Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs

BACKGROUND Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). RESULTS We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. CONCLUSION This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

Chromosomal assignment of the canine melanophilin gene (MLPH): a candidate gene for coat color dilution in Pinschers

Pinschers affected by coat color dilution show a specific pigmentation phenotype. The dilute pigmentation phenotype leads to a silver-blue appearance of the eumelanin-containing fur and a pale sandy color of pheomelanin-containing fur. In Pinscher breeding, dilute black-and-tan dogs are called "blue," and dilute red or brown animals are termed "fawn" or "Isabella fawn." Coat color dilution in Pinschers is sometimes accompanied by hair loss and a recurrent infection of the hair follicles. In human and mice, several well-characterized genes are responsible for similar pigment variations. To investigate the genetic cause of the coat color dilution in Pinschers, we isolated BAC clones containing the canine ortholog of the known murine color dilution gene Mlph. RH mapping of the canine MLPH gene was performed using an STS marker derived from BAC sequences. Additionally, one MLPH BAC clone was used as probe for FISH mapping, and the canine MLPH gene was assigned to CFA25q24.

A duplication in the canine beta-galactosidase gene GLB1 causes exon skipping and GM1-gangliosidosis in Alaskan huskies

GM(1)-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the beta-galactosidase gene (GLB1). The molecular cause of GM(1)-gangliosidosis in Alaskan huskies was investigated and a novel 19-bp duplication in exon 15 of the GLB1 gene was identified. The duplication comprised positions +1688-+1706 of the GLB1 cDNA. It partially disrupted a potential exon splicing enhancer (ESE), leading to exon skipping in a fraction of the transcripts. Thus, the mutation caused the expression of two different mRNAs from the mutant allele. One transcript contained the complete exon 15 with the 19-bp duplication, while the other transcript lacked exon 15. In the transcript containing exon 15 with the 19-bp duplication a premature termination codon (PTC) appeared, but due to its localization in the last exon of canine GLB1, nonsense-mediated RNA decay (NMD) did not occur. As a consequence of these molecular events two different truncated GLB1 proteins are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active enzyme to prevent clinical signs of disease. In affected homozygous dogs no functional GLB1 is synthesized and G(M1)-gangliosidosis occurs.

A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12.

Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds-the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10-11). Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018-5,081,823 bp), tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8) and does not extend to the dog leukocyte antigen (DLA) class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.

In vitro effects of 3 % hypertonic saline and 20 % mannitol on canine whole blood coagulation and platelet function.

BACKGROUND: Hyperosmolar therapy, using either mannitol or hypertonic saline (HTS), is considered the treatment of choice for intracranial hypertension. However, hyperosmolar agents may impair coagulation and platelet function, limiting their use in patients at risk for hemorrhage. Despite this, studies evaluating the effects of mannitol compared to other hyperosmolar agents in dogs are largely lacking. The aim of this study was to compare the in vitro effects on global hemostasis and platelet function of 20 % mannitol and 3 % HTS on canine blood. METHODS: Citrated whole blood from 15 healthy dogs was diluted with 0.9 % saline, 20 % mannitol and 3 % HTS in ratios of 1:16 and 1:8. Rotational thromboelastometry (ROTEM) was used to assess clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) following extrinsic activation (Ex-tem) and after platelet inhibition (Fib-tem). A platelet function analyzer (PFA-100) was used to assess closure time (CtPFA). RESULTS: No significant differences were observed between untreated whole blood and samples diluted with saline. Samples diluted with both mannitol and HTS were hypocoagulable compared to untreated whole blood samples. At a dilution of 1:16, no significant differences were found between any measured parameter in samples diluted with saline compared to mannitol or HTS. At a 1:8 dilution, CtPFA was prolonged in samples diluted with mannitol and HTS compared to saline, and CtPFA was prolonged more with mannitol than HTS. Ex-tem CT was increased at a 1:8 dilution with mannitol compared to HTS. Ex-tem CFT was prolonged at a 1:8 dilution with both agents compared to saline, and was prolonged more with mannitol than HTS. Ex-tem MCF was reduced at a 1:8 dilution with both agents compared to saline. DISCUSSION AND CONCLUSIONS: Data in this study indicate that both mannitol and HTS affect canine platelet function and whole blood coagulation in vitro in a dose-dependent fashion. The most pronounced effects were observed after high dilutions with mannitol, which impaired platelet aggregation, clot formation time, clot strength, and fibrin formation significantly more than HTS. Further in vivo studies are necessary before recommendations can be made

Analytic validation of a gas chromatography-mass spectrometry method for quantification of six amino acids in canine serum samples.

OBJECTIVE To analytically validate a gas concentration of chromatography-mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at -80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at -80°C for 1 week had no effect on GC-MS results.

Acute kidney injury in three dogs after ingestion of a descaling agent containing maleic acid

Maleic acid (MA) is a common component of descaling products and is widely used in daily life. Accidental ingestion in relevant amounts does not play a major role in human beings; however, it seems to be highly toxic for dogs. It has been commonly used experimentally to induce Fanconi syndrome in dogs or small rodents. Two dogs were presented for acute kidney injury (AKI) after accidental ingestion of a descaling agent containing MA at an estimated amount of 70 mg/kg each. The third dog involved was euthanased by the referring veterinarian, and postmortem pathological analysis revealed severe acute tubular necrosis consistent with toxic nephropathy. The other dogs received symptomatic therapy for AKI including treatment with haemodialysis and showed complete normalisation of serum creatinine at a follow-up after five months. Renal damage can be very severe, but seems to be at least partially reversible and an attempt to treatment is warranted.

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection.

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

Biomechanical Evaluation of the Stabilizing Function of Three Atlantoaxial Implants Under Shear Loading: A Canine Cadaveric Study

OBJECTIVE: To compare the biomechanical properties of a ventral transarticular lag screw fixation technique, a new dorsal atlantoaxial instability (AAI) clamp, and a new ventral AAI hook plate under sagittal shear loading after transection of the ligaments of the atlantoaxial joint. STUDY DESIGN: Cadaveric biomechanical study. ANIMALS: Canine cadavers (n = 10). MATERIALS AND METHODS: The occipitoatlantoaxial region of Beagles euthanatized for reasons unrelated to the study was prepared leaving only ligamentous structures and the joint capsules between the first 2 cervical vertebrae (C1 and C2). The atlanto-occipital joints were stabilized with 2 transarticular diverging positive threaded K-wires. The occipital bone and the caudal end of C2 were embedded in polymethylmethacrylate and loaded in shear to a force of 50 Newtons. The range of motion (ROM) and neutral zone (NZ) of the atlantoaxial joint were determined after 3 loading cycles with atlantoaxial ligaments intact, after ligament transection, and after fixation with each implant. The testing order of implants was randomly assigned. The implants tested last were subjected to failure testing. RESULTS: All stabilization procedures decreased the ROM and NZ of the atlantoaxial joint compared to transected ligament specimens. Only stabilization with transarticular lag screws and ventral plates produced a significant reduction of ROM compare to intact specimens. CONCLUSION: Fixation with transarticular lag screws and a ventral hook plate was biomechanically similar and provided more rigidity compared to dorsal clamp fixation. Further load cycling to failure tests and clinical studies are required before making clinical recommendations.

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury

BACKGROUND CONTEXT In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

Minimally invasive approach to lumbosacral decompression in a cadaveric canine model

AIMS To determine efficacy of a minimally invasive (MI) surgical approach using a human MI lumbar retractor for canine lumbosacral dorsal laminectomy and partial discectomy and to compare this technique to the standard open surgical (OS) approach. METHODS Lumbosacral dorsal laminectomy and partial discectomy was performed on 16 large-breed canine cadavers using either a standard OS (n=8) or MI (n=8) approach. Skin and fascial incision length, procedure time, and intraoperative complications were recorded. Postoperatively specimens were evaluated for laminectomy and discectomy dimensions, and visible damage to the cauda equina and exiting nerve roots. RESULTS Median length of skin and fascial incisions in the OS group were longer than in the MI group (p<0.001). Median laminectomy length was similar between both approaches (p=0.234) but width was greater for the MI than OS approach (p=0.002). Both approaches achieved similar partial discectomy width (p=0.279). Overall surgical time was longer for MI approaches compared to OS, with a median of 18.5 (min 15.5, max 21.8) minutes for MI compared to 14.6 (min 13.1, max 16.9) minutes for OS (p=0.001). CONCLUSIONS The MI approach reduced incision lengths while retaining comparable laminectomy and discectomy dimensions. For this in vitro model the MI approach required more time to complete, but this difference may not be relevant in clinical cases. CLINICAL RELEVANCE Dogs undergoing lumbosacral dorsal laminectomy are commonly large-breed dogs. The traditional open approach requires a large skin incision and soft tissue dissection, especially in overweight animals. A MI approach accomplishing the same surgical result while minimising soft tissue trauma could reduce post-operative pain and recovery time, and may lower wound-related complications. Clinical studies are needed to confirm postoperative benefit and assess operating times in vivo.

Does Stepwise Voltage Ramping Protect the Kidney from Injury During Extracorporeal Shockwave Lithotripsy? Results of a Prospective Randomized Trial

BACKGROUND Renal damage is more frequent with new-generation lithotripters. However, animal studies suggest that voltage ramping minimizes the risk of complications following extracorporeal shock wave lithotripsy (SWL). In the clinical setting, the optimal voltage strategy remains unclear. OBJECTIVE To evaluate whether stepwise voltage ramping can protect the kidney from damage during SWL. DESIGN, SETTING, AND PARTICIPANTS A total of 418 patients with solitary or multiple unilateral kidney stones were randomized to receive SWL using a Modulith SLX-F2 lithotripter with either stepwise voltage ramping (n=213) or a fixed maximal voltage (n=205). INTERVENTION SWL. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS The primary outcome was sonographic evidence of renal hematomas. Secondary outcomes included levels of urinary markers of renal damage, stone disintegration, stone-free rate, and rates of secondary interventions within 3 mo of SWL. Descriptive statistics were used to compare clinical outcomes between the two groups. A logistic regression model was generated to assess predictors of hematomas. RESULTS AND LIMITATIONS Significantly fewer hematomas occurred in the ramping group(12/213, 5.6%) than in the fixed group (27/205, 13%; p=0.008). There was some evidence that the fixed group had higher urinary β2-microglobulin levels after SWL compared to the ramping group (p=0.06). Urinary microalbumin levels, stone disintegration, stone-free rate, and rates of secondary interventions did not significantly differ between the groups. The logistic regression model showed a significantly higher risk of renal hematomas in older patients (odds ratio [OR] 1.03, 95% confidence interval [CI] 1.00-1.05; p=0.04). Stepwise voltage ramping was associated with a lower risk of hematomas (OR 0.39, 95% CI 0.19-0.80; p=0.01). The study was limited by the use of ultrasound to detect hematomas. CONCLUSIONS In this prospective randomized study, stepwise voltage ramping during SWL was associated with a lower risk of renal damage compared to a fixed maximal voltage without compromising treatment effectiveness. PATIENT SUMMARY Lithotripsy is a noninvasive technique for urinary stone disintegration using ultrasonic energy. In this study, two voltage strategies are compared. The results show that a progressive increase in voltage during lithotripsy decreases the risk of renal hematomas while maintaining excellent outcomes. TRIAL REGISTRATION ISRCTN95762080.

Whole-Genome Sequencing of a Canine Family Trio Reveals a FAM83G Variant Associated with Hereditary Footpad Hyperkeratosis

Over 250 Mendelian traits and disorders, caused by rare alleles have been mapped in the canine genome. Although each disease is rare in the dog as a species, they are collectively common and have major impact on canine health. With SNP-based genotyping arrays, genome-wide association studies (GWAS) have proven to be a powerful method to map the genomic region of interest when 10-20 cases and 10-20 controls are available. However, to identify the genetic variant in associated regions, fine-mapping and targeted re-sequencing is required. Here we present a new approach using whole-genome sequencing (WGS) of a family trio without prior GWAS. As a proof-of-concept, we chose an autosomal recessive disease known as hereditary footpad hyperkeratosis (HFH) in Kromfohrl änder dogs. To our knowledge, this is the first time this family trio WGS-approach, has successfully been used to identify a genetic variant that perfectly segregates with a canine disorder. The sequencing of three Kromfohrl änder dogs from a family trio (an affected offspring and both its healthy parents) resulted in an average genome coverage of 9.2X per individual. After applying stringent filtering criteria for candidate causative coding variants, 527 single nucleotide variants (SNVs) and 15 indels were found to be homozygous in the affected offspring and heterozygous in the parents. Using the computer software packages ANNOVAR and SIFT to functionally annotate coding sequence differences and to predict their functional effect, resulted in seven candidate variants located in six different genes. Of these, only FAM83G:c155G>C (p.R52P) was found to be concordant in eight additional cases and 16 healthy Kromfohrl änder dogs.

Proliferative kidney disease in brown trout – infection level, pathology and mortality under field conditions

Proliferative kidney disease (PKD) is an emerging disease threatening wild salmonid populations. In temperature-controlled aquaria, PKD can cause mortality rates of up to 85% in rainbow trout. So far, no data about PKD-related mortality in wild brown trout Salmo trutta fario are available. The aim of this study was to investigate mortality rates and pathology in brown trout kept in a cage within a natural river habitat known to harbor Tetracapsuloides bryosalmonae. Young-of-the-year (YOY) brown trout, free of T. bryosalmonae, were exposed in the River Wutach, in the northeast of Switzerland, during 3 summer months. Samples of wild brown trout caught by electrofishing near the cage location were examined in parallel. The incidence of PKD in cage-exposed animals (69%) was not significantly different to the disease prevalence of wild fish (82 and 80% in the upstream and downstream locations, respectively). The mortality in cageexposed animals, however, was as low as 15%. At the termination of the exposure experiment, surviving fish showed histological lesions typical for PKD regression, suggesting that many YOY brown trout survive the initial infection. Our results at the River Wutach suggest that PKD in brown trout does not always result in high mortality under natural conditions.