Molecular evaluation of five cardiac genes

in Doberman Pinschers with dilated

cardiomyopathy
Kathryn M. Meurs, DVM, PhD; Kristina P. Hendrix, BS; Michelle M. Norgard, BS

Objective—To sequence the exonic and splice site regions of 5 cardiac genes associated
with the human form of familial dilated cardiomyopathy (DCM) in Doberman Pinschers with
DCM and to identify a causative mutation.
Animals—5 unrelated Doberman Pinschers with DCM and 2 unaffected Labrador Retriev-
ers (control dogs).
Procedures—Exonic and splice site regions of the 5 genes encoding the cardiac proteins
troponin C, lamin A/C, cysteine- and glycine-rich protein 3, cardiac troponin T, and the β-
myosin heavy chain were sequenced. Sequences were compared for nucleotide changes
between affected dogs and the published canine sequences and 2 control dogs. Base pair
changes were considered to be causative for DCM if they were present in an affected dog
but not in the control dogs or published sequences and if they involved a conserved amino
acid and changed that amino acid to a different polarity, acid-base status, or structure.
Results—A causative mutation for DCM in Doberman Pinschers was not identified, al-
though single nucleotide polymorphisms were detected in some dogs in the cysteine- and
glycine-rich protein 3, β-myosin heavy chain, and troponin T genes.
Conclusions and Clinical Relevance—Mutations in 5 of the cardiac genes associated with
the development of DCM in humans did not appear to be causative for DCM in Doberman
Pinschers. Continued evaluation of additional candidate genes or a focused approach with
an association analysis is warranted to elucidate the molecular cause of this important
cardiac disease in Doberman Pinschers. (Am J Vet Res 2008;69:1050–1053)

D ilated cardiomyopathy is a familial disease in Do-

berman Pinschers and is inherited as an autosomal
dominant trait.1–3 Onset of DCM is in adulthood, and af-
fected dogs may have myocardial dysfunction, tachyar-
rhythmias, and congestive heart failure.4–6 At this time,
the molecular basis for the disease in Doberman Pin-
schers is unknown.
A similar form of cardiomyopathy exists in humans
and is inherited in at least 20% to 50% of cases.7 Famil-
ial DCM in humans has been associated with mutations
in > 20 genes and is most commonly inherited in an
autosomal dominant pattern similar to that of Dober-
man Pinschers. The most common causes of the auto-
somal dominant form of familial DCM are single base
pair changes in the coding region of a gene that en-
codes for the one of the following proteins: lamin A/C,
MYH7, troponin T, CSRP3, troponin C, δ-sarcoglycan,
desmin, actin, troponin I, actinin-α2, α-tropomyosin,
and titin-cap, among others.7-13 A similarity between
many of these genes is that they encode for cytoskeletal
proteins with structural functions that include main-
taining structural integrity, preserving cell shape, orga-
nizing the contractile apparatus, and enabling the cell
Received October 22, 2007.
Accepted December 7, 2007.
From the Department of Veterinary Clinical Sciences, College of
Veterinary Medicine, Washington State University, Pullman, WA
99164.
Address correspondence to Dr. Meurs.
AECG
Abbreviations
Ambulatory electrocardiogram
CSRP3 Cysteine- and glycine-rich protein 3
DCM Dilated cardiomyopathy
FS Fractional shortening
LVIDD Left ventricular internal diameter during
diastole
LVIDS Left ventricular internal diameter during
systole
MYH7 β-myosin heavy chain
VPC Ventricular premature complex
to withstand mechanical stress.13 One proposed theory
for the development of DCM is that an abnormality of
a cytoskeletal protein may be a common factor in the
development of the disease and that without the struc-
tural support provided by these proteins, dilation and
dysfunction of the heart develop.11
The etiology of familial DCM in Doberman Pin-
schers is unknown. Previous studies14,15 have excluded
some of the genes responsible for the disease in humans,
including the desmin and δ-sarcoglycan genes, by the
typing of adjacent genetic markers in affected and un-
affected Doberman Pinschers. Additional studies16,17
have excluded the promoter and coding regions of the
phospholamban gene and coding region of the actin
gene via direct sequencing in affected and unaffected
dogs. Exclusion of these 4 genes still leaves several oth-
er genes associated with the autosomal dominant form

1050 AJVR, Vol 69, No. 8, August 2008

of the human disease yet to be evaluated. Additionally,
it is possible that Doberman Pinscher DCM does not
have the same genetic cause as in humans and that a
mutation will be identified in a novel gene. A linkage
analysis approach was performed to attempt to identify
a statistical relationship between the disease and a spe-
cific genetic region to focus the evaluation of additional
cardiac genes.6 Linkage analysis was not able to identify
a significant region of interest, but the strongest, yet
not significantly, associated marker was found on ca-
nine chromosome 20.6
The objectives of the study reported here were to
evaluate the splice site and coding regions of 5 addi-
tional cardiac genes in Doberman Pinschers with DCM.
The genes evaluated were the 4 most commonly re-
ported as causative in humans, including lamin A/C,
MYH7, troponin T, and CSRP3. A fifth gene, troponin
C, was selected for evaluation because of its location
on canine chromosome 20, the chromosome with the
highest linkage score in a previous linkage study.6
Materials and Methods
This study was conducted under the guidelines of
the Animal Care and Use Committee of The Ohio State
University. Written consent authorizing study partici-
pation was obtained from each client.
Phenotypic classification and pedigree analysis—
Five unrelated Doberman Pinschers with a diagnosis of
DCM as determined via physical examination, 24-hour
AECG, and an echocardiogram (2 dimensional and M-
mode) and 2 unrelated Labrador Retrievers (control
dogs) were evaluated. The AECG was recorded with a
3-channel transthoracic systema by use of a previously
described technique.18 The number of VPCs was tabu-
lated. Echocardiographic examination was performed
via transthoracic echocardiography and included 2-
dimensional and M-mode evaluation.b Left ventricular
internal diameter during systole, LVIDD, and FS(%)
were determined from the M-mode study at the left
ventricular level. The examinations were conducted
without sedation via standard clinical techniques de-
scribed for dogs.19 Participating dogs were classified
as affected on the basis of echocardiographic measure-
ments of an LVIDD > 4.6 cm or LVIDS > 3.8 cm with
an FS < 25%.20
Genotyping—Genomic DNA samples were pre-
pared from blood samples, as described.21 Briefly, cells
were osmotically lysed in 2X sucrose-Triton and Tris-
NH4Cl buffer, and nuclei were pelleted by centrifuga-
tion at 800 X g for 20 minutes at 4°C. Pellets were re-
suspended in saline (0.9% NaCl) solution–EDTA with
1% SDS and 50 µg of proteinase K/mL and incubated
overnight at 56°C. The samples were subjected to 2
successive phenol:chloroform:isoamyl (25:24:1; pH, 8)
extractions and 1 chloroform extraction. The DNA was
ethanol precipitated, washed with 75% ethanol, and re-
suspended in 250 µL of Tris EDTA buffer (10mM Tris-
HCl and 1mM EDTA; pH, 8).
Polymerase chain reaction amplification prim-
ers were designed for all exons of lamin A/C, MYH7,
troponin T, troponin C, and CSRP3 genes by use
of computer software and the canine nucleotide se-
quence information22 (ENSCAFG00000009404, EN-
SCAFG00000010798, ENSCAFG00000009424, EN-
SCAFG00000016863, and ENSCAFG0000001161923).
Standard PCR amplifications were carried out via
NH4SO4 amplification buffer, 0.1 U of Taq DNA poly-
merasec/µL of reaction volume, 2.5mM MgCl2, 12.5µM
of each dNTP, 2.5mM of each PCR amplification primer,
and 100 ng of template DNA. Samples were denatured
for 5 minutes at 94°C, followed by 40 cycles of 94°C for
20 seconds, 58°C for 30 seconds, 72°C for 30 seconds,
and 72°C for 7 minutes. The annealing temperature
was optimized to accommodate the respective primer
requirement.
Residual amplification primers and dNTPs were
removed from the PCR product. Amplicons were then
subjected to nucleotide sequence determination and
analyzed on a sequencerd by use of a forward and re-
verse primer for each reaction for every sample.
The sequences were compared for nucleotide se-
quence changes between affected dogs and the pub-
lished normal canine sequence or the control dogs.24
Base pair changes were considered to be possibly caus-
ative for DCM when they were present in any of the af-
fected dogs. When a base pair alteration was detected in
any of the affected dogs, the alteration was evaluated to
determine whether it changed a conserved amino acid
and whether that amino acid was changed to one of a
different polarity, acid-base status, or structure because
these could be evidence for a causative change.7
Results
The Doberman Pinscher group consisted of 3 fe-
males and 2 males (age range, 6 to 10 years; mean, 9
years). Affected dogs had a median FS of 12% (range,
9% to 17%), median LVIDD of 5.4 cm (range, 5.0 to

Table 1—Single nucleotide polymorphisms detected in the canine CSRP3, MYH7, and troponin T genes
in a group of 5 Doberman Pinschers with DCM and 2 unaffected Labrador Retrievers.

Gene Exon Codon Amino acid Dog group Dogs

CSRP3 4 9 Threonine Affected, controls 1 Female control
1 Female affected
1 Male affected

Troponin T 7 15 Alanine Affected 1 Female affected

1 Male affected

MYH7 31 54 Lysine Affected, controls 1 Male control

2 Male affected
2 Female affected

AJVR, Vol 69, No. 8, August 2008 1051

8.0), median LVIDS of 4.6 cm (range, 4.2 to 7.1), and
median number of VPCs/24 h of 250 (range, 77 to
15,419/24 h) The Labrador Retriever group (control
group) consisted of 2 unrelated dogs (a 4-year-old male
and a 7-year-old female).
No base pair differences that met the criteria for a
causative change were identified in the exonic or splice
site regions of the lamin A/C, MYH7, troponin T, tropo-
nin C, or CSRP3 genes between the affected Doberman
Pinschers and the control dogs or the published canine
sequence. A single nucleotide polymorphism that did
not segregate with disease was evident in the CSRP3,
MYH7, and troponin T genes (Table 1).
Discussion
In the study reported here, a causative mutation
was not identified in the coding regions of the 5 cardiac
genes evaluated. The criterion for a causative mutation
was defined as a single base pair change that segregated
with affected dogs, occurred in a highly conserved re-
gion, and changed an amino acid.7 Although a few base
pair changes were detected, none of them changed an
amino acid or consistently segregated with disease. The
genes evaluated in this study have not been completely
excluded, however, because it is still possible that a
causative error may exist in the promoter or noncoding
regions of the gene. We believe this to be unlikely given
that the reported mutations associated with the human
disease are all single base pair changes or small dele-
tions that exist in the coding regions of the genes.7–13
The gene sequences from the affected Doberman
Pinschers were compared with the published canine se-
quence (derived from a Boxer) as well as 2 adult Labra-
dor Retrievers. Because the published canine sequence
is from a Boxer, a breed known to develop DCM, DNA
from 2 Labrador Retrievers was also compared as a con-
trol because this breed has a low prevalence of DCM.4
Additional Doberman Pinschers were not used as con-
trols because we did not want to risk the accidental in-
clusion of any affected dogs that had not yet developed
this adult-onset phenotype as control dogs.
The 5 genes evaluated for this study were selected
predominantly on the basis of their importance in the
development of autosomal dominant DCM in humans.
The lamin A/C gene is one of the most common genes
reported as containing a mutation causative for DCM in
humans.7 The lamin A/C gene encodes for lamin A and
lamin C, which are nuclear envelope proteins that ap-
pear to have structural and regulatory functions.25 The
genes that encode for the sarcomeric proteins MYH7,
troponin T, and troponin C were evaluated, although
mutations in the genes that encode for sarcomeric pro-
teins are more commonly associated with development
of hypertrophic cardiomyopathy. More recently, how-
ever, point mutations in the these genes have also been
associated with development of autosomal dominant
DCM.26–29 We also had increased interest in the evalua-
tion of the troponin C gene because it is located on ca-
nine chromosome 20 (36,113,191–36,114,151 bp). We
have identified a positive, but not significant, linkage
score on chromosome 20 in the 29,594,835–29,595,016
bp region via linkage analysis, so we considered tropo-
nin C to be of increased interest as a candidate for the
1052 AJVR, Vol 69, No. 8, August 2008
disease.6 In addition, the gene for CSRP3 was evaluated.
Cysteine- and glycine-rich protein 3 is an LIM domain
protein that appears to be important in the stabilization
of the titin/Z disc complex, which is part of the myocyte
cytoskeleton.30
One limitation of this study was the small number
of dogs in the affected group. It is possible that a muta-
tion may be identified in one of these genes in some Do-
berman Pinschers with DCM, although it was not iden-
tified in the dogs evaluated here. However, this would
seem unlikely. Doberman Pinschers are purebred dogs
with a closed gene pool. It has been estimated that by
the 1950s, 50% of the registered Doberman Pinschers
in the United States were descendents from a single
family of dogs.31 Therefore, it is reasonable to assume that
most of the affected dogs in this breed will share a com-
mon mutation because of the founder effect. Although a
causative mutation was not identified in this study, the
clinical importance of this disease as well as its strong fa-
milial nature warrants continued evaluation of additional
candidate genes or an association analysis.
a. Delmar Medical Systems, Irvine, Calif.
b. GE Healthcare BioSciences Corp, Piscataway, NJ.
c. Fermentas, Hanover, Md.
d. Applied Biosystems, Foster City, Calif.
References
1. Hammer TA, Venta PJ, Eyster GE. The genetic basis of di-
lated cardiomyopathy in Doberman pinschers. Anim Genet
1996;27:101–119.
2. Calvert CA. Dilated congestive cardiomyopathy in Doberman
pinschers. Compend Contin Educ Pract Vet 1986;8:417–430.
3. Meurs KM, Fox PR, Norgard M, et al. A prospective genetic
evaluation of familial dilated cardiomyopathy in the Doberman
pinscher. J Vet Intern Med 2007;21:1016–1020.
4. Buchanan JW. Prevalence of cardiovascular disorders. In:
Fox PR, Sisson DD, Moise NS, eds. Textbook of canine and
feline cardiology. 2nd ed. Philadelphia: WB Saunders Co,
1999;457–470.
5. Tidholm A, Jönsson L. A retrospective study of canine di-
lated cardiomyopathy (189 cases). J Am Anim Hosp Assoc
1997;33:544–550.
6. Monnet E, Orton EC, Salman M, et al. Idiopathic dilated car-
diomyopathy in dogs: survival and prognostic indicators. J Vet
Intern Med 1995;9:2–7.
7. Burkett EL, Hershberger RE. Clinical and genetic issues in fa-
milial dilated cardiomyopathy. J Am Coll Cardiol 2005;45:969–
981.
8. Fatkin D, Graham RM. Molecular mechanisms of inherited car-
diomyopathies. Physiol Rev 2002;82:945–980.
9. Osterziel KJ, Hassfeld S, Geier C, et al. Familial dilated cardio-
myopathy [in German]. Herz 2005;30:529–534.
10. Olson TM, Michels VV, Thibodeau SN, et al. Actin mutations in
dilated cardiomyopathy, a heritable form of heart failure. Science
1998;280:750–752.
11. Towbin JA. The role of cytoskeletal proteins in cardiomyopa-
thies. Curr Opin Cell Biol 1998;10:131–139.
12. Towbin JA, Hejtmancik F, Brink P, et al. X-linked dilated cardio-
myopathy. Molecular genetic evidence of linkage to the Duch-
enne muscular dystrophy (dystrophin) gene at the Xp21 locus.
Circulation 1993;87:1854–1865.
13. Maeda M, Holder E, Lowes B, et al. Dilated cardiomyopathy as-
sociated with deficiency of the cytoskeletal protein metavincu-
lin. Circulation 1997;95:17–20.
14. Stabej P, Imholz S, Versteeg SA, et al. Characterization of the ca-
nine desmin gene (DES) and evaluation as a candidate gene for
dilated cardiomyopathy in the Dobermann. Gene 2004;340:241–
249.
15. Stabej P, Leegwater PA, Imholz S, et al. The canine sarcoglycan
delta gene: BAC clone contig assembly, chromosome assignment
and interrogation as a candidate gene for dilated cardiomyopathy
in Dobermann dogs. Cytogenet Genome Res 2005;111:140–146.
16. Stabej P, Leegwater PA, Stokhof AA, et al. Evaluation of the
phospholamban gene in purebred large-breed dogs with dilated
cardiomyopathy. Am J Vet Res 2005;66:432–436.
17. Meurs KM, Magnon AL, Spier AW, et al. Evaluation of the car-
diac actin gene in Doberman Pinschers with dilated cardiomy-
opathy. Am J Vet Res 2001;62:33–36.
18. Meurs KM, Spier AW, Wright NA, et al. Use of ambulatory elec-
trocardiography for detection of ventricular premature complex-
es in healthy dogs. J Am Vet Med Assoc 2001;218:1291–1292.
19. Thomas WP, Gaber CE, Jacobs GJ, et al. Recommendations for
standards in transthoracic two-dimensional echocardiography
in the dog and cat. J Vet Intern Med 1993;7:247–252.
20. O’Grady MR, Horne R. Occult dilated cardiomyopathy in the
Doberman pinscher, in Proceedings. 13th Annu Meet Am Coll
Vet Intern Med 1995;298–299.
21. Meurs KM, Kittleson MD, Spangler E, et al. Nine polymor-
phisms within the head and hinge region of the feline cardiac
beta-myosin heavy chain gene. Anim Genet 2000;31:231.
22. Rozen S, Skaletsky HJ. Primer 3 on the WWW for general users
and for biologist programmers. In: Krawetz SA, Misener S, eds.
Bioinformatics methods and protocols: methods in molecular biol-
ogy. Totowa, NJ: Human Press, 2000;365–386.
AJVR, Vol 69, No. 8, August 2008 1053
23. e!Ensembl. Available at: www.ensembl.org/index.html. Ac-
cessed Dec 1, 2007.
24. Lindblad-Toh K, Wade CM, Mikkelsen TS, et al. Genome se-
quence, comparative analysis and haplotype structure of the
domestic dog. Nature 2005;438:803–819.
25. Rankin J, Ellard S. The laminopathies: a clinical review. Clin
Genet 2006;70:261–274.
26. Daehmlow S, Erdmann J, Knueppel T, et al. Novel mutations
in sarcomeric protein genes in dilated cardiomyopathy. Biochem
Biophys Res Commun 2002;298:116–120.
27. Kamisago M, Sharma SD, DePlama SR, et al. Mutations in sarco-
mere protein genes as a cause of dilated cardiomyopathy. N Engl
J Med 2000;343:1688–1696.
28. Li D, Czernuszewicz GZ, Gonzalez O, et al. Novel cardiac tro-
ponin T mutation as a cause of familial dilated cardiomyopathy.
Circulation 2001;104:2188–2193.
29. Mogensen J, Murphy RT, Shaw T, et al. Severe disease expression
of cardiac troponin C and T mutations in patients with idio-
pathic dilated cardiomyopathy. J Am Coll Cardiol 2004;44:2033–
2040.
30. Knöll R, Hoshijima M, Hoffman HM, et al. The cardiac mechan-
ical stretch sensor machinery involves a Z disc complex that
is defective in a subset of human dilated cardiomyopathy. Cell
2002;111:943–955.
31. Walker J. New Doberman Pinscher. Hoboken, NJ: Howell Book
House, 1981;18–25.