Dilated cardiomyopathy is the most common myocardial disease in dogs and is characterized by reduced contractility (systolic dysfunction) and eccentric hypertrophy (dilation) of the left or both ventricles. It is responsible for a considerable degree of morbidity and death in this species, with the primary consequences being CHF or arrhythmic sudden death.1 Most cases are idiopathic, and an underlying cause remains to be identified. Comparatively few cases are associated with known underlying causes, such as nutritional deficiencies (eg, taurine deficiency), drugs or toxins (eg, doxorubicin), or primary arrhythmias (tachycardiomyopathy).1,2 The high prevalence of DCM in specific dog breeds strongly suggests that genetic factors have a role in the pathogenesis of some and probably most forms of DCM in dogs.3
The Doberman Pinscher is one such highly affected breed, with almost 50% of dogs affected over their lifetime in some populations.a Prospective pedigree analyses in Doberman Pinschers have revealed that DCM is indeed a familial disease inherited as an autosomal dominant trait.4 The Doberman Pinscher breed in North America is a relatively closed breed; by the 1950s, approximately half of the registered Doberman Pinschers in the United States were descended from a single family.5 Thus, it is possible that the genetic basis for DCM in that breed stems from the same causative mutation or mutations. The fact that familial forms of DCM are known to exist in humans further suggests a genetic cause, and several genetic mutations responsible for these forms of DCM have been described.6–8
Dilated cardiomyopathy in humans is characterized by the same structural and functional changes in affected Doberman Pinschers, and at least 30% to 50% of cases are familial (genetic).7 Mutations that affect genes encoding proteins of the contractile apparatus may cause defective force generation, and genetic mutations that affect actin, myosin, MYBP-C, and troponin T have been described.6–8 Similarly, mutations that affect genes encoding cardiac structural proteins may cause defective force transmission and mutations affecting titin, desmin, dystrophin, dystroglycans, and sarcoglycans have been described.6–8 Other genetic mutations may affect proteins involved in calcium handling, thereby altering availability of or sensitivity to calcium (eg, phospholamban and SERCA2), or proteins involved in regulating energy use, thereby causing energy deficits. Evaluation of genetic mutations known to be associated with human familial DCM may provide a framework within which to begin investigating the causes of DCM in dogs.
Comparatively little information exists with respect to the genetics of DCM in dogs; however, mutations of genes encoding for contractile, regulatory, and structural cardiac proteins are all potential candidates. With respect to isolated DCM (in the absence of skeletal muscle involvement), studies investigating genetic causes in dogs have revealed neither mutations in specific segments of the cardiac actin, desmin, sarcoglycan delta, phospholamban, troponin C, lamin A/C, cysteine- and glycine-rich protein 3, cTnT, or MYHC7 genes in Doberman Pinschers9–13 nor abnormalities in myocardial dystrophin, α-sarcoglycan, or β-dystroglycan in various breeds, including Doberman Pinschers.14 No changes were found in the sequences of Tcap, α-tropomyosin, vinculin, or taffazin in Newfoundlands or Irish Wolfhounds with DCM.15–17 Results of other research have suggested that DCM in a European line of Doberman Pinschers may be associated with a mutation of the titin gene.b To our knowledge, this was the first evidence supporting a potential specific genetic cause for DCM in Doberman Pinschers.
The purpose of the study reported here was to identify a causative mutation for DCM in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes associated with familial DCM in humans. The 10 genes were selected on the basis of known mutations associated with DCM in humans who had the characteristics of the disease that develops in Doberman Pinschers, combined with information from the scarce literature regarding genetic linkage to DCM in dogs and from the recently available dog genome.18–20
Congestive heart failure
Myosin-binding protein C
β-Myosin heavy chain
National Center for Biotechnology Information
Single nucleotide polymorphism
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RNAlater-ICE solution, Ambion Inc, Streetsville, ON, Canada.
PureYield RNA Midiprep System, Promega Corp, Madison, Wis.
RNeasy Midi Kit, Qiagen, Mississauga, ON, Canada.
Gene Tools Software, BioTools, Edmonton, AB, Canada.
ABI 3900 High-Throughput DNA Synthesizer, Applied Biosystems, Foster City, Calif.
SuperScript First-Strand Synthesis System, Invitrogen, Carlsbad, Calif.
AccuPrime Taq DNA Polymerase High Fidelity kit, Invitrogen, Carlsbad, Calif.
GeneAmp PCR System 9700, Invitrogen, Carlsbad, Calif.
Bioshop, Burlington, ON, Canada.
NucleoFast 96 PCR clean-up plate, Biolynx, Brockville, ON, Canada.
BigDye Terminator Cycle Sequencing Ready Reaction kit, version 3.1, Invitrogen, Carlsbad, Calif.
GeneAmp PCR System 2720 Thermal Cycler, Invitrogen, Carlsbad, Calif.
Multiscreen-HV plates, Millipore, Mississauga, ON, Canada.
Sephadex G-50 superfine, Sigma-Aldrich, Oakville, ON, Canada.
3730 DNA Analyzer, Invitrogen, Carlsbad, Calif.
ABI Prism DNA Sequencing Analysis Software, version 3.7, Invitrogen, Carlsbad, Calif.
N-SCAN gene prediction system, UCSC Genome Bioinformatics, University of California-Santa Cruz, Santa Cruz, Calif. Available at: genome.ucsc.edu/. Accessed Apr 16, 2009.
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Primers used for amplification of the coding regions of 10 cardiac genes (associated with familial DCM in humans) in ventricular myocardial samples obtained from dogs of breeds other than Doberman Pinscher without overt signs of cardiac disease (control dogs) and Doberman Pinschers with DCM and CHF in a study to identify a causative mutation for DCM in Doberman Pinschers.
|Untranslated region covered (bp)†|
|Gene||Accession No.||Primer 5′–3′*||Amplicon size (kbp)||5′||3′|
|β-Cardiac myosin||XM_854082||Forward: TGTCTTTCCTTGCTGCTCTC||5.8||65||11|
|α-Cardiac actin||XM_535424||Forward: CGCAGCTGAGCCGGGATGTG||1.1||(6)||9|
|Cardiac troponin T||NM_001003012||Forward: GGAGGATGTCTGACGTGGAAGA||0.8||(18)||(21)|
|Myosin binding protein-C||NM_001048106||Forward: CTCTTTGGGCGGCCTGTGACTG||4.0||28||85|
|Muscle LIM protein||XM_859394||Forward: AGCGGTCTCTGCCCTCTCC||2.0||42||42|
The sizes of each amplicon and the length of the 5′ and 3′ untranslated region covered by each primer pair are shown.
Start and stop codons are in bold.
When the primer includes coding sequence, the last base pair in the coding sequence covered by the primer is listed in parentheses.
Primer was designed against the α-cardiac myosin gene.
Primer failed to provide good sequence.
Coding sequence was truncated at exon 15, compared with predicted sequence in database.
Coverage of primers used for sequencing each reading frame of 10 cardiac genes (associated with familial DCM in humans) in ventricular myocardial samples obtained from dogs of breeds other than Doberman Pinscher without overt signs of cardiac disease (control dogs) and Doberman Pinschers with DCM and CHF in a study to identify a causative mutation for DCM in Doberman Pinschers.
|Forward primers||Reverse primers|
|Gene||Amplicon size (kbp)||No. of primers||Mean coverage (bp)||No. of primers||Mean coverage (bp)|
|Myosin binding protein-C||4.0||5||805||3||767*|
|Muscle LIM protein||2.0||3||630||2||NA*|
The primers used for amplification were also used for sequencing and are included. Sequences < 1 kbp are not listed because only the primers used for amplification were used for sequencing and provided complete coverage.
Some primers produced sequences that did not overlap on that strand but provided complement sequences that were obtained by use of primers for the opposite strand.
NA = Not applicable.