Whole exome sequencing analysis of canine urothelial carcinomas without BRAF V595E mutation: Short in-frame deletions in BRAF and MAP2K1 suggest alternative mechanisms for MAPK pathway disruption.

Molecular profiling studies have shown that 85% of canine urothelial carcinomas (UC) harbor an activating BRAF V595E mutation, which is orthologous to the V600E variant found in several human cancer subtypes. In dogs, this mutation provides both a powerful diagnostic marker and a potential therapeutic target; however, due to their relative infrequency, the remaining 15% of cases remain understudied at the molecular level. We performed whole exome sequencing analysis of 28 canine urine sediments exhibiting the characteristic DNA copy number signatures of canine UC, in which the BRAF V595E mutation was undetected (UDV595E specimens). Among these we identified 13 specimens (46%) harboring short in-frame deletions within either BRAF exon 12 (7/28 cases) or MAP2K1 exons 2 or 3 (6/28 cases). Orthologous variants occur in several human cancer subtypes and confer structural changes to the protein product that are predictive of response to different classes of small molecule MAPK pathway inhibitors. DNA damage response and repair genes, and chromatin modifiers were also recurrently mutated in UDV595E specimens, as were genes that are positive predictors of immunotherapy response in human cancers. Our findings suggest that short in-frame deletions within BRAF exon 12 and MAP2K1 exons 2 and 3 in UDV595E cases are alternative MAPK-pathway activating events that may have significant therapeutic implications for selecting first-line treatment for canine UC. We developed a simple, cost-effective capillary electrophoresis genotyping assay for detection of these deletions in parallel with the BRAF V595E mutation. The identification of these deletion events in dogs offers a compelling cross-species platform in which to study the relationship between somatic alteration, protein conformation, and therapeutic sensitivity.

Comparison of urine fibrinogen and interleukin-6 concentrations between healthy dogs and dogs with risk factors for enterococcal bacteriuria.

OBJECTIVE: To compare urine concentrations of fibrinogen (uFIB) and interleukin-6 (uIL-6) between dogs with risk factors for enterococcal bacteriuria and healthy dogs. SAMPLE: Banked urine samples with negative aerobic culture results from 8 dogs with urolithiasis, 9 dogs with anatomic abnormalities of the lower portion of the urinary tract (LUT), 10 dogs with LUT neoplasia, and 21 healthy control dogs. PROCEDURES: Urine creatinine concentration (uCrea) was determined by an automated biochemical analyzer, and uFIB and uIL-6 were determined by dog-specific ELISAs. The uFIB:uCrea and uIL-6:uCrea ratios were calculated for each sample to normalize intersample differences in urine concentration and were compared among the 4 experimental groups. RESULTS: Median uFIB:uCrea ratios for dogs with urolithiasis (0.72; interquartile [25th to 75 percentile] range [IQR], 0.46 to 3.48) and LUT neoplasia (6.16; IQR, 3.89 to 12.75), but not for dogs with LUT anatomic abnormalities (0.48; IQR, 0.27 to 0.69), were significantly greater than that for control dogs (0.17; IQR, 0.07 to 0.39). Median uIL-6: uCrea ratios for dogs with urolithiasis (0.48; IQR, 0.18 to 1.61), LUT anatomic abnormalities (0.25; IQR, 0.17 to 0.33), and LUT neoplasia (0.25; IQR, 0.12 to 1.01) were significantly greater than that for control dogs (0.08; IQR, 0.06 to 0.11). CONCLUSIONS AND CLINICAL RELEVANCE: The uFIB and uIL-6 in dogs with risk factors for enterococcal bacteriuria were generally greater than corresponding values in control dogs. Further investigation is necessary to determine the role of fibrinogen in enterococcal colonization of the urinary tract of dogs.

Glomerulopathy and mutations in NPHS1 and KIRREL2 in soft-coated Wheaten Terrier dogs.

Dogs of the soft-coated wheaten terrier breed (SCWT) are predisposed to adult-onset, genetically complex, protein-losing nephropathy (average onset age = 6.3 ± 2.0 years). A genome-wide association study using 62 dogs revealed a chromosomal region containing three statistically significant SNPs (p(raw) ≤ 4.13 × 10(-8); p(genome) ≤ 0.005) when comparing DNA samples from affected and geriatric (≥14 years) unaffected SCWTs. Sequencing of candidate genes in the region revealed single nucleotide changes in each of two closely linked genes, NPHS1 and KIRREL2, which encode the slit diaphragm proteins nephrin and Neph3/filtrin, respectively. In humans, mutations in nephrin and decreased expression of Neph3 are associated with podocytopathy and protein-losing nephropathy. The base substitutions change a glycine to arginine in the fibronectin type 3 domain of nephrin and a proline to arginine in a conserved proline-rich region in Neph3. These novel mutations are not described in other species, nor were they found in 550 dogs of 105 other breeds, except in 3 dogs, including an affected Airedale terrier, homozygous for both substitutions. Risk for nephropathy is highest in dogs homozygous for the mutations (OR = 9.06; 95 % CI = 4.24-19.35). This is the first molecular characterization of an inherited podocytopathy in dogs and may serve as a model for continued studies of complex genetic and environmental interactions in glomerular disease.