was extracted from normal tissues on FFPE sections using a QIAamp DNA FFPE Tissue Kit (QIAGEN). The concentration and purity of the extracted DNA were measured using NanoDropTM Lite.

Samples from canine genomic bank

Genomic DNA samples from dogs with GI epithelial tumors were also obtained from the canine genome bank at Azabu University, Kanagawa, Japan. The samples contained 39 dogs from 19 different breeds, including four JRTs, and four mixed-breed dogs (Table 4 and Supplemental Table 3).

Genotyping assays

PCR-RFLP assay

PCR-RFLP assay was performed as previously described [9]. Briefly, DNA fragments of 156 bp containing the variant site at nucleotides 462 and 463 in exon 4 of the canine APC gene were amplified by PCR using the primers listed in Table 5, and the products were digested with the restriction enzyme MseI (RspRSII) (Takara Bio). The digested products were separated by electrophoresis on a 15% polyacrylamide gel (SuperSep™ Ace; Fujifilm Wako Pure Chemical Industries, Osaka, Japan) and visualized with a UV transilluminator after staining with ethidium bromide.

PCR-direct sequencing

PCR-direct sequencing was performed as previously described [6, 9]. PCR amplification was performed to amplify a 385-bp fragment containing the entire exon 4 of the canine APC gene from the blood samples of JRTs and canine genomic bank samples, as well as a 156-bp fragment containing a variant site in exon 4 from the FFPE samples, as previously reported [6]. The primers used are listed in Table 5. After PCR amplification, the purified PCR products were subjected to sequencing analysis using an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).

TaqMan duplex real-time PCR assay

Real-time PCR with TaqMan probe was performed as previously described [9]. The primers and probes used are listed in Table 5. The ABI StepOnePlus system (Applied Biosystems) was used to amplify and quantify the PCR products. Data were analyzed using StepOne software v2.3 (Applied Biosystems). Allelic type was determined based on a real-time amplification plot constructed based on FAM and VIC fluorescence intensities monitored during each PCR cycle, as well as an allelic discrimination plot constructed based on the signal intensity ratio of FAM and VIC at the end points of PCR amplification.

Statistical analysis

For statistical analysis, the chi-square test was used to compare the prevalence between/among the groups. P < 0.05 was considered statistically significant.

APC, adenomatous polyposis coli; EDTA, ethylenediaminetetraacetic acid; GI, gastrointestinal; JRT, Jack Russell Terrier; FFPE, formalin-fixed paraffin-embedded; PCR, polymerase chain reaction; PCR-RFLP, PCR-restriction fragment length polymorphism.

Ethics approval and consent to participate

Blood samples and pedigree certificates were collected with written informed consent from all dog owners and with approval of the Committee of Animal Research and Welfare of Gifu University (approval number: 2019-156).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. DNA sequence data of the germline APC variant is available in GenBank repository (ID: LC598892.1).

Competing interests

The authors declared that they have no competing interests.

Authors' contributions

AH designed the study. KY, WY, KO, and AH contributed to the collection of samples from animal hospitals, KY, HM, and AH at the Laboratory of Veterinary Pathology at Gifu University, and MS at the canine genome bank at Azabu University. KY, HM, and AH collected and interpreted data. KY and AH drafted the manuscript. HS supervised the study. All the authors have read and approved the final manuscript.

Funding

This work was supported by JSPS KAKENHI (grant numbers 18K05969 and 22K05985). The funding body had no role in the design of the study; collection, analysis, and interpretation of data; or in writing the manuscript.

Acknowledgements

We thank all clinical veterinarians and staff of animal hospitals for their contribution in collecting blood samples from JRTs and Ms. Chikako Iriyama for technical assistance.

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Tables 1 to 5 are available in the Supplementary Files section.

No competing interests reported.

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Molecular Epidemiological Study of Germline APC Variant Associated with Hereditary Gastrointestinal Polyposis in Dogs: Current Prevalence in Jack Russell Terriers in Japan and Breed Distribution

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