Characterization of putative germline pathogenic variants in 27 candidate cancer-predisposing genes in 813 cats using a feline-specific multiplex targeted sequencing

Abstract

In humans, about 5–10% of all cancers are caused by germline pathogenic variants (PVs) in cancer-predisposing genes, and their identification enables precision oncology approaches, such as surveillance for early detection, preventive medicine, and targeted therapy. Although cancer is a leading cause of death in cats, PVs have not been investigated for precision oncology. We developed a feline-specific multiplex targeted sequencing method to analyze 813 cats for putative PVs in 27 candidate feline cancer-predisposing genes. A total of 784 variants were identified, 13 of which were classified as putative PVs based on predicted truncating impact of amino acid sequence, clinical interpretation of corresponding variants in human, and in silico prediction on amino acid functions. Among 18 cats with one of the 13 putative PVs, seven (38.9%) had various types of confirmed or suspected tumor. Although PV carriers do not always develop cancer even in humans, putative PV carriers without tumors tended to be younger (1.83–16.58, years old, 9.16 years old on average) than the median age of tumor-bearing putative PV carriers (11.83 years old), suggesting that the proportion of affected cats may increase over time. Moreover, five cats with putative PVs in homologous recombination repair genes (BRCA2, RAD51C, or ATM) and two cats with those in mismatch repair genes (MSH2 and MSH6) may be candidates for targeted therapy with PARP inhibitors and immunotherapy with immune checkpoint inhibitors, respectively. These findings provide the first characterization of putative PVs in feline candidate cancer-predisposing genes, representing an important step toward genomics-informed oncology and risk stratification in cats.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-61718-w.

Introduction

Familial aggregation of cancer has been recognized for approximately 150 years¹. Currently, 5–10% of human cancers are considered to be hereditary cancer caused by pathogenic variants (PVs) in cancer-predisposing genes², such as homologous recombination repair genes (e.g. BRCA1 and BRCA2) and mismatch repair genes (e.g. MLH1, MSH2 and MSH6). Detection of carriers with PVs via genetic testing has enabled precision oncology, such as regular surveillance for early detection, preventive medicine, and targeted therapies, all of which contribute to improved clinical outcomes. PVs in cancer-predisposing genes have also been identified in other species. In dogs, where tumors are also the leading cause of death as in humans, PVs in APC causing familial adenomatous polyposis in Jack Russell Terriers³ and those in FLCN causing renal cystadenocarcinoma in German Shepherds⁴ have been documented. In Jack Russell Terriers, genetic testing could contribute to a reduction in disease incidence. In horses, a PV in STX17 gene, responsible for the gray coat color, has also been linked to increased risk of malignant melanoma⁵. These observations highlight not only the veterinary importance of identifying high-risk animals for clinical management, but also their scientific value in elucidating shared and species-specific mechanisms of tumorigenesis through comparative oncology with humans. Unlike artificially induced models, naturally occurring tumors in companion animals provide a complex and biologically relevant setting for comparative oncology⁶. Characterizing the hereditary cancer predispositions in these species may provide valuable insights into tumorigenesis, potentially contributing to precision medicine in both veterinary and human oncology.

Such discoveries and their associated benefits are also anticipated in cats, where cancer is also a leading cause of death worldwide. Studies suggest that 30–40% of cats develop tumors (benign or malignant) during their lifetime, with 53–85% of these tumors being malignant⁷. Moreover, cats have an approximately four-fold higher risk of developing a malignant tumor than dogs⁸. Although surgery, chemotherapy, and radiotherapy are standard treatments, the prognosis for many high-grade or advanced-stage malignant tumors remains poor, with the median overall survival often limited to approximately one year^9,10. However, to date, hereditary cancers in cats remain poorly characterized. Consequently, the potential for integrating germline risk assessment into feline precision oncology has been largely unexplored, limiting our ability to provide comprehensive, genomics-informed care. Moreover, in contrast to dogs, cats tend to conceal clinical signs, which often delay cancer diagnosis and worsen clinical outcomes¹¹. As a result, this diagnostic challenge contributes to limited progress in improving long-term survival outcomes for many feline malignancies.

Thus, in this study, we aimed to characterize hereditary cancer in cats. However, since there are few reports of familial aggregation of cancer in cats, forward genetics (a phenotype-first approach) could be challenging, despite the fact that cancer-predisposing genes in humans have largely been identified through this approach. We tried to overcome this difficulty by another approach, reverse genetics (a genotype-first approach), to systematically identify putative PVs in feline candidate cancer-predisposing genes. We developed a novel, feline-specific multiplex targeted sequencing method to efficiently analyze feline orthologs of 27 human cancer-predisposing genes commonly used in clinical genetic testing in human. Using this method, we examined 813 cats recruited from a veterinary hospital, including both tumor-affected and unaffected individuals, to identify those carrying putative PVs. We then assessed the clinical features of putative PV carriers to explore potential genotype-phenotype associations.

Results

Patient characteristics

A total of 813 cats were included in this study. The most common breed was mixed (59%), followed by Scottish Fold (8%), and American Shorthair (7%). Following them, British Shorthair, Munchkin, Norwegian Forest Cat, and Ragdoll were observed in relatively similar, lower proportions (each representing approximately 2–3%). This breed distribution aligns with the reported ranking of popular cat breeds in Japan (Fig. 1a). Females accounted for 47%, of which 82.5% were spayed and 90.3% of male cats were castrated (Fig. 1b). The age of cats ranged from 0 to 20 years old.

Fig. 1.

Characteristics of cats in this study. (a) A pie chart of cat breed distribution in this study. Others breed included Abyssinian, American, Curl, Bengal, Birman, Bombay, Burmese, Chartreux, Cornish Rex, Devon Rex, Egyptian Mau, Exotic, Shorthair, Himalayan, Japanese Bobtail, Khao Manee, Kurilian Bobtail, Lambkin, Maine Coon, Minuet, Ocicat, Persian, Ragamuffin, Russian Blue, Savannah, Siamese, Siberian, Singapura, Snowshoe, Somali, and Tonkinese. (b) A pie chart of sex and reproductive status in this study.

Development of a multiplex PCR–based targeted sequencing method for cats

In this study, we established a multiplex polymerase chain reaction (PCR)–based targeted sequencing method for cats to analyze the 27 candidate cancer-predisposing genes by modifying those previously developed for the human or dog genome in our laboratory^12,13. In total, 99.6% of all targeted regions across the 27 genes (85,629 bp) were covered by at least 20 sequencing reads (≥ 20×), indicating that nearly all germline variants within the targeted regions could be reliably detected. Across individual cats, the average proportion of target regions with ≥ 20× coverage varied between 5.22% and 99.2% (98.86% ± 4.47%, mean ± SD). As quality control, 792 cats with at least 95% of the target regions covered at ≥ 20× were retained for reliable variant detection for further analysis.

Finally, 784 germline variants were identified in the 792 cats. The total number of variants in each of the 27 genes varied substantially between 1 variant in PTEN and 125 variants in ATM (Fig. 2a). A statistically significant positive correlation was observed between transcript length and the number of variants (Spearman’s ρ = 0.813, P = 7.93 × 10^− 7), consistent with findings from our previous human study¹⁴, and supporting the validity of the multiplex PCR–based targeted sequencing approach.

Fig. 2.

Association between each gene length and the number of germline variants in each gene, and workflow for defining putative pathogenic variants (PVs). (a) Variant counts and transcript lengths per gene. Red bar and black dashed line present the total number of variants identified in 27 genes and transcript lengths, respectively. This statistically significant positive correlation between transcript length and the number of variants detected demonstrated the validity and effectiveness of the multiplex targeted sequencing method developed in this study. (b) Workflow for putative PV identification. First, among the 784 input variants, protein-truncating variants were identified using SnpEff as criterion 1. All 784 variants were nonetheless subjected to LiftOver for genome coordinate conversion; 318 failed conversion and were excluded, leaving 466 converted variants. Comparison of pre- and post-LiftOver coordinates removed a further 143 non-matching calls, yielding 323 matched variants used for downstream analyses. For missense variants within this matched set, putative PV identification then applied criterion 2 and criterion 3.

Determination of pathogenic variants

Among the 784 variants identified, 11 were loss-of-function variants including nonsense variants, frame-shift variants, and variants in 2 bp-canonical splice sites, 427 were non-synonymous variants, and 346 were synonymous variants. From this list, putative PVs were determined according to the three criteria described in the Materials and Methods section (Fig. 2b) because there are no standard guidelines in cats to determine PV among germline variants such as the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines used in human¹⁵. According to criterion 1, eleven loss-of-function variants were classified as PVs initially, however, three were excluded for the following reasons. One ATM variant (c.7539 + 2C > T) could affect a canonical splice donor site (5’ splice site); however, the substitution from C to T in this splice donor site created a more common consensus splice motif (GT), this variant would be unlikely to be a putative PV due to splicing abnormality. One TP53 variant (c.1222 C > T), although located within the coding region of the canonical transcript (ENSFCAT00000065820.2) used in this study, was excluded because it fell within an intronic region in alternative transcript isoforms (ENSFCAT00000055487.1 and ENSFCAT00000009625.4). A third variant in NBN (c.28 C > T) was also excluded because it was annotated within an NBN transcript in the felCat9 reference but had no transcript annotation at the corresponding genomic position in the alternative assembly (F.catus_Fca126_mat1.0), resulting in inconsistent gene assignment. In addition, three distinct TP53 variants and one CHEK2 variant classified as putative PVs were examined carefully because somatic variants related to clonal hematopoiesis of indeterminate potential can occur in these genes could be observed in genomic DNA from peripheral blood DNA¹⁶. All these variants showed variant allele fractions close to 0.5 as shown in IGV (Supplementary Fig. S1), while clonal hematopoiesis of indeterminate potential is generally characterized by variant allele fractions that are markedly lower than those of germline variants. Therefore, these were considered germline putative PVs. Finally, we identified eight variants as pathogenic.

Two variants met criterion 2. One BRCA1 variant (p.Ala1791Val) in cats corresponded to p.Ala1789Val in humans, classified as likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar Variation ID: 865385). The other was an MSH6 variant (p.Arg1074Cys), which corresponded to p.Arg1076Cys in humans and classified as likely pathogenic for Lynch syndrome (ClinVar Variation ID: 89357). The latter classification was reviewed by an expert panel in ClinVar. This designation indicates that the variant was curated and classified by an international consortium of experts, thus ensuring a high level of confidence in the pathogenicity assessment based on established consensus criteria.

For criterion 3, only non-synonymous variants that resulted in amino acid substitutions corresponding to those in humans were evaluated with three in silico prediction tools. Variants were considered pathogenic if predicted to be deleterious by at least two of the three in silico tools. Thresholds were optimized using human variations classified of 27 cancer-predisposing genes in ClinVar by comparing pathogenic/likely pathogenic variants (n = 6,324) with benign/likely benign variants (n = 1,022). The optimal cut-offs were 0.637 for AlphaMissense (AUC = 0.953), − 11.594 for ESM1b (AUC = 0.910), and 26.1 for CADD (AUC = 0.983), which provided the best discrimination between pathogenic and benign variants registered in ClinVar (Supplementary Fig. S2). Variants were considered potentially deleterious if their scores exceeded the AlphaMissense or CADD thresholds, or fell below the ESM1b threshold. In total, 21 missense variants exceeded the deleteriousness threshold in at least one of the three in silico tools. Of these, three exceeded the threshold in at least two tools, and one variant exceeded the threshold in all three tools. (Supplementary Fig. S3). One of the four variants (p.Ala1791Val in BRCA1) also fulfilled criterion 2. The three remaining variants met criterion 3. The CDKN2A variant (p.Pro148Leu) was predicted to be deleterious by AlphaMissense (score: 0.683) and ESM1b (–12.102) but did not exceed the threshold in CADD (score:23.5). The MSH2 variant (p.Arg711Gln) exceeded the threshold in all three tools: AlphaMissense (0.987), ESM1b (–11.744), and CADD (33). In contrast, the ATM variant (p.Thr2861Met) exceeded the thresholds in ESM1b (–12.477) and CADD (30) but not in AlphaMissense (0.467). Consequently, 13 variants were classified as putative PVs in this study (Table 1), including eight under criterion 1, two under criterion 2, and three additional variants under criterion 3.

Table 1.

Pathogenic variants and clinical characteristics of the carrier cats. The criteria refer to the numbered criteria described in the Materials and Methods section. A genotype of 0/1 indicates the presence of the variant in a heterozygous state. The diseases indicated in bold are confirmed or suspected tumors. CM, castrated male; F, Intact female; SF spayed female.

Chr	Pos	Ref	Alt	Gene	HGVS.c	HGVS.p	Criteria	Genotype	Sex	Breed	Age	Disease
A1	11,592,703	CTAAA	C	BRCA2	c.7072_7075delAAAT	p.Lys2358fs	1	0/1	CM	Mix	9.92	Multicentric lymphoma
D3	24,038,210	G	GT	CHEK2	c.1503dupT	p.Thr502fs	1	0/1	CM	Mix	11.5	Chronic kidney disease
								0/1	CM	Mix	12.2	Renal calculi (left and right)
												Ureteral calculi (left)
								0/1	CM	Mix	3.25	Foreign body ingestion
								0/1	SF	Mix	9	Chronic kidney disease
												Renal cell carcinoma (right kidney)
								0/1	SF	Mix	16.6	Chronic cholangitis (Suspected)
E1	2,541,383	G	A	TP53	c.1105 C > T	p.Arg369*	1	0/1	SF	Mix	11.8	Mass (Right iliac wing)
												Enlarged right medial iliac lymph node
E1	25,428,85	TA	T	TP53	c.617delT	p.Leu206fs	1	0/1	SF	Mix	8.58	Primary hyperparathyroidism (Suspected)
E1	2,543,653	GTT	G	TP53	c.82_83delAA	p.Asn28fs	1	0/1	SF	Bengal	13.3	Follicular thyroid adenomas (left and right)
												Mast cell tumor (skin)
E1	30,209,703	CAG	C	RAD51C	c.154_155delCT	p.Leu52fs	1	0/1	SF	Mix	8.58	Giant cell tumor (left hind leg)
F2	39,289,669	C	T	NBN	c.2322-1G > A	.	1	0/1	CM	Ragdoll	8.75	Idiopathic hypercalcemia
								0/1	CM	Ragdoll	1.83	Feline infectious peritonitis (Suspected)
F2	39,347,744	T	TG	NBN	c.237dupC	p.Arg80fs	1	0/1	CM	Bengal	10.3	Rhinitis
E1	43,503,797	G	A	BRCA1	c.5372 C > T	p.Ala1791Val	2, 3	0/1	SF	Mix	3	Subcutaneous tissue inflammation (right side of anus)
A3	69,675,642	C	T	MSH6	c.3220 C > T	p.Arg1074Cys	2	0/1	SF	Mix	9.17	Maxillary fracture
A3	69,440,942	G	A	MSH2	c.2132G > A	p.Arg711Gln	3	0/1	SF	Scottish Fold	9.16	Bladder calculi
D1	6,832,452	C	T	ATM	c.8582 C > T	p.Thr2861Met	3	0/1	CM	Mix	14.4	Mass (left kidney)
D4	48,180,098	G	A	CDKN2A	c.443 C > T	p.Pro148Leu	3	0/1	F	Scottish Fold	13.8	Mammary gland tumor (simple type)

Associations between pathogenic variant carriers and cancer status

To investigate the association between the 13 putative PVs and feline clinical characteristics, we analyzed the clinical data from 18 putative PV-carrier cats. All putative PVs were heterozygous state, and 7 of 18 cats (38.9%) had various types of confirmed or suspected tumors. Two putative PVs were found in multiple cats: a CHEK2 frameshift variant (p.Thr502fs) in five cats and an NBN splice-site variant (positioned 1 bp downstream of exon 15 and predicted to disrupt normal splicing; c.2322-1G > A) in two cats. All five carriers of the CHEK2 variant were mixed-breed cats (three castrated males and two spayed females) with diverse clinical presentations, including only one case of renal cell carcinoma (a 9-year-old spayed female mixed-breed cat); the others had renal disease, suspected chronic cholangitis, or foreign body ingestion, respectively. The NBN variant was found in two castrated male Ragdolls diagnosed with idiopathic hypercalcemia and suspected feline infectious peritonitis, respectively, without a shared clinical phenotype.

The other 11 putative PVs were each observed in only one cat. Of these, 6 were associated with confirmed or suspected tumors: multicentric lymphoma in a BRCA2 variant (p.Lys2358fs) carrier (castrated male mixed breed); suspected osteosarcoma in a TP53 variant (p.Arg369*) carrier (spayed female mixed breed); cutaneous mast cell tumor in another TP53 variant (p.Asn28fs) carrier (spayed female Bengal); giant cell tumor in a RAD51C variant (p.Leu52fs) carrier (spayed female mixed breed); mammary carcinoma in a CDKN2A variant (p.Pro148Leu) carrier (intact female Scottish Fold); and suspected renal cell carcinoma in an ATM variant (p.Thr2861Met) carrier (castrated male mixed breed). Although cutaneous mast cell tumors are exceedingly rare among the 18 putative PV carriers and have no documented association with TP53 variants, osteosarcoma is a sentinel cancer of Li-Fraumeni syndrome¹⁷, a cancer predisposition syndrome caused by germline TP53 variants. Therefore, the spayed female mixed-breed cat carrying the TP53 variant (p.Arg369*) with suspected osteosarcoma may have developed the tumor via a mechanism analogous to Li-Fraumeni syndrome in humans. The remaining five putative PVs, each located in a different gene (TP53, NBN, BRCA1, MSH6, and MSH2), were associated with non-tumor-related clinical symptoms, specifically hyperparathyroidism, purulent nasal discharge, subcutaneous inflammation, maxillary fracture, and bladder stones, respectively.

Among the 18 cats with one of the 13 putative PVs, 38.9% had various types of confirmed or suspected tumors. Since not all putative PV carriers develop cancer in humans, this proportion could be considered reasonable but, we investigated whether demographic factors, such as breed, sex, neuter status, and age, might account for differences in cancer development among the feline putative PV carriers. Most putative PV carriers were mixed-breed cats (n = 12), with Bengal, Ragdoll, and Scottish Fold each represented by two cats. The group consisted of eight castrated males, nine spayed females, and one intact female, with no apparent trend by breed, sex, or neuter status. Of the seven tumor-bearing putative PV carriers (age, 9.00–14.41 years old; median, 11.83 years), five were older than the median feline cancer diagnosis age (approximately 9.5 years old)¹⁸, whereas 7 of the 11 tumor-free putative PV carriers (age, 1.83–16.58 years old; median, 9.16 years) were younger than this threshold. Although the difference in age between tumor-bearing and tumor-free putative PV carriers was not statistically significant (P = 0.082, Welch’s t-test), tumor-bearing putative PV carriers tended to be older than tumor-free putative PV carriers. A similar age difference was observed among non-carriers, in whom tumor-bearing cats (n = 47; median age, 12.75 years) were older than tumor-free cats (median age, 6.58 years) (Supplementary Table 1 for age and comorbidities of tumor-bearing cats among non-carriers). It should be noted, however, that the reference age of approximately 9.5 years was derived from a Korean feline population; therefore, comparison with our Japanese cohort should be interpreted with caution.

Discussion

In this study, we established a novel multiplex PCR–based targeted sequencing method to analyse 27 candidate cancer-predisposing genes in 813 cats. Among the 784 germline variants identified, 13 putative PVs were classified according to three criteria. Of the 18 cats carrying these putative PVs, seven (38.9%) cats were diagnosed with or suspected of having tumors. The median age of tumor-bearing putative PV carriers tended to be older than tumor-free carriers.

The identification of putative PVs in genes associated with DNA repair or mismatch repair pathways suggests new opportunities for applying molecular targeted therapies in feline oncology. BRCA2, CHEK2, RAD51C, and ATM are central to the homologous recombination repair pathway, and PVs in these genes can result in homologous recombination deficiency. BRCA2 and RAD51C, in particular, are well established as homologous recombination repair genes whose loss confers strong sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors via synthetic lethality^19,20. For instance, a cat diagnosed with multicentric lymphoma carried a BRCA2 putative PV. Since feline multicentric lymphoma is considered analogous to human diffuse large B-cell lymphoma, as shown in our previous human study²¹, the affected feline case may also benefit from such treatment²². Furthermore, two tumor-free cats carried putative PVs in mismatch repair genes (MSH2 and MSH6). Should they develop tumors in the future, immune checkpoint inhibitors such as anti-PD-1/PD-L1 antibodies—recently developed for cats in Japan²³ could prove effective, as seen in human MMR-deficient tumors²⁴. A similar rationale may apply to the cats carrying a CDKN2A variant, as CDKN2A putative PVs have been associated with immune checkpoint inhibitor efficacy in human malignant melanoma²⁵. Collectively, these findings highlight the potential of germline variant profiling to assist in guiding the application of molecular targeted therapies, such as PARP inhibitors, and immunotherapies, such as immune checkpoint inhibitors, as useful approaches for individualized cancer treatment in cats. Nevertheless, given the absence of functional characterization of the identified variants and clinical response data in feline patients, the efficacy of the proposed molecular targeted therapies and immunotherapies warrants further investigation in preclinical models and clinical trials.

Among the 18 putative PV carriers, 11 cats were tumor-free at the time of investigation but had other common feline diseases, such as renal or internal disorders, metabolic abnormalities, or bone fractures. This is consistent with the observations in human populations, where PV carriers often exhibit incomplete penetrance, with lifetime cancer risks varying widely across genes. For example, the highest risk for breast cancer associated with BRCA1 was estimated at 72.5% (95% CI, 20.4%–90.5%) and with BRCA2 at 58.3% (95% CI, 38.3%–71.9%)^26,27. For carriers of MSH6 PVs, the cumulative risk of any cancer has been reported as 60.9% (95% CI, 42.7%–79.0%)²⁸, and lower for some moderate-risk genes such as NBN²⁹. In humans, sex hormonal influences are known to significantly modulate the penetrance of PVs. For instance, early menarche³⁰ and shorter breastfeeding³¹ are established risk factors for cancer in women with PVs. Based on this, we evaluated whether castration and spaying status could explain the incomplete penetrance observed in our feline cohort. However, no such association was found in this study. Another possible explanation is age at sampling. Tumor-bearing putative PV carriers were older (median, 11.83 years) than tumor-free putative PV carriers (median, 9.16 years). A similar age difference was also observed among the non-carriers, in whom tumor-bearing cats were older (median, 12.75 years) than tumor-free cats (median, 6.58 years). These findings suggest that the observed difference between tumor-bearing and tumor-free putative PV carriers may reflect the broader age structure of the cohort rather than a PV-specific effect. Therefore, further studies with larger, age-matched cohorts are essential to validate the association between putative PV carriage and tumor development independently of age. Nonetheless, considering the tendency of cats to mask clinical signs, recognizing these tumor-free putative PV carriers as a population at potentially elevated risk for subsequent tumor development and performing continuous and rigorous surveillance remain clinically beneficial. However, the current tools for early cancer detection in cats are limited. Our study suggests the existence of hereditary cancers in cats and highlights the necessity for scientifically validated early detection tools for feline cancers.

This study also demonstrates the utility of reverse genetics’ strategy because we could successfully characterize putative PVs in feline candidate cancer-predisposing genes. Traditionally, human oncology has relied on forward genetics, which starts with clinical phenotypes and identifies causal genes³². This strategy has revealed germline variants in oncogenes, tumor suppressors, and genes associated with metastasis or drug resistance^33,34. However, in veterinary medicine, the forward genetic approach is inherently challenging due to several factors. These include fragmented clinical datasets, incomplete pedigree information, and lack of standardized health registries. Furthermore, the late onset of cancer in many feline populations often complicates longitudinal follow-up and the assessment of penetrance, thereby hindering precise phenotypic classification. Although several single-gene disorders have been reported in cats, hereditary cancers have not been recognized. In this study, we instead applied reverse genetic approach with the feline developed multiplex PCR–based targeted sequencing method, beginning with germline variants to identify candidates associated with inherited cancer. This approach may also be extended to other feline diseases with suspected genetic predisposition, such as breed-specific hypertrophic cardiomyopathy and common kidney diseases.

This study has several limitations. First, pathogenicity classification relied on human resources and in silico tools. Although we recognize that evolutionary divergence and differences in protein interaction landscapes may affect variant function, as highlighted by recent guidelines for animal variant interpretation³⁵, our approach serves as a robust preliminary strategy in the absence of feline-specific databases. Nevertheless, these classifications remain putative and further feline-specific functional validation is essential. Second, the single-center study design potentially introduced an ascertainment bias. Third, the small sample size of pathogenic variant carriers limited the statistical power to establish robust genotype-phenotype associations. Larger cohorts and independent validation studies are therefore required to confirm our findings. Fourth, our clinical phenotyping relied partly on medical records, which included a small number of “suspected” tumor cases that were not histopathologically confirmed. Specifically, these cases were limited to individuals identified with distinct masses through clinical or diagnostic imaging, although we believe that the impact of these suspected cases is minimal, given that they represent only a minor fraction of the affected group (2/7).

In conclusion, this study established a novel multiplex PCR–based targeted sequencing method and, for the first time, revealed that 18 of 792 (2.3%) cats carried putative PVs in one of the 27 candidate cancer-predisposing genes. Among these carriers, 38.9% developed confirmed or suspected tumor. These results highlight the potential of precision oncology in cats, including routine surveillance for early detection, preventive medicine, and application of molecular targeted therapies for cancer—the leading cause of feline mortality worldwide.

Materials and methods

Sample collection and DNA isolation

Between 2022 and 2024, 813 surplus EDTA blood samples from cats with various diseases were collected at the University of Tokyo Veterinary Medical Center. We obtained written informed consent at the initial consultation, prior to any medical intervention, for sample collection and secondary research use. Genomic DNA was isolated using NucleoSpin^® Blood DNA extraction kit (MACHEREY-NAGEL). The clinical characteristics were extracted from medical records. We classified diagnoses as “confirmed” if supported by histopathological or cytological evidence, or “suspected” if diagnostic imaging or physical examination confirmed the presence of a distinct mass. The study was approved by the RIKEN Center for Integrative Medical Sciences (AEY2024-028). No animal experiments, experimental infections, anesthesia, or euthanasia were performed for the purpose of this study. All methods were performed in accordance with the relevant guidelines and regulations.

Development of multiplex PCR–based targeted sequencing method for cats

To enable scalable and cost-effective genetic analysis in cats, we developed a feline multiplex PCR–based targeted sequencing method to sequence specific genes of suspected clinical significance. Based primarily on the hereditary cancer panel used in our previous study³⁶, we adapted and extended the design for feline genetics by targeting all coding regions and 2 bp flanking intronic sequences of 27 candidate cancer-predisposing genes (APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDK4, CDKN2A, CDH1, CHEK2, EPCAM, HOXB13, NBN, NF1, MLH1, MSH2, MSH6, MUTYH, PALB2, PMS2, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53). Exon regions were determined based on canonical transcripts retrieved from the Ensembl database (Ensembl 111: Jan 2024), and the primers for the coding regions and 2-bp flanking intronic sequences of the 27 genes were designed based on felCat9 using Primer 3 (ver. 2.4.0)³⁷. The final primer design consisted of 1036 amplicons distributed across 4 multiplex PCR pools.

A feline multiplex PCR–based targeted sequencing method for cat genomes was developed by modifying methods previously developed for the human genome in our laboratory¹²,with the target regions redefined to cover feline orthologous protein-coding exons and splice-site boundaries based on the felCat9 reference genome and translated-region annotations. The sizes of the PCR products were designed to be 180–295 bp to cover the amplicon with sequencing reads. Multiplex PCR was performed using Platinum Multiplex PCR Master Mix (Life Technologies), followed by additional PCR using KOD One PCR Master Mix (TOYOBO) to attach flow cell binding sequences. The final libraries were pooled and purified with Agencourt AMPure XP (Beckman Coulter) to eliminate primer dimers for one sequencing. The Illumina NovaSeq and MiSeq instrument were used to generate 2 × 150-bp paired-end reads.

Sequence reads were aligned to the cat reference sequence (felCat9) using the Burrows-Wheeler Aligner (ver. 0.7.17)³⁸, and then applied to RealignerTargetCreator and IndelRealigner using GATK (ver. 3.7)³⁹ for each BAM file. For quality control, cat samples were excluded from further analysis if the proportion of covered bases of ≥ 20 reads in the target region was < 95%. Variants of each cat were called separately using UnifiedGenotyper and HaplotypeCaller in GATK, and listed all variants detected using either method were retained. We calculated alternative allele frequencies for each variant using SAMtools (ver. 1.6)⁴⁰. All variants were manually inspected in the BAM files using IGV (ver. 2.16.0) to confirm that the alternative allele was supported by multiple sequencing reads and to exclude apparent sequencing or alignment artifacts. Orthogonal validation by Sanger sequencing was not performed.

Pathogenicity assignment of variants

Among the genetic variants detected via the multiplex PCR-based targeted sequencing method, we need to determine putative PVs. However, there is no standard guidelines in cats such as the ACMG/AMP guidelines used in human¹⁵. Putative PVs were defined according to the following criteria. (1) Variants annotated as having a “HIGH” impact by SnpEff (ver. 4.3), including loss-of-function variants, such as nonsense and frameshift variants, that are predicted to severely disrupt protein function because this is considered very strong evidence of pathogenicity in the ACMG/AMP guidelines, based on the canonical transcripts of each gene as defined in the Ensembl database (Supplementary Table 2 for transcript IDs used in the annotation). Among loss-of-function variants (stop-gained, frameshift, and canonical splice site variants within 2 bp of an exon–intron junction), variants were excluded if there were inconsistencies between the canonical transcript of Felis_catus_9.0 genome used in this study and the F.catus_Fca126_mat1.0 reference genome. Canonical splice site variants were considered putative PVs only if they were predicted to disrupt consensus splice site sequences (AG-GT). (2) Missense variants that resulted in amino acid substitutions identical to those observed in the human ortholog and were annotated as “pathogenic” or “likely pathogenic” in the ClinVar database, under the assumption that such variants may exert comparable functional effects in cats because ClinVar serves as a reasonably reliable source of pathogenic variant information⁴¹. (3) Missense variants with identical amino acid substitutions predicted to be deleterious by at least two out of three high-performance in silico tools: AlphaMissense⁴², ESM1b⁴³, and CADD⁴⁴ because each tool showed reasonable precision to determine putative PV.

For criteria 2 and 3, concordant amino acid substitutions were identified through LiftOver coordinate conversion from the feline genome (felCat9) to the human reference genome (GRCh38) using the UCSC felCat9ToHg38.over.chain.gz file. The accuracy of this conversion was validated by confirming that the predicted amino acid changes remained consistent before and after conversion. For criterion 2, clinical significance (i.e., pathogenic or likely pathogenic) was assigned based on annotations in ClinVar (v20241021)⁴⁵. For criterion 3, cut-off values for deleteriousness scores were established by scoring ClinVar-annotated variants (pathogenic, likely_pathogenic, benign, and likely_benign) in the target genes using each in silico prediction tools to predict their pathogenicity. Based on these scores, the thresholds were then optimized to maximize true positive rate while maintaining false positive rates of 5% or less.

Clinical characteristics of cat patients with putative PVs

For cats carrying putative PVs, electronic medical records at the University of Tokyo Veterinary Medical Center were reviewed to evaluate the clinical relevance of these variants. Detailed clinical characteristics were extracted for each case, including demographic information (breed, age, sex, neuter status), disease-related information (diagnoses, suspected tumors, medical history), and clinical course (treatments, treatment responses, and prognosis). All medical records were reviewed by experienced veterinarians.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

N.I. and Y.M. designed research; N.I., H.Toyoda., T.A., M.E., Y.I., D.K., T.N., R.N., and H.Tomiyasu performed research; N.I., K.M., and R.Y. analyzed data; N.I. and Y.M. wrote the paper.

Funding

This work was supported by JSPS KAKENHI Grant Number 25K18384.

Data availability

The multiplex PCR–based targeted sequencing data generated in this study from samples of 813 cats have been deposited in the DNA Data Bank of Japan (DDBJ) and are publicly available under BioProject accession number PRJDB37916 (https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB37916).

Declarations

Competing interests

The authors declare no competing interests.