Kathryn M. Meurs

Genome-wide association identifies a deletion in the 3' untranslated region of striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins. We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine mapping and direct DNA sequencing identified an 8-bp deletion in the 3′ untranslated region (UTR) of the Striatin gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3′ UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in Striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized Striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, Plakophilin-2, Plakoglobin and Desmoplakin, all involved in the pathogenesis of ARVC in human beings. We suggest that Striatin may serve as a novel candidate gene for human ARVC.

A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher.

Familial dilated cardiomyopathy is a primary myocardial disease that can result in the development of congestive heart failure and sudden cardiac death. Spontaneous animal models of familial dilated cardiomyopathy exist and the Doberman pinscher dog is one of the most commonly reported canine breeds. The objective of this study was to evaluate familial dilated cardiomyopathy in the Doberman pinscher dog using a genome-wide association study for a genetic alteration(s) associated with the development of this disease in this canine model. Genome-wide association analysis identified an area of statistical significance on canine chromosome 14 (prawxa0=xa09.999e−05 corrected for genome-wide significance), fine-mapping of additional SNPs flanking this region localized a signal to 23,774,190–23,781,919 (pxa0=xa00.001) and DNA sequencing identified a 16-base pair deletion in the 5′ donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 gene in affected dogs (pxa0<xa00.0001). Electron microscopy of myocardium from affected dogs demonstrated disorganization of the Z line, mild to moderate T tubule and sarcoplasmic reticulum dilation, marked pleomorphic mitochondrial alterations with megamitochondria, scattered mitochondria with whorling and vacuolization and mild aggregates of lipofuscin granules. In conclusion, we report the identification of a splice site deletion in the PDK4 gene that is associated with the development of familial dilated cardiomyopathy in the Doberman pinscher dog.

A prospective genetic evaluation of familial dilated cardiomyopathy in the Doberman pinscher.

BACKGROUNDnThe Doberman Pinscher is one of the most common breeds of dogs to develop dilated cardiomyopathy (DCM), a primary heart muscle disorder characterized by myocardial dysfunction, cardiac arrhythmias, and congestive heart failure. In the Doberman Pinscher, the disease is typically adult onset, and a familial etiology has been suggested.nnnHYPOTHESISnDCM in the Doberman Pinscher, is a familial disease linked to a specific genetic marker.nnnANIMALSnThe study comprised an extended family of Doberman Pinschers with a history of DCM.nnnMETHODSnParticipating dogs were prospectively evaluated over an 8-year period. Phenotype of participating dogs was determined by annual echocardiography and ambulatory electrocardiography, and the pedigree was evaluated to determine a specific mode of inheritance. Three hundred seventy-two microsatellite markers were selected and genotyped to cover the 38 autosomal chromosomes. Phenotyping, genotyping, and pedigree information was entered into a database, and parametric, 2-point analysis was performed. Markers were considered to be linked to the development of DCM if the logarithm of odds LOD score was >/= 3.0.nnnRESULTSnAn autosomal dominant mode of inheritance was defined by the appearance of the disease in multiple generations, equal gender representation (P = .973) and male-to-male transmission. A maximum LOD score of 1.31 was obtained for I marker on chromosome 20, a score not high enough to be associated with DCM.nnnCONCLUSIONnDCM in the Doberman Pinscher is a familial disease inherited as an autosomal dominant trait. The causative gene(s) responsible for this condition remain unresolved. Association studies by means of array technology may provide new insights into gene identification.

Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency

OBJECTIVEnTo examine the presence and effect of calstabin2-deficiency in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy (ARVC).nnnANIMALSnThirteen Boxer dogs with ARVC.nnnMATERIALS AND METHODSnTissue samples were collected for histopathology, oligonucleotide microarray, PCR, immunoelectrophoresis, ryanodine channel immunoprecipitation and single-channel recordings, and calstabin2 DNA sequencing.nnnRESULTSnIn cardiomyopathic Boxer dogs, myocardial calstabin2 mRNA and protein were significantly decreased as compared to healthy control dogs (calstabin2 protein normalized to tetrameric cardiac ryanodine receptor (RyR2) complex: affected, 0.51+/-0.04; control, 3.81+/-0.22; P<0.0001). Calstabin2 deficiency in diseased dog hearts was associated with a significantly increased open probability of single RyR2 channels indicating intracellular Ca(2+) leak. PCR-based sequencing of the promoter, exonic and splice site regions of the canine calstabin2 gene did not identify any causative mutations.nnnCONCLUSIONSnCalstabin2 deficiency is a potential mechanism of Ca(2+) leak-induced ventricular arrhythmias and heart disease in Boxer dogs with ARVC.

Prevalence of the Myosin-Binding Protein C Mutation in Maine Coon Cats

BACKGROUNDnAn autosomal dominant mutation has been identified in the myosin-binding protein C (MYBPC3) gene of Maine Coon cats. This mutation changes a conserved amino acid and computationally alters the protein conformation of this gene in Maine Coon cats with hypertrophic cardiomyopathy. The prevalence of this mutation is unknown.nnnOBJECTIVEnTo determine the genetic prevalence of the MYBPC3 mutation in a large cohort of predominantly Maine Coon cats.nnnANIMALSnThree thousand three hundred and ten DNA samples (blood or buccal swab) from cats.nnnMETHODSnThis retrospective study reviewed the Veterinary Cardiac Genetics Laboratory database at Washington State University for samples submitted for evaluation of the Maine Coon MYBPC3 mutation. The data were analyzed with respect to the breed of cat, mutation status (negative, heterozygous, homozygous), and geographic origin of the submission.nnnRESULTSnIn the population of cats studied, Maine Coon cats accounted for 100% of all cats positive for the mutation, and the worldwide percentage of Maine Coon cats carrying the MYBPC3 mutation was 34%.nnnCONCLUSIONS AND CLINICAL IMPORTANCEnThe prevalence of the mutation (heterozygous or homozygous) was very similar among countries of submission, suggesting that the 34% mutation rate of the tested samples is a reasonable estimate of the true prevalence of the mutation within the breed. Because of the high prevalence of this mutation, a breeding recommendation to eliminate all cats with the mutation could have a substantial impact on the gene pool. Additional studies are indicated to explore the relationship between genotype and clinical outcome in affected cats.

Tissue Doppler Imaging in Maine Coon Cats with a Mutation of Myosin Binding Protein C with or without Hypertrophy

BACKGROUNDnThe cardiac myosin binding protein C gene is mutated in Maine Coon (MC) cats with familial hypertrophic cardiomyopathy.nnnHYPOTHESESnEarly diastolic mitral annular velocity is incrementally reduced from normal cats to MC cats with only an abnormal genotype to MC cats with abnormal genotype and hypertrophy.nnnANIMALSnGroup 1 consisted of 6 normal domestic shorthair cats, group 2 of 6 MC cats with abnormal genotype but no hypertrophy, and group 3 of 15 MC cats with hypertrophy and abnormal genotype.nnnMETHODSnThe genotype and echocardiographic phenotype of cats were determined, and the cats were divided into the 3 groups. Tissue Doppler imaging (TDI) of the lateral mitral annulus from the left apical 4-chamber view was performed. Five nonconsecutive measurements of early diastolic mitral annular velocity (EM) or summated early and late diastolic velocity (EAsum) and heart rate were averaged.nnnRESULTSnThere was an ordered reduction in Em-EAsum as group number increased (group 1, range 9.7-14.7 cm/s; group 2, range 7.5-13.2 cm/s; group 3, range 4.5-14.1 cm/s; P = .001). Using the lower prediction limit for normal Em-EAsum, the proportion of cats with normal Em-EAsum decreased as the group number increased (P = .001). However, Em-EAsum was reduced in only 3 of 6 cats in group 2.nnnCONCLUSIONnThe incremental reduction of Em-EAsum as group severity increased indicates that diastolic dysfunction is an early abnormality that occurs before hypertrophy development. TDI measurement of Em or EAsum of the lateral mitral annulus is an insensitive screening test for identification of phenotypically normal, genotypically affected cats.

Analysis of 8 sarcomeric candidate genes for feline hypertrophic cardiomyopathy mutations in cats with hypertrophic cardiomyopathy.

BACKGROUNDnHypertrophic cardiomyopathy (HCM) is the most common heart disease in cats. Causative mutations have been identified in the Maine Coon (MC) and Ragdoll breed in the cardiac myosin binding protein C gene (MYBPC3). HCM is thought to be inherited in other breeds.nnnHYPOTHESISnThat a causative mutation for HCM in the British Shorthair (BSH), Norwegian Forest (NWF), Siberian, Sphynx, or MC cats would be identified in the exonic and splice site regions of 1 of 8 genes associated with human familial HCM.nnnANIMALSnThree affected BSH, NWF, Siberians, Sphynx, 2 MC (without the known MC mutation), and 2 Domestic Shorthair cats (controls) were studied.nnnMETHODSnProspective, observational study. Exonic and splice site regions of the genes encoding the proteins cardiac troponin I, troponin T, MYBPC3, cardiac essential myosin light chain, cardiac regulatory myosin light chain, alpha tropomyosin, actin, and beta-myosin heavy chain were sequenced. Sequences were compared for nucleotide changes between affected cats, the published DNA sequences, and control cats. Changes were considered to be causative for HCM if they involved a conserved amino acid and changed the amino acid to a different polarity, acid-base status, or structure.nnnRESULTSnA causative mutation for HCM was not identified, although several single nucleotide polymorphisms were detected.nnnCONCLUSIONS AND CLINICAL IMPORTANCEnMutations within these cardiac genes do not appear to be the only cause of HCM in these breeds. Evaluation of additional cardiac genes is warranted to identify additional molecular causes of this feline cardiac disease.

Finding cardiovascular disease genes in the dog

Recent advances in canine genomics are changing the landscape of veterinary biology, and by default, veterinary medicine. No longer are clinicians locked into traditional methods of diagnoses and therapy. Rather, major advances in canine genetics and genomics from the past five years are now changing the way the veterinarian of the 21st century practices medicine. First, the availability of a dense genome map gives canine genetics a much-needed foothold in comparative medicine, allowing advances made in human and mouse genetics to be applied to companion animals. Second, the recently released 7.5x whole genome sequence of the dog is facilitating the identification of hereditary disease genes. Finally, development of genetic tools for rapid screening of families and populations at risk for inherited disease means that the cost of identifying and testing for disease loci will significantly decrease in coming years. Out of these advances will come major changes in companion animal diagnostics and therapy. Clinicians will be able to offer their clients genetic testing and counseling for a myriad of disorders. In this review we summarize recent findings in canine genomics and discuss their application to the study of canine cardiac health.

Correlation of heart rate to body weight in apparently normal dogs

OBJECTIVEnTo evaluate the correlation between heart rate and body weight in the apparently healthy dog.nnnANIMALSnSixty dogs weighing between 2 and 80 kg.nnnMETHODSnHeart rate was evaluated with a 24-h ambulatory electrocardiogram. Minimum, average, maximum heart rate, ventricular premature complex (VPC) number and supraventricular premature complex (SVC) number were tabulated for each dog.nnnRESULTSnMinimum, maximum and average heart rate did not correlate to body weight. For all dogs, the median minimum heart rate was 42 bpm (beats per minute), median average heart rate was 73 bpm, and median maximum heart rate was 190. The median number of VPCs and SVC was zero.nnnCONCLUSIONSnThe present study does not support a correlation between heart rate and body weight in apparently healthy dogs.

Comparison of Polymerase Chain Reaction with Bacterial 16s Primers to Blood Culture to Identify Bacteremia in Dogs with Suspected Bacterial Endocarditis

BACKGROUNDnIdentification of the bacterial organism in dogs with endocarditis is challenging. Human studies have reported the utility of the polymerase chain reaction (PCR) to amplify and identify bacterial nucleic acid from infected valvular tissue and blood.nnnHYPOTHESIS/OBJECTIVESnWe hypothesized that PCR using primers designed to amplify the bacterial 16s gene would identify circulating bacteria in dogs with suspected bacterial endocarditis more consistently than standard blood culture techniques.nnnANIMALSnEighteen dogs with suspected bacterial endocarditis based upon clinical and echocardiographic findings. Fifteen clinically normal dogs served as negative controls.nnnMETHODSnProspective study of dogs evaluated for suspect endocarditis at 6 veterinary hospitals. A blood sample was drawn from all dogs and evaluated with both a single-sample PCR and standard 3-sample blood culture techniques.nnnRESULTSnBlood culture identified noncontaminant bacteria in 6/18 study animals (33%) and 1 control dog; PCR identified noncontaminant bacteria in 7/18 study animals (39%). There were no study animals in which the 2 tests identified different bacteria (κ = 1.0). However, bacteria were identified by both techniques in only 2/18 study animals. When results from both PCR and blood culture were considered together, a noncontaminant bacterial organism was identified in 11/18 study animals (61%).nnnCONCLUSION AND CLINICAL IMPORTANCEnThe results of this study suggest that although single sample PCR with 16s primers was not more sensitive than blood culture for detection of bacteremia in dogs with suspect endocarditis, performing both techniques simultaneously did increase the likelihood of identification of bacteria in blood.