- Open Access
Canine candidate genes for dilated cardiomyopathy: annotation of and polymorphic markers for 14 genes
© Wiersma et al; licensee BioMed Central Ltd. 2007
- Received: 09 March 2007
- Accepted: 19 October 2007
- Published: 19 October 2007
Dilated cardiomyopathy is a myocardial disease occurring in humans and domestic animals and is characterized by dilatation of the left ventricle, reduced systolic function and increased sphericity of the left ventricle. Dilated cardiomyopathy has been observed in several, mostly large and giant, dog breeds, such as the Dobermann and the Great Dane. A number of genes have been identified, which are associated with dilated cardiomyopathy in the human, mouse and hamster. These genes mainly encode structural proteins of the cardiac myocyte.
We present the annotation of, and marker development for, 14 of these genes of the dog genome, i.e. α-cardiac actin, caveolin 1, cysteine-rich protein 3, desmin, lamin A/C, LIM-domain binding factor 3, myosin heavy polypeptide 7, phospholamban, sarcoglycan δ, titin cap, α-tropomyosin, troponin I, troponin T and vinculin. A total of 33 Single Nucleotide Polymorphisms were identified for these canine genes and 11 polymorphic microsatellite repeats were developed.
The presented polymorphisms provide a tool to investigate the role of the corresponding genes in canine Dilated Cardiomyopathy by linkage analysis or association studies.
- Dilate Cardiomyopathy
- Polymorphism Information Content
- ACTC Protein
- Human Exon
- Genomic Contig
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by dilatation of the left ventricle, reduced systolic function and increased sphericity of the left ventricle. This disease has been described in different species and multiple genes have been found in the human , mouse  and hamster  causing DCM. These genes mainly encode cyto-skeletal components of the cardiac myocytes and can be divided into sarcomeric and extra-sarcomeric proteins. The identified sarcomeric proteins involved in DCM include α-cardiac actin, encoded by ACTC , cysteine-rich protein 3 (CSRP3) , LIM-domain binding factor 3 (LDB3, also known as Cypher or ZASP) , myosin heavy polypeptide 7 (MYH7) , titin cap (TCAP) , α-tropomyosin (TPM1), troponin I (TNNI3) , troponin T (TNNT2) , titin (TTN)  and vinculin (VCL) . The extra-sarcomeric proteins implicated in DCM are encoded by the genes including caveolin 1 (CAV1) , desmin (DES) , lamin A/C (LMNA) , phospholamban (PLN)  and sarcoglycan δ (SGCD) . The genes encoding all of the above proteins are located on the autosomal chromosomes. X-linked genes implicated in DCM include dystrophin (DYS)  and tafazzin (TAZ) . In addition, mitochondrial dysfunction and mitochondrial DNA (mtDNA) mutations have been associated with maternally inherited DCM . Furthermore, DCM has also been described with arrhythmias, with mutations in genes encoding sodium  and potassium channels .
DCM has been described in many different breeds of mostly giant and large dogs, including the Dobermann , Great Dane , Newfoundland  and Irish Wolfhound . Clinical variation exists in the presentation and progression of DCM between different dog breeds and breed specific variation has also been found in histological findings in DCM-affected hearts tissue . Since clinical DCM may be a late onset disease, following a long pre-symptomatic course, dogs are often used for breeding before the disease becomes apparent . So far, no causative mutation has been discovered in canine DCM. The phenotype of the adult onset forms of canine DCM in most breeds is consistent with a defect in components of the cytoskeleton.
Of the 14 autosomal DCM candidate genes for the dog, ACTC, CAV1, CSRP3, DES, LDB3, LMNA, MYH7, PLN, SGCD, TCAP, TNNI3, TNNT2, TPM1 and VCL, genomic information and/or polymorphic markers were already available for ACTC [26, 27], DES , PLN , SGCD  and TPM1 . In this article, we describe a complete set of polymorphic markers for these 14 candidate genes for canine DCM. The markers, both microsatellites and Single Nucleotide Polymorphisms (SNPs), provide a useful tool to perform linkage and association studies between each of these genes and DCM in the different dog breeds. Furthermore, we present the annotation of 14 candidate genes in the canine genome, which will facilitate mutation screening of these genes.
Assignment, genomic location and the degree of sequence conservation compared to human of the canine DCM candidate genes.
Annotation of canine gene
Similarity to human 4
Identification sequence 1
Dog prot. (a.a.)
16582, 16583(5), 16584
16848, 16849(5), 16850(5), 16851, 16852
The conservation of the coding region of each gene was assessed by BLAST comparison of the cDNA and derived amino acid sequences with those of human (at the website of NCBI , BLASTN and TBLASTX analysis, respectively). The percentages of identity at the nucleotide level varied between 88 and 95% (Table 1). At the amino acid level, the percentages of identity varied in general between 90–100%, except for the canine LDB3 protein, that was 79% identical to the human protein. The canine ACTC protein appeared to be identical to the human protein. In LDB3, a relatively low percentage of identity was found between the canine and human gene, both at the cDNA and the protein level. This was caused by the large (inframe) loss of part of exons (i.e. 4, 7, 8 and 9) compared to the human gene: the canine gene had 660 codons, the human gene had 734 codons.
The chromosomal position of the 14 canine candidate genes can be found in Table 1.
Single Nucleotide Polymorphism detection
Single Nucleotide Polymorphisms in the DCM candidate genes. For each SNP its origin, its primers and the PCR conditions, and its informativity are listed.
Prod. size (bp)
30,312 A/G b,5
31,216 A/G 6
25,753 T/C 6
25,446 A/G 6
28,779 A/G 6
28,742 C/A 6
28,737 G/A 6
28,642 T/A 6
15,228 C/T 7
15,224 C/G 7
15,166 G/A 7
15,006 C/T 7
19,903 T/C 7
19,196 C/T c,7,8
19,105 G/A 7,8
14,090 C/T 11
25,205 T/C 12,d
25,452 A/G 12
51,818 A/G 13
30,703 G/C 14
151,312 A/G 14
29,656 C/G 14
116,470 A/G 14
28,606 C/T 15,e
29,957 T/C 15,f
30,330 A/G 15,g
30,687 C/T 15,h
10,466 C/T 16
10,577 T/C 16
10,671 T/C 16
Twenty-eight SNPs were discovered by WAVE analysis (Table 2). No indication of the presence of a SNP was found in WAVE fragments of LMNA, MYH7 and TNNI3 (3, 5 and 3 fragments analyzed, respectively). One new SNP, TCAP SNP 29,957 T/C in genomic contig [Genbank: AAEX01022011], was found when we resequenced a TCAP fragment in a group of Newfoundland dogs. WAVE analysis of this fragment had not indicated presence of a potential SNP – although the obtained DNA sequences showed that both homozygous and heterozygous animals were among the dogs used for WAVE analysis. Conversely, sometimes WAVE analysis indicated potential presence of SNPs, yet sequencing of dogs with different WAVE patterns did not confirm these. This could be due to the sequencing procedure used.
In search of additional SNPs for canine ACTC and DES, genomic DNA fragments containing SNPs annotated by others (Table 2) were resequenced. After PCR amplification of these fragments, 1 μl of 1:15 diluted PCR product was used in a Tercycle big dye reaction with the F-PCR-primer for the ACTC SNP and a HPLC-purified M13 F-primer (5'-GTTTTCCCAGTCACGAC-3') for the DES SNPs. The Tercycle consisted of 25 cycles of 30 sec at 96°C, 15 sec at 55°C and 2 min at 60°C. After purification (Sephadex TM G50 Superfine, Amersham Biosciences), each product was processed with an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Five SNPs (ACTC 5,452G/A; DES 19,196C/T and 19,105G/A; LDB3 25,452A/G and TCAP 29.957 T/C) were identified by resequencing areas of earlier described SNPs (Table 2).
Of the total of 33 identified SNPs, 4 were in coding regions (DES 15,006C/T, LDB3 14,090C/T, TCAP 29,957T/C and TNNT2 10,466C/T). These exonic SNPs, however, did not cause polymorphisms at the amino acid level. Comparing the 33 newly discovered SNPs to the dog SNP database of the Broad Institute  showed 25 of our SNPs to be new, the remaining 8 SNPs matched SNPs present in the Broad database (see Table 2). This indicates that, in addition to the many SNPs that have become available by random sequencing of the dog genome, many more canine SNPs exist. Our limited search for SNPs in 14 DCM candidate genes took place in a single breed, the Newfoundland dog. However, a high percentage of SNPs found in one breed can be expected to be polymorphic in other breeds too . All identified SNPs were submitted to dbSNP and the respective accession numbers are listed in Table 2.
Detection of microsatellite polymorphisms
Polymorphic microsatellite markers for canine DCM candidate genes1
Origin (bp ... of contig AAEX 010...)
Distance to gene3
69.2 kb downstr. Stop
intragenic (intron 4)
8.2 kb downstr. Stop
2.8 kb upstr. Start
9.3 kb downstr. Stop
22.6 kb downstr. Stop
36.6 kb downstr. Stop
119.3 kb upstr. Start
110.2 kb downstr. Stop
179.8 kb upstr. Start
6.5 kb downstr. Stop
88.6 kb downstr. Stop
The distance between the microsatellite and the corresponding gene was derived from the dog genome build 1.1  and can be found in Table 3. This distance varied from zero for an intragenic microsatellite to 179.8 kb. The genomic locations of polymorphic microsatellites, already available for DES, SGCD, TPM1 and VCL, were determined. For DES a CA-repeat  was located at position 5,688 of [Genbank: AAEX01055032], 9.0 kb downstream of the stop codon. For SGCD both a GAAA-repeat and a CA-repeat were available . The first was located at position 76,364 of [Genbank: AAEX4801016848], the second at position 42,047 of the same genomic contig and both markers are in intron 7 of SGCD. For TPM1 a GA-repeat  was located at position 88,113 of [Genbank: AAEX01008742], 6.5 kb downstream of the stop codon. A polymorphic GAAA-repeat for VCL showed to be located at position 12,680 of [Genbank: AAEX01016406] in the dog genome, 88.6 kb downstream of the stop codon.
With the annotation of these 14 candidate genes for DCM and the identification of polymorphic markers, the genes can be evaluated for the involvement in breed specific DCM. The SNPs and microsatellites presented in this paper are a powerful tool to analyse linkage between the fourteen candidate genes encoding cytoskeletal proteins and DCM in the dog. The annotation of each gene facilitates screening of these genes for mutations in naturally occurring canine DCM in specific breeds, potential models for forms of human DCM.
This study and A.C. Wiersma were supported by a grant from the Kennel Club Charitable Trust Canine Health Foundation Fund, United Kingdom, and by the Faculty of Veterinary Science, University of Liverpool, United Kingdom. Special thanks to Francine Jury of the Centre for Integrated Genomic Medical Research (CIGMR, University of Manchester, United Kingdom) for her help with the WAVE analyses. Thanks to Polona Stabej (Faculty of Veterinary Medicine, University of Utrecht, The Netherlands) for the VCL microsatellite data.
- Burkett EL, Hershberger RE: Clinical and Genetic Issues in Familial Dilated Cardiomyopathy. Journal of the American College of Cardiology. 2005, 45: 969-981. 10.1016/j.jacc.2004.11.066.PubMedView ArticleGoogle Scholar
- Zhao YY, Liu Y, Stan RV, Fan L, Gu Y, Dalton N, Chu PH, Peterson K, Ross JJ, Chien KR: Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice. Proceedings of the National Academy of Sciences of the United States of America. 2002, 99: 11375-11380. 10.1073/pnas.172360799.PubMedPubMed CentralView ArticleGoogle Scholar
- Nigro V, Okazaki Y, Belsito A, Piluso G, Matsuda Y, Politano L, Nigro G, Ventura C, Abbondanza C, Molinari AM, Acampora D, Nishimura M, Hayashizaki Y, Puca GA: Identification of the Syrian hamster cardiomyopathy gene. Human Molecular Genetics. 1997, 6: 601-607. 10.1093/hmg/6.4.601.PubMedView ArticleGoogle Scholar
- Olson TM, Michels VV, Thibodeau SN, Tai YS, Keating MT: Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science. 1998, 280: 750-752. 10.1126/science.280.5364.750.PubMedView ArticleGoogle Scholar
- Knoll R, Hoshijima ML, Bang ML, Hayashi H, Shiga N, Yasukawa H, Schaper W, McKenna W, Yokoyama M, Schork NJ, Omens JH, McCulloch AD, Kimura A, Gregorio CC, Poller W, Schaper J, Schultheiss HP, Chien KR: The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell. 2002, 111: 943-955. 10.1016/S0092-8674(02)01226-6.PubMedView ArticleGoogle Scholar
- Arimura T, Hayashi T, Terada H, Lee SY, Zhou Q, Takahashi M, Ueda K, Nouchi T, Hohda S, Shibutani M, Hirose M, Chen J, Park JE, Yasunami M, Hayashi H, Kimura A: A Cypher/ZASP mutation associated with dilated cardiomyopathy alters the binding affinity to protein kinase C. The Journal of Biological Chemistry. 2004, 279: 6746-6752. 10.1074/jbc.M311849200.PubMedView ArticleGoogle Scholar
- Kamisago M, Sharma SD, DePalma SR, Solomon S, Sharma P, McDonough R, Smoot L, Mullen MP, Woolf PK, Wigle ED, Seidman JG, Seidman CE: Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. The New England journal of medicine. 2000, 343: 1688-1696. 10.1056/NEJM200012073432304.PubMedView ArticleGoogle Scholar
- Hayashi T, Arimura T, Itoh-Satoh M, Ueda K, Hohda S, Inagaki N, Takahashi M, Hori H, Yasunami M, Nishi H, Koga Y, Nakamura H, Matsuzaki M, Choi BY, Bae SW, You CW, Han KH, Park JE, Knoll R, Hoshijima M, Chien KR, Kimura A: Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy. Journal of the American College of Cardiology. 2004, 44: 2192-2201. 10.1016/j.jacc.2004.08.058.PubMedView ArticleGoogle Scholar
- Murphy RT, Mogensen J, Shaw A, Kubo T, Hughes SS, McKenna WJ: Novel mutation in cardiac troponin I in recessive idiopathic dilated cardiomyopathy. Lancet. 2004, 363 (9406): 371-372. 10.1016/S0140-6736(04)15468-8.PubMedView ArticleGoogle Scholar
- Itoh-Satoh M, Hayashi H, Nishi H, Koga Y, Arimura T, Koyanagi T, Takahashi M, Hohda S, Ueda K, Nouchi T, Hiroe M, Marumo F, Imaizumi T, Yasunami M, Kimura A: Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem Biophys Res Commun. 2002, 291 (2): 385-393. 10.1006/bbrc.2002.6448.PubMedView ArticleGoogle Scholar
- Olson TM, Illenberger S, Kishimoto NY, Huttelmaier S, Keating MT, Jockusch BM: Metavinculin mutations alter actin interaction in dilated cardiomyopathy. Circulation. 2002, 105: 431-437. 10.1161/hc0402.102930.PubMedView ArticleGoogle Scholar
- Li D, Tapscoft T, Gonzalez O, Burch PE, Quinones MA, Zoghbi WA, Hill R, Bachinski LL, Mann DL, Roberts R: Desmin mutation responsible for idiopathic dilated cardiomyopathy. Circulation. 1999, 100 (5): 461-464.PubMedView ArticleGoogle Scholar
- Burke B, Stewart CL: Life at the edge: the nuclear envelope and human disease. Nature reviews Molecular Cell Biology. 2002, 3: 575-585. 10.1038/nrm879.PubMedView ArticleGoogle Scholar
- MacLennan DH, Kranias EG: Phospholamban: a crucial regulator of cardiac contractility. Nature Reviews Molecular cell biology. 2003, 4: 566-577. 10.1038/nrm1151.PubMedView ArticleGoogle Scholar
- Cohen N, Muntoni F: Multiple pathogenetic mechanisms in X linked dilated cardiomyopathy. Heart. 2004, 90: 835-841. 10.1136/hrt.2003.023390.PubMedPubMed CentralView ArticleGoogle Scholar
- D'Adamo P, Fassone L, Gedeon A, Janssen EA, Bione S, Bolhuis PA, Barth PG, Wilson M, Haan E, Orstavik KH, Patton MA, Green AJ, Zammarchi E, Donati MA, Toniolo D: The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies. American Journal of Human Genetics. 1997, 61: 862-886.PubMedPubMed CentralView ArticleGoogle Scholar
- Suomalainen A, Paetau A, Leinonen A, Majander A, Peltonen L, Somer H: Inherited idiopathic dilated cardiomyopathy with multiple deletions of mitochondrial DNA. Lancet. 1992, 340: 1319-1320. 10.1016/0140-6736(92)92496-3.PubMedView ArticleGoogle Scholar
- McNair WP, Ku L, Taylor MR, Fain PR, Dao D, Wolfel E, Mestroni L, Group FCRR: SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia. Circulation. 2004, 110: 2163-2167. 10.1161/01.CIR.0000144458.58660.BB.PubMedView ArticleGoogle Scholar
- Bienengraeber M, Olson TM, Selivanov VA, Kathmann EC, O'Cochlain F, Gao F, Karger AB, Ballew JD, Hodgson DM, Zingman LV, Pang YP, Alekseev AE, Terzic A: ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating. Nature Genetics. 2004, 36: 382-387. 10.1038/ng1329.PubMedPubMed CentralView ArticleGoogle Scholar
- Domanjko-Petric A, Stabej P, Zemva A: Dilated cardiomyopathy in Dobermanns, survival, causes of death and pedigree review in a related line. Journal of Veterinary Cardiology. 2002, 4: 17-24. 10.1016/S1760-2734(06)70019-4.View ArticleGoogle Scholar
- Meurs KM, Miller MW, Wright NA: Clinical features of dilated cardiomyopathy in Great Danes and results of a pedigree analysis. Journal of the American Veterinary Medical Association. 2001, 218: 729-732. 10.2460/javma.2001.218.729.PubMedView ArticleGoogle Scholar
- Tidholm A, Jonsson L: Dilated cardiomyopathy in the Newfoundland: a study of 37 cases (1983-1994). Journal of the American Animal Hospital Association. 1996, 32: 465-471.PubMedView ArticleGoogle Scholar
- Brownlie SE, Cobb MA: Observations on the development of congestive heart failure in Irish wolfhounds with dilated cardiomyopathy. Journal of Small Animal Practice. 1999, 40: 371-377.PubMedView ArticleGoogle Scholar
- Tidholm A, Haggstrom J, Jonsson L: Detection of attenuated wavy fibers in the myocardium of Newfoundlands without clinical or echocardiographic evidence of heart disease. American journal of veterinary research. 2000, 61: 238-241. 10.2460/ajvr.2000.61.238.PubMedView ArticleGoogle Scholar
- Dukes-McEwan J, Borgarelli M, Tidholm A, Vollmar AC, Häggström J: Proposed Guidelines for the Diagnosis of Canine Idiopathic Dilated Cardiomyopathy. Journal of Veterinary Cardiology. 2003, 5: 7-19. 10.1016/S1760-2734(06)70047-9.PubMedView ArticleGoogle Scholar
- Brouillette JA, Andrew JR, Venta PJ: Estimate of nucleotide diversity in dogs with a pool-and-sequence method. Mammalian Genome. 2000, 11: 1079-1086. 10.1007/s003350010220.PubMedView ArticleGoogle Scholar
- Meurs KM, Magnon AL, Spier AW, Miller MW, Lehmkuhl LB, Towbin JA: Evaluation of the cardiac actin gene in Doberman Pinschers with dilated cardiomyopathy. American journal of veterinary research. 2001, 62: 33-36. 10.2460/ajvr.2001.62.33.PubMedView ArticleGoogle Scholar
- Stabej P, Imholz S, Versteeg SA, Zijlstra C, Stokhof AA, Domanjko-Petric A, Leegwater PA, Van Oost BA: Characterization of the canine desmin (DES) gene and evaluation as a candidate gene for dilated cardiomyopathy in the Dobermann. Gene. 2004, 340: 241-249. 10.1016/j.gene.2004.06.050.PubMedView ArticleGoogle Scholar
- Stabej P, Leegwater PA, Stokhof AA, Domanjko-Petric A, Van Oost BA: Evaluation of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy. American journal of veterinary research. 2005, 66: 432-436. 10.2460/ajvr.2005.66.432.PubMedView ArticleGoogle Scholar
- Stabej P, Leegwater PA, Imholz S, Versteeg SA, Zijlstra C, Stokhof AA, Domanjko-Petric A, Van Oost BA: 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 (2): 140-146. 10.1159/000086383.PubMedView ArticleGoogle Scholar
- Stabej P: Molecular Genetics of Dilated Cardiomyopathy in the Dobermann Dog. PhD thesis. University of Utrecht, Faculty of Veterinary Medicine, Dept of Clinical Sciences of Companion Animals; 2005:183-Google Scholar
- BLAST Dog Sequences section of NCBI BLAST. [http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/genome/seq/BlastGen/BlastGen.cgi?taxid=9615]
- Ensembl. [http://www.ensembl.org]
- NCBI Trace Archive database Mega BLAST search. [http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/blast/mmtrace.shtml]
- Schott EJ, Robledo JA, Wright AC, Silva AM, Vasta GR: Gene organization and homology modeling of two iron superoxide dismutases of early branching protist Perkinsus marinus. Gene. 2003, 309: 1-9. 10.1016/S0378-1119(03)00469-4.PubMedView ArticleGoogle Scholar
- NCBI BLAST Assembled Genomes. [http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/BLAST/]
- Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Quignon P, Lowe JK, Renier C, Gelfenbeyn B, Vignaux F, DeFrance HB, Gloux S, Mahairas GG, Andre C, Galibert F, Ostrander EA: A 1-Mb resolution radiation hybrid map of the canine genome. Proceedings of the National Academy of Sciences of the United States of America. 2003, 100: 5296-5301. 10.1073/pnas.0831002100.PubMedPubMed CentralView ArticleGoogle Scholar
- Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by: Krawetz S and Misener S. Totowa, NJ, Humana Press; 2000:365-386.Google Scholar
- Dog SNPs - CanFam 1.0 database, BROAD Institute. [http://www.broad.mit.edu/mammals/dog/snp/]
- Parker HG, Kim LV, Sutter NB, Carlson S, Lorentzen TD, Malek TB, Johnson GS, DeFrance HB, Ostrander EA, Kruglyak L: Genetic structure of the purebred domestic dog. Science. 2004, 304: 1160-1164. 10.1126/science.1097406.PubMedView ArticleGoogle Scholar
- Botstein D, White RL, Skolnick M, Davis RW: Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics. 1980, 32: 314-331.PubMedPubMed CentralGoogle Scholar
- NCBI Genomic Biology. [http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/Genomes/]
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