Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite.

BACKGROUND: As a population genetic tool, mitochondrial DNA is commonly divided into the ~ 1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes. RESULTS: To increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes in our dataset) and defines a phylogeny showing similar starlike topologies within all major clades (haplogroups), likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups. CONCLUSIONS: In defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

Defining cat mitogenome variation and accounting for numts via multiplex amplification and Nanopore sequencing.

Hair shed by domestic cats is a potentially useful source of forensic evidence. Analysable hair DNA is predominantly mitochondrial, but the recent domestication history of cats means that mtDNA diversity is low. A 402-bp control region segment is usually sequenced, defining only a small number of distinct haplotypes in populations. Previously, we used a long-amplicon approach to sequence whole mitogenomes in a sample of blood DNAs from 119 UK cats, greatly increasing observed diversity and reducing random match probabilities. To exploit this variation for forensic analysis, we here describe a multiplex system that amplifies the cat mitogenome in 60 overlapping amplicons of mean length 360 bp, followed by Nanopore sequencing. Variants detected in multiplex sequence data from unrooted hair completely mirror those from long-amplicon data from blood from the same individuals. However, applying the multiplex to matched blood DNA reveals additional sequence variants which derive from the major feline nuclear mitochondrial insertion sequence (numt), which covers 7.9 kb of the 17-kb mitogenome and exists in multiple tandem copies. We use long-amplicon Nanopore sequencing to investigate numt variation in a set of cats, together with an analysis of published genome sequences, and show that numt arrays are variable in both structure and sequence, thus providing a potential source of uncertainty when nuclear DNA predominates in a sample. Forensic application of the multiplex was demonstrated by matching hairs from a cat with skeletal remains from its putative mother, both of which shared a globally common haplotype at the control region. The random match probability in this case with the CR 402-bp segment was 0.21 and this decreased to 0.03 when considering the whole mitogenome. The developed multiplex and sequencing approach, when applied to cat hair where nuclear DNA is scarce, can provide a reliable and highly discriminating source of forensic genetic evidence from a single hair. The confounding effect of numt co-amplification in degraded samples where mixed sequences are observed can be mitigated by variant phasing, and by comparison with numt sequence diversity data, such as those presented here.

Cutaneous spindle cell tumors with features of peripheral nerve sheath tumors and concurrent cardiac involvement: neurofibromatosis type 1-like presentation in a Labrador Retriever dog.

An 8-y-old intact male Labrador Retriever dog developed cutaneous masses over the entire body. On histologic evaluation, the masses were composed of bundles of fusiform neoplastic cells arranged around adnexa, with mild atypia and no mitoses, consistent with peripheral nerve sheath tumors (PNSTs). Immunohistochemically, neoplastic cells were immunoreactive for vimentin, glial fibrillary acidic protein (GFAP), and S100, confirming their perineurial origin. The dog was euthanized because of deteriorating clinical signs. In addition to the cutaneous masses, a cardiac mass was identified at postmortem examination. The histopathologic and immunohistochemical features of the cardiac mass were similar to those of the cutaneous masses. To our knowledge, the combination of multiple cutaneous masses with features of PNSTs and a concurrent cardiac lesion has not been reported previously in a dog. We suggest "neurofibromatosis type 1-like" presentation for this unique combination of cutaneous and cardiac masses. Further studies are required to investigate the etiopathogenesis of this condition and explore its genetic background.

Validation of a Liquid Biopsy Protocol for Canine BRAFV595E Variant Detection in Dog Urine and Its Evaluation as a Diagnostic Test Complementary to Cytology.

A significant proportion of canine urothelial carcinomas carry the driver valine to glutamic acid variation (V595E) in BRAF kinase. The detection of V595E may prove suitable to guide molecularly targeted therapies and support non-invasive diagnosis of the urogenital system by means of a liquid biopsy approach using urine. Three cohorts and a control group were included in this multi-step validation study which included setting up a digital PCR assay. This was followed by investigation of preanalytical factors and two alternative PCR techniques on a liquid biopsy protocol. Finally, a blind study using urine as diagnostic sample has been carried out to verify its suitability as diagnostic test to complement cytology. The digital PCR (dPCR) assay proved consistently specific, sensitive, and linear. Using the dPCR assay, the prevalence of V595E in 22 urothelial carcinomas was 90.9%. When compared with histopathology as gold standard in the blind-label cases, the diagnostic accuracy of using the canine BRAF (cBRAF) variation as a surrogate assay against the histologic diagnosis was 85.7% with 92.3% positive predictive value and 80.0% negative predictive value. In all the cases, in which both biopsy tissue and the associated urine were assayed, the findings matched completely. Finally, when combined with urine sediment cytology examination in blind-label cases with clinical suspicion of malignancy, the dPCR assay significantly improved the overall diagnostic accuracy. A liquid biopsy approach on urine using the digital PCR may be a valuable breakthrough in the diagnostic of urothelial carcinomas in dogs.

Quality documentation challenges for veterinary clinical pathology laboratories.

An increasing number of veterinary laboratories worldwide have obtained or are seeking certification based on international standards, such as the International Organization for Standardization/International Electrotechnical Commission 17025. Compliance with any certification standard or quality management system requires quality documentation, an activity that may present several unique challenges in the case of veterinary laboratories. Research specifically addressing quality documentation is conspicuously absent in the veterinary literature. This article provides an overview of the quality system documentation needed to comply with a quality management system with an emphasis on preparing written standard operating procedures specific for veterinary laboratories. In addition, the quality documentation challenges that are unique to veterinary clinical pathology laboratories are critically evaluated against the existing quality standards and discussed with respect to possible solutions and/or recommended courses of action. Documentation challenges include the establishment of quality requirements for veterinary tests, the use or modification of human analytic methods for animal samples, the limited availability of quality control materials satisfactory for veterinary clinical pathology laboratories, the limited availability of veterinary proficiency programs, and the complications in establishing species-specific reference intervals.

Application of a mitochondrial DNA control region frequency database for UK domestic cats.

DNA variation in 402bp of the mitochondrial control region flanked by repeat sequences RS2 and RS3 was evaluated by Sanger sequencing in 152 English domestic cats, in order to determine the significance of matching DNA sequences between hairs found with a victim's body and the suspect's pet cat. Whilst 95% of English cats possessed one of the twelve globally widespread mitotypes, four new variants were observed, the most common of which (2% frequency) was shared with the evidential samples. No significant difference in mitotype frequency was seen between 32 individuals from the locality of the crime and 120 additional cats from the rest of England, suggesting a lack of local population structure. However, significant differences were observed in comparison with frequencies in other countries, including the closely neighbouring Netherlands, highlighting the importance of appropriate genetic databases when determining the evidential significance of mitochondrial DNA evidence.

Establishment of canine hematology reference intervals for the Sysmex XT-2000iV hematology analyzer using a blood donor database.

BACKGROUND: The Sysmex XT-2000iV is a hematology analyzer that combines impedance and optical techniques and has been previously validated for dogs. Specific reference intervals (RIs) are useful when interpreting results. OBJECTIVES: The aim of this study was to determine hematologic RIs for the Sysmex XT-2000iV using a large reference population of client-owned clinically healthy blood donor dogs, adopting an indirect sampling method. METHODS: Dogs were screened for breed, size, health, travel history, and previous blood transfusions, and the quality of blood specimens was also reviewed. Results from specimens that met inclusion criteria were used to determine RIs using a nonparametric method. Specimens from Akitas and sighthounds were excluded from the study. RESULTS: Of 992 specimens that had been collected from blood donors and analyzed, 297 were initially included in the RI study. An additional 38 specimens were excluded as outliers, and hematologic RIs for the Sysmex XT-2000iV were based on analysis of specimens from 259 clinically healthy dogs. Measurands evaluated had variable distributions, and intervals obtained were generally comparable to previously reported RIs. Differences observed included higher lower and upper reference limits (LRL and URL, respectively) for MCV and lower URL for WBC count. Reticulocyte count and the LRL of the absolute lymphocyte count were also higher than previously reported, and the RI for platelet count was narrower and lower. CONCLUSIONS: Canine RIs for the Sysmex hematology analyzer were established using an indirect sampling method with reference individuals selected from a large database of client-owned clinically healthy blood donor dogs. For specimens included in this study, time from collection to analysis was similar to what veterinary commercial laboratories experience.

Reference intervals for Greyhounds and Lurchers using the Sysmex XT-2000iV hematology analyzer.

BACKGROUND: The need for breed-specific reference intervals (RIs) for Greyhounds has been identified. As Lurchers are a sighthound cross-breed, specific RIs may also be needed for these dogs. Hematologic RIs for Greyhounds and Lurchers using the Sysmex XT-2000iV hematology analyzer have not been established. OBJECTIVES: The aims of this study were to establish RIs for Greyhounds using the Sysmex XT-2000iV, to investigate whether RIs for Greyhound and nonsighthound dogs could be transferred to Lurchers, and to establish new RIs for Lurchers if transference was not possible. METHODS: Data were retrieved retrospectively from a database of blood donor dogs. Greyhound RIs were established using nonparametric methods based on a reference population of 179 dogs. For the RI transference study, 38 Lurchers were selected, following guidelines proposed by the Clinical and Laboratory Standards Institute. When transference was not appropriate, new RIs were generated using the robust method. RESULTS: Greyhound RIs for the Sysmex hematology analyzer reflected known differences in this breed with a tendency toward higher RBC mass and lower WBC and platelet counts. RIs for hemoglobin concentration, HCT, MCV, MCH, MCHC, and WBC, neutrophil, lymphocyte, monocyte, and platelet counts for Greyhounds were suitable for transference to Lurchers. For RBC and eosinophil counts, new RIs were established. CONCLUSION: Our study suggests that Lurchers share many hematologic characteristics with Greyhounds, but had higher reference limits for RBC and eosinophil counts.