Selective intestinal cobalamin malabsorption with proteinuria (Imerslund-Gräsbeck syndrome) in juvenile Beagles.

BACKGROUND: Selective intestinal cobalamin malabsorption with mild proteinuria (Imerslund-Gräsbeck syndrome; I-GS), is an autosomal recessive disorder of dogs caused by mutations in AMN or CUBN that disrupt cubam function and which can present as a medical emergency. OBJECTIVES: To describe the clinical, metabolic, and genetic bases of I-GS in Beagles. ANIMALS: Four cobalamin-deficient and 43 clinically normal Beagles and 5 dogs of other breeds. METHODS: Clinical description and candidate gene genetic study. Urinary organic acid and protein excretion were determined by gas-chromatography and SDS-PAGE, respectively. Renal cubilin protein expression was assessed on immunoblots. Mutation discovery was carried out by PCR amplification and DNA sequencing of exons with flanking splice sites and cDNA of CUBN and AMN. Genotyping was performed by restriction enzyme digestion of PCR amplicons. RESULTS: Juvenile-affected Beagles exhibited failure to thrive, dyshematopoiesis with neutropenia, serum cobalamin deficiency, methylmalonic aciduria, hyperammonemia, and proteinuria. Affected dogs' kidneys lacked detectable cubilin protein. All affected dogs were homozygous for a single-base deletion in CUBN exon 8 (CUBN c.786delC), predicting a translational frameshift, and the 2 parents tested were heterozygous. CONCLUSIONS: The CUBN mutation in juvenile I-GS Beagles causes a more severe cobalamin malabsorption than in Border Collies with a different CUBN defect, but is similar to I-GS caused by AMN mutations in Giant Schnauzers and Australian Shepherds. Awareness of the disorder and breed predispositions to I-GS is crucial to precisely diagnose and promptly treat hereditary cobalamin malabsorption and to prevent disease in those dogs at risk in future generations.

Congenital ureteral ectopia in continent and incontinent-related Entlebucher mountain dogs: 13 cases (2006-2009).

BACKGROUND: Ectopic ureters (EUs) associated with varying combinations of urinary incontinence, hydronephrosis, and urinary tract infection have been identified in related North American Entlebucher Mountain Dogs. OBJECTIVES: To characterize the disease phenotype in affected dogs and evaluate possible modes of inheritance. ANIMALS: Twenty client-owned Entlebucher Mountain Dogs. Nine dogs had clinical signs of urinary tract disease. METHODS: Prospective case series in which 17 dogs were evaluated with excretory urography, ultrasonography, and urethrocystoscopy. Three additional dogs were evaluated by necropsy alone. Clinical and pedigree histories from 165 North American Entlebuchers were compiled for analysis. RESULTS: Eleven female and 2 male dogs were found to have EUs. Six females and 1 male were continent. Bilateral intravesicular ectopic ureters (IVEUs) were identified in 9 dogs, bilateral extravesicular ectopic ureters (EVEUs) in 3 dogs, and 1 dog had IVEU and EVEU. Hydronephrosis was identified in 5 dogs, 3 of which had bilateral IVEUs. Two necropsied dogs had bilateral hydronephrosis with presumed ureterovesical junction obstruction associated with chronic granulation tissue or lymphoplasmacytic inflammation. Twenty-six dogs with EUs were identified in the pedigree. Because of incomplete penetrance, mode of inheritance could not be determined. CONCLUSIONS AND CLINICAL IMPORTANCE: Ureteral ectopia is common in North American Entlebucher Mountain Dogs and clinical signs alone could not reliably predict disease phenotype. EVEUs were associated with urinary incontinence and occasionally hydronephrosis. IVEUs were clinically silent or associated with hydronephrosis. Further analyses are necessary to confirm and characterize the hereditary nature of the disorder.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Expression of toll-like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO-affected horses.

REASONS FOR PERFORMING STUDY: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables.It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll-like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)-8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. HYPOTHESIS: TLR2 and IL-8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. OBJECTIVES: To determine TLR2, IL-8 and A20 mRNA expression in lungs of stabled and pastured RAO-affected and control horses. METHODS: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR2, IL-8 and A20 mRNA expression quantified by qRT-PCR in 6 RAO-affected and 6 control horses, during and after exposure to hay and straw. RESULTS: Airway obstruction and neutrophils were increased in RAO-affected horses during stabling. While stabling increased IL-8, TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. POTENTIAL RELEVANCE: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal-induced airway inflammation. Equine epithelial-derived A20 may be involved in modulation of airway inflammation.

The long and the short of it: evidence that FGF5 is a major determinant of canine 'hair'-itability.

Hair length in dogs has been known for many years to be primarily controlled by a limited number of genes, but none of the genes have been identified. One of these genes produces a recessively inherited long-haired phenotype that has been thought to explain the bulk of hair-length variation among many breeds. Sequence analysis of the FGF5 gene in short and long-haired corgis resulted in the identification of two coding region differences: a duplication in a relatively non-conserved region of the gene and a missense mutation, resulting in the substitution of Phe for Cys, in a highly conserved region. Genotyping of 218 dogs from three breeds fixed for long hair, eight breeds fixed for short hair and five breeds in which long hair is segregating provided evidence that the missense mutation is associated with the hair-length differences among these breeds.

A von Willebrand's factor genomic nucleotide variant and polymerase chain reaction diagnostic test associated with inheritable type-2 von Willebrand's disease in a line of german shorthaired pointer dogs.

Heritable, type-2 von Willebrand's disease (vWD) was studied in a line of German Shorthaired Pointers (GSPs) in which some members had a nucleotide variant in exon 28 of the von Willebrand factor (VWF) gene. A polymerase chain reaction (PCR) diagnostic test for the nucleotide variant was developed to establish the disorder's mode of inheritance and to eliminate it from the line. Thirty-six of the 49 GSPs in the line, 14 unrelated GSP controls, and 71 unrelated dogs of various breeds were tested for the presence of the variant nucleotide. All the dogs with a vWF antigen deficiency (<70% of normal) were either homozygous or heterozygous for the nucleotide variant. The variant was not located in any tested dog in the line or outside of the line with a vWF antigen value greater than 68%. Of the GSPs in the line tested, two were homozygous for the variant, 15 were heterozygous, and 19 were variant free. The collective evidence of this and other studies is consistent with the variant nucleotide being the cause of the type-2 vWD in this line of GSPs and German Wirehaired Pointers. The PCR diagnostic test for the variant nucleotide was successfully used to select and produce progeny that were variant free and vWD free. This test should be effective in the subsequent elimination of this same variant from other lines of dogs.

Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig.

Genes located on human chromosome 12 (HSA12) are conserved on pig chromosomes 5 and 14 (SSC5 and SSC14), with HSA12q23.3-->q24.11 harboring the evolutionary breakpoint between these chromosomes. For this study, pig sequence-tagged sites (STS) were developed for nine HSA12 genes flanking this breakpoint. Radiation hybrid (RH) mapping using the IMpRH panel revealed that COL2A1, DUSP6, KITLG, PAH and STAB2 map to SSC5, while PXN, PLA2G1B, SART3 and TCF1 map to SSC14. Polymorphisms identified in COL2A1, DUSP6, PAH, PLA2G1B and TCF1 were used for genetic linkage mapping and confirmed the map locations for these genes. Our results indicate that the HSA12 evolutionary breakpoint occurs between STAB2 and SART3 in a region spanning less than five million basepairs. These results refine the comparative map of the HSA12 evolutionary breakpoint region and help to further elucidate the extensive gene order rearrangements between HSA12 and SSC5 and 14.

Digging up the canine genome--a tale to wag about.

There is incredible morphological and behavioral diversity among the hundreds of breeds of the domestic dog, CANIS FAMILIARIS. Many of these breeds have come into existence within the last few hundred years. While there are obvious phenotypic differences among breeds, there is marked interbreed genetic homogeneity. Thus, study of canine genetics and genomics is of importance to comparative genomics, evolutionary biology and study of human hereditary diseases. The most recent version of the map of the canine genome is comprised of 3,270 markers mapped to 3,021 unique positions with an average intermarker distance of approximately 1 Mb. The markers include approximately 1,600 microsatellite markers, about 1,000 gene-based markers, and almost 700 bacterial artificial chromosome-end markers. Importantly, integration of radiation hybrid and linkage maps has greatly enhanced the utility of the map. Additionally, mapping the genome has led directly to characterization of microsatellite markers ideal for whole genome linkage scans. Thus, workers are now able to exploit the canine genome for a wide variety of genetic studies. Finally, the decision to sequence the canine genome highlights the dog's evolutionary and physiologic position between the mouse and human and its importance as a model for study of mammalian genetics and human hereditary diseases.

Within-breed heterozygosity of canine single nucleotide polymorphisms identified by across-breed comparison.

Identification of single nucleotide polymorphisms (SNPs) by DNA sequence comparison across breeds is a strategy for developing genetic markers that are useful for many breeds. However, the heterozygosity of SNPs identified in this way might be severely reduced within breeds by inbreeding or genetic drift in the small effective population size of a breed (population subdivision). The effect of inbreeding and population subdivision on heterozygosity of SNPs in dog breeds has never been investigated in a systematic way. We determined the genotypes of dogs from three divergent breeds for SNPs in four canine genes (ACTC, LMNA, SCGB, and TYMS) identified by across-breed DNA sequence comparison, and compared the genotype frequencies to those expected under Hardy-Weinberg equilibrium (HWE). Although population subdivision significantly skewed allele frequencies across breeds for two of the SNPs, the deviations of observed heterozygosities compared with those expected within breeds were minimal. These results indicate that across-breed DNA sequence comparison is a reasonable strategy for identifying SNPs that are useful within many canine breeds.

Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.

Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.

Inducible nitric oxide expression in equine articular chondrocytes: effects of antiinflammatory compounds.

OBJECTIVE: To determine the effects of recombinant equine IL-1beta and a number of antiinflammatory compounds on the expression and activity of inducible nitric oxide synthase (iNOS) in cultured equine chondrocytes. DESIGN: RT-PCR methods were used to amplify a portion of the equine iNOS message to prepare an RNA probe. Northern blot analysis was used to quantify the expression of iNOS in first passage cultures of equine articular chondrocytes propagated in the presence or absence of recombinant equine interleukin-1beta (reIL-1beta), dexamethasone (DEX), polysulfated glycosaminoglycan (PSGAG), hyaluronan (HA), and phenylbutazone (PBZ), each at concentrations of 10 and 100 microg/ml. Nitrite concentrations in conditioned media of similarly treated cells were used to quantify iNOS activity. RESULTS: Recombinant equine IL-1beta increased the expression of iNOS in a dose-dependent manner. This result was paralleled by an increased concentration of nitrite in the culture media of reIL-1beta-treated cells. DEX and PSGAG significantly reduced iNOS gene expression and media supernatant nitrite concentrations in cytokine-stimulated cultures. HA and PBZ had no consistent effect on the expression of iNOS and did not significantly influence nitrite content of conditioned media. CONCLUSIONS: NO is considered an important mediator in the pathophysiologic processes of arthritis and an inducible NOS is expressed by equine chondrocytes. Pre-translational regulation of the iNOS gene by DEX and PSGAG appears to contribute to the cartilage-sparing properties of these compounds.

SCID in Jack Russell terriers: a new animal model of DNA-PKcs deficiency.

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in "leaky" coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.

DNA sequence and physical mapping of the canine transglutaminase 1 gene.

The transglutaminase 1 gene (TGM1) encodes an enzyme necessary for cross-linking the structural proteins that form the cornified envelope, an essential component of the outermost layer of the skin, the stratum corneum. Reported here is the complete coding region of canine TGM1, its chromosome localization, and its map position in the integrated canine linkage-radiation hybrid map. Canine TGM1 consists of 2,448 nucleotides distributed over 15 exons. The nucleotide sequence has 90% identity to human TGM1. The deduced canine TGM1 protein is 816 amino acids long and is 92% identical to human TGM1. Using fluorescence in situ hybridization, we localized canine TGM1 to dog (Canis familiaris) chromosome 8 (CFA 8q). Canine TGM1 localized to CFA 8 on the integrated linkage-radiation hybrid map in the interval FH2149-MYH7. Characterizing the coding region of canine TGM1 is a first step in examining the role of this enzyme in normal and defective cornification in the dog.

Polymorphism identification within 50 equine gene-specific sequence tagged sites.

The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 bp. Several of the putative SNPs were tested and verified by restriction enzyme analysis using natural restriction sites or ones created by primer mutagenesis. The lowest allele frequency for a SNP detected in pooled samples was 10%. Three of the SNPs verified in the diverse horse pool were further tested in six breed-specific horse pools and were found to be reasonably variable within breeds. The pool-and-sequence method allows identification of polymorphisms in horse populations and will be a valuable tool for future disease gene and comparative mapping in horses.

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan.

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.

Estimate of nucleotide diversity in dogs with a pool-and-sequence method.

Nucleotide diversity (pi), the average number of base differences per site for two homologous sequences randomly selected from a population, is an important parameter used to understand the structure and history of populations. It is also important for determining the feasibility of developing a genetic map for a species from single nucleotide polymorphisms (SNPs). Nucleotide diversity has never been estimated for dogs. Segments of twelve canine genes from ten diverse dog breeds were examined for nucleotide variation by using a pool-and-sequence method. We identified three SNPs in the coding regions (2501 bp) and 11 SNPs in the introns (2953 bp). Each of these putative SNPs was tested by restriction enzyme analysis, and all were verified. Six additional SNPs were identified in a single SINE contained in one gene. Using these data, canine sequence diversity across breeds was estimated to be 0.001 and 0.0004 in intronic and coding regions, respectively, with SNPs spaced every 400 bp on average. Discovery of useful SNPs in 7 of the 12 genes suggests that construction of a canine SNP-based map can be accomplished with current technology. Thirteen polymorphic SNPs were also found in 5847 bp in the cat, horse, ox, and pig, by using four of the same genes from which canine nucleotide diversity was estimated. These results suggest that these species may have similar amounts of nucleotide diversity.

Mutation causing von Willebrand's disease in Scottish Terriers.

Von Willebrand's Disease (vWD) in the Scottish Terrier breed is a serious, often fatal, hereditary bleeding disorder. Elimination of the mutated gene by selective breeding is an important goal for the health of this breed. Although the standard protein-based tests are accurate for identification of affected Scottish Terriers, they are not reliable for the identification of carriers of the mutant gene unless multiple replicate assays are performed. A simple, highly accurate test for carriers of the disease is needed so that veterinarians can counsel clients on which animals to use in their breeding programs. The complete coding region of von Willebrand factor (vWF) complementary DNA (cDNA) was sequenced from an affected animal, and a single base deletion in the codon for amino acid 85 of the prepro-vWF cDNA that leads to Scottish Terrier vWD was identified. A highly accurate polymerase chain reaction assay was developed that can distinguish homozygous normal animals from those that are homozygous affected or heterozygous. In a voluntary survey of 87 animals provided by Scottish Terrier owners, 15 were carriers and 4 were affected with vWD, 2 of which had previously been shown to have undetectable vWF. The determination of the complete canine vWF cDNA sequence should facilitate the identification of additional vWD alleles in other breeds and other species.