Open Access

Canine candidate genes for dilated cardiomyopathy: annotation of and polymorphic markers for 14 genes

  • Anje C Wiersma1, 2, 3Email author,
  • Peter AJ Leegwater3,
  • Bernard A van Oost4,
  • William E Ollier2 and
  • Joanna Dukes-McEwan1
BMC Veterinary Research20073:28

https://doi.org/10.1186/1746-6148-3-28

Received: 09 March 2007

Accepted: 19 October 2007

Published: 19 October 2007

Abstract

Background

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.

Results

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.

Conclusion

The presented polymorphisms provide a tool to investigate the role of the corresponding genes in canine Dilated Cardiomyopathy by linkage analysis or association studies.

Background

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 [1], mouse [2] and hamster [3] 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 [4], cysteine-rich protein 3 (CSRP3) [5], LIM-domain binding factor 3 (LDB3, also known as Cypher or ZASP) [6], myosin heavy polypeptide 7 (MYH7) [7], titin cap (TCAP) [8], α-tropomyosin (TPM1), troponin I (TNNI3) [9], troponin T (TNNT2) [7], titin (TTN) [10] and vinculin (VCL) [11]. The extra-sarcomeric proteins implicated in DCM are encoded by the genes including caveolin 1 (CAV1) [2], desmin (DES) [12], lamin A/C (LMNA) [13], phospholamban (PLN) [14] and sarcoglycan δ (SGCD) [3]. The genes encoding all of the above proteins are located on the autosomal chromosomes. X-linked genes implicated in DCM include dystrophin (DYS) [15] and tafazzin (TAZ) [16]. In addition, mitochondrial dysfunction and mitochondrial DNA (mtDNA) mutations have been associated with maternally inherited DCM [17]. Furthermore, DCM has also been described with arrhythmias, with mutations in genes encoding sodium [18] and potassium channels [19].

DCM has been described in many different breeds of mostly giant and large dogs, including the Dobermann [20], Great Dane [21], Newfoundland [22] and Irish Wolfhound [23]. 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 [24]. 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 [25]. 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 [28], PLN [29], SGCD [30] and TPM1 [31]. 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.

Genomic Annotation

The 14 canine DCM candidate genes were identified on the canine genome by means of a BLAST analysis [32], using available canine and human DNA sequences as a query (Table 1). The exons were identified based on the corresponding human exon sequence (retrieved from [33], Table 1). Each gene was found to be covered by 1 to 5 contigs of the Canis familiaris genome build 1.1. (Additional file 1 and Table 1). CAV1 was covered by 2 neighbouring contigs and the 3 coding exons matched the human ones. Exon 1 of the dog seemed to have an extra nucleotide (T, position 336 of [Genbank: AAEX01048547]) compared to human exon 1 of CAV1. However, this nucleotide was not present in the single trace file of the Canis familiaris Trace Archive [34] covering this sequence. Canine DES had 1 amino acid less than the human protein. The canine LDB3 protein is 67 amino acids shorter than human LDB3. Canine LMNA had 1 amino acid extra compared to the human protein. Exon 24 of canine MYH7 seemed to have 1 bp extra (G, bp 7,902 of [Genbank: AAEX01041100]), however, this nucleotide was not present in any of 11 Canis familiaris trace sequences covering this position. Without this extra nucleotide, canine exon 24 matched the human exon. Canine TNNI3 had 1 amino acid extra compared to the human protein. For TNNT2, coding exons 1, 15 and 16 could not be recognized in canine genomic contigs. TNNT2 exon 6 showed 1 extra bp compared to human (G, bp 5622 of [Genbank: AAEX01013360]), however, this nucleotide was not found in the 2 traces covering this DNA sequence. Without this additional bp, exon 6 matched the corresponding human exon exactly in length. Exon 12 had 1 codon less than the human gene. Exon 13 was located at the end of genomic contig [Genbank: AAEX01013360] and although its terminal 2 putative bp were not included in this contig, exon 12 seemed to match the human exon. For the remaining candidate genes, ACTC, CSRP3, PLN, SGCD, TCAP, TPM1 and VCL, the annotated canine exons matched the corresponding human exons exactly. We could not identify non-coding exons. Apparently, the conservation of these exons is too low for identification purposes. Complementary DNA sequencing is necessary to identify these non-coding exons. All of the predicted introns of the 14 candidate genes started and ended with the canonical GT and AG dinucleotides, respectively [35]. Even though a high quality DNA sequence of the canine genome has recently become available, it has not yet been fully annotated.
Table 1

Assignment, genomic location and the degree of sequence conservation compared to human of the canine DCM candidate genes.

Gene

Annotation of canine gene

CFA

Similarity to human 4

 

Identification sequence 1

ENST 00000..2

AAEX010... 3

Dog prot. (a.a.)

  

ACTC

AF203019 (C),

290378

13478

377

30

100% AAB59619

 

AF203020 (C)

     

CAV1

U47060 (C)

341049

48546, 48547

178

14

96% NP_001744

CSRP3

BC024010 (H)

265968

17412

194

21

99% AAH24010

DES

BK005142 (C)

273074

55032

469

37

97% NP_001918

LDB3

NM_007078 (H)

361816

16582, 16583(5), 16584

660

4

79% AB014513

LMNA

AF427092 (C)

310777

12733, 12734

665

7

98% CAI15522

MYH7

NM_000257 (H)

355349

41099, 41100

1935

8

98% NP_000248

PLN

Y00399 (C)

357525

14037

52

1

96% CAI21610

SGCD

NM_000337 (H)

303006

16848, 16849(5), 16850(5), 16851, 16852

289

4

98% NP_000328

TCAP

NM_003673 (H)

309889

22011

167

9

90% CAA09479

TNNI3

AF506750 (C)

344887

53923

211

1

95% CAG46782

TNNT2

(ex 2–14)

NM_000364 (H)

367317

13359, 13360

254

7

90% NP_000355

TPM1

NM_000366 (H)

288398

08742

284

30

99% AAH07433

VCL

NM_003373 (H)

211998

16404

1134

4

99% NP_054706

1 Sequence used to identify the canine gene in the dog genome, Genbank accession numbers; C = canine sequence, H = human sequence; 2 Transcript ID numbers of human annotation [33] used to annotate the canine gene; 3 canine genomic contig in which the gene's coding exons were identified; 4 the percentage identity of each canine protein compared to the human protein (Genbank accession number is listed); 5 canine genomic contig containing only intronic sequence.

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 [36], 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.

When analysing the location of the genes in the dog genome (Table 1), using the canine-human comparative map of Guyon et al. [37], each was found to be syntenic to the human location.

Polymorphisms

Single Nucleotide Polymorphism detection

We used denaturing high-performance liquid chromatography (DHPLC) analysis for the detection of SNPs in amplified genomic canine DNA fragments. Polymorphisms were assessed in DNA from Newfoundland dogs. For each gene, several DNA fragments of approximately 500 bp were selected based on melting profile (analyzed with WAVEMAKER™ software from Transgenomic) with a maximum of 2 melting temperatures covering each product. The melting behaviour of a fragment depends on the fragment's DNA sequence. Primers were designed using Primer3 [38] and annealing temperatures of the PCRs were optimized (Table 2). Touchdown PCR amplification of these fragments was performed with DNA of Newfoundland dogs (n = 16; 8 unrelated founders of a pedigree of Newfoundland dogs and 8 family members), using HotStartTaq DNA Polymerase (Qiagen). The Touchdown (TD) PCR program consisted of a denaturing step of 5 min at 95°C, followed by 14 cycles of 95°C 30 sec, Ta +7°C 30 sec, 72°C 20 sec, with a Ta decrease of 0.5°C/cycle, followed by 25 cycles of 30 sec at 94°C, 30 sec at Ta°C, 30 sec at 72°C, followed by a final extension at 72°C for 2 min (Ta in Table 2). Subsequently, a heteroduplex formation step was carried out to allow formation of hetero- and homo-duplex products; the PCR products were heated 5 min at 95°C, after which the temperature was decreased gradually (38 cycles of 1 min, temperature decreasing 1.5°C/cycle), followed by a final step of 5 min at 10°C. Mutation analysis of the PCR products, based on the presence of heteroduplexes, followed on a WAVE instrument (WAVE Nucleic Acid Fragment Analysis System, Transgenomic). Multiple WAVE patterns of a single PCR fragment in different dogs pointed at existence of both homoduplexes and heteroduplexes and, therefore, indicated potential presence of SNPs in the fragment. In that case, the PCR fragment (of at least of 2 dogs per WAVE pattern) was cleaned (Shrimp Alkaline Phosphatase/ExoI) and the DNA sequence was obtained to determine the identity of the SNPs, by a commercial company (Lark Technologies™, UK).
Table 2

Single Nucleotide Polymorphisms in the DCM candidate genes. For each SNP its origin, its primers and the PCR conditions, and its informativity are listed.

Gene

dbSNP

access. no.

ss4985...

SNP

Primers (5'-3')

Forward;

Reverse

Ta (°C)1

Prod. size (bp)

Informativeness2

      

PIC

#chr

ACTC

2973

5,452 G/Aa,3,4

gccctggattttgagaatgagat

acgatcagcaataccagggtaca

62.0 1

1067

0.14

12

CAV1

2978

30,312 A/G b,5

tgagtgccttgcttgtgg

gcatcattggaacttgttgg

62.0

565

0.28

24

 

2979

30,088 G/A5

tgagtgccttgcttgtgg

gcatcattggaacttgttgg

62.0

565

0.24

24

CSPR3

2980

31,216 A/G 6

ggaggccaggatgagaac

gtttattgtactgaatgatggtcag

62.0

507

0.15

22

 

2981

25,753 T/C 6

aatcatcctcccattgttcc

cagaagtgctcatagtctttaccc

58.0

510

0.37

24

 

2982

25,446 A/G 6

aatcatcctcccattgttcc

cagaagtgctcatagtctttaccc

58.0

510

0.24

24

 

2983

28,779 A/G 6

atggacctttgtatctccag

tctgtaggtttcattcattgg

58.0

455

0.19

24

 

2984

28,742 C/A 6

atggacctttgtatctccag

tctgtaggtttcattcattgg

58.0

455

0.19

24

 

2985

28,737 G/A 6

atggacctttgtatctccag

tctgtaggtttcattcattgg

58.0

455

0.19

24

 

2986

28,642 T/A 6

atggacctttgtatctccag

tctgtaggtttcattcattgg

58.0

455

0.19

24

DES

2989

15,228 C/T 7

cgtcacaacccccacaag

gctgggtgccatgaggtc

67.0

530

0.30

8

 

2990

15,224 C/G 7

cgtcacaacccccacaag

gctgggtgccatgaggtc

67.0

530

0.19

8

 

2991

15,166 G/A 7

cgtcacaacccccacaag

gctgggtgccatgaggtc

67.0

530

0.19

8

 

2992

15,006 C/T 7

cgtcacaacccccacaag

gctgggtgccatgaggtc

67.0

530

0.19

8

 

2993

19,903 T/C 7

agggcagagggagaccag

gacctaatggtgggctttacc

66.0

575

0.30

8

 

2975

19,196 C/T c,7,8

ttgcttgaccactaccagga9

agatgttcttagccgcgatg10

57.0 1

402

0.35

12

 

2976

19,105 G/A 7,8

ttgcttgaccactaccagga9

agatgttcttagccgcgatg10

57.0 1

402

0.30

12

LDB3

2987

14,090 C/T 11

tgttaatcacctctgcggatagt

ggctccctacacgttgatg

58.0

540

0.33

24

 

2988

25,205 T/C 12,d

gcctcctccatcctgacc

cctcccagtaccctgtaggc

66.0

566

0.19

24

 

s2974

25,452 A/G 12

gcctcctccatcctgacc

cctcccagtaccctgtaggc

66.0

566

0.38

24

PLN

2994

51,818 A/G 13

tggtttgccttcatacactacaac

tgtcttcatctgtgggattttg

64.0

573

0.21

14

SGCD

2995

30,703 G/C 14

ccttcagacccccatctagg

ccacctgacataatcccactttag

66.0

521

0.36

8

 

2996

151,312 A/G 14

ggaggtagcaaagtatagtgctc

atgttcatgccaacaagc

62.0

558

0.30

8

 

2997

29,656 C/G 14

ttccagccaactgagaagc

cactgtcatttccatgtcaacc

58.0

525

0.30

8

 

2998

116,470 A/G 14

gcaatctcctcctccagacc

tcatggcctcactctgatctc

58.0

529

0.38

8

TCAP

2999

28,606 C/T 15,e

gctgcttcccttgaatgc

cagacagtggcaggaatcg

64.0

588

0.28

24

 

2977

29,957 T/C 15,f

gtagagggtagcagatttcagg

ctctgggcaaactacaaagc

69.0

555

0.26

16

 

3000

30,330 A/G 15,g

tgctttgtagtttgcccagag

agccagccaccctgtttac

64.0

557

0.30

8

 

3001

30,687 C/T 15,h

tgctttgtagtttgcccagag

agccagccaccctgtttac

64.0

557

0.30

8

TNNT2

3002

10,466 C/T 16

tgaccctcacttggggaac

cgcagggctcttccagac

58.0

519

0.38

24

 

3003

10,577 T/C 16

tgaccctcacttggggaac

cgcagggctcttccagac

58.0

519

0.38

24

 

3004

10,671 T/C 16

tgaccctcacttggggaac

cgcagggctcttccagac

58.0

519

0.38

24

VCL

3005

177,743 G/A17

tgcaggccacagagatgc

ggaatgagggcggagcag

62.0

491

0.30

8

1 All PCR program were Touchdown (TD) at the listed Ta, accept for the ACTC SNP: 94°C 5 min, 35× (94°C 30 sec, 62°C 1 min, 72°C 1 min), 72°C 10 min, 20°C ∞; and for the DES SNPs 19,196 C/T and 19,105 G/A: 94°C 10 min, 35× (94°C 30 sec, 57°C 30 sec, 72°30 sec), 72°C 10 min, 20°∞; 2 The informativeness of each SNP was described by its polymorphism information content (PIC), based on the number of genotyped chromosomes (#chr) listed; 3 SNP detected while sequencing available ACTC SNPs (166C/T and 38C/T of [Genbank: AF203019] and 289T/A of [Genbank: AF203020], these were in the dog genome, respectively, 4,871G/A, 5,000 G/A and 5,454 A/T in [Genbank: AAEX01013478)] [26]; 4 in genomic contig AAEX01013478; 5 AAEX01048546; 6 AAEX01017412; 7 AAEX01055032; 8 SNP detected while sequencing available DES SNPs (1,808C/T and 1,851G/C of [Genbank: BK005142], these were in the dog genome, respectively, 19,262 G/A and 19,218C/G in [Genbank: AAEX01055032]) [28]; 9 M13-tailed F-primer: 5'- GTTTTCCCAGTCACGAC---- 3'; 10 M13-tailed R-primer: 5'- CAGGAAACAGCTATGAC----3'; 11 in genomic contig AAEX01016584; 12 AAEX01016582; 13 AAEX01014037; 14 AAEX01016848; 15 AAEX01022011; 16 AAEX01013360; 17 AAEX01016404; a is identical to SNP BICF237J37997 (Broad, at [39]); b identical to BICFPJ1220038; c identical to BICFPJ152241; d identical to BICF231J18538; e identical to BICFG630J165218; f identical to BIFG630J165217; g identical to BICFG630J165215; h identical to BICFG630J165213.

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 [39] 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 [40]. All identified SNPs were submitted to dbSNP and the respective accession numbers are listed in Table 2.

Detection of microsatellite polymorphisms

Simple DNA sequences composed of CA, GAAA or GA repeats were identified in the genomic contigs that contain the candidate genes or in neighbouring contigs. For VCL, a polymorphic microsatellite became available through personal communication with P.Stabej (Table 3; a repeat was obtained from BAC RP81-251B5, isolated using methods as described in [28] with an overgo probe based on murine VCL exon 17, F-overgo CCAAGGTCAGAGAAGCCTTCCAAC, R-overgo AAGTCAGGCTCCTGAGGTTGGAAG). Primers were designed from the DNA sequence flanking the repeats and the forward primer was fluorescently labelled with 6-FAM or HEX. For some microsatellites, a 3-primer protocol was used for the PCR amplification (Table 3), using an M13-tailed (GTTTTCCCAGTCACGAC----- (5'-3')) F-primer, a 6-FAM-labelled M13 primer (GTTTTCCCAGTCACGAC (5'-3')) and a R-primer. Genotyping PCR reactions were incubated 12 min at 94°C, followed by 35 cycles of 10 sec at 94°C, 15 sec at Ta°C and 30 sec at 72°C, and a final step of 20 min at 72°C (Ta in Table 3). An ABI PRISM ® 3100 Genetic Analyzer (Applied Biosystems) was used for genotyping and allele sizes were determined with Genescan Analysis 3.7 and Genotyper 3.7 software (Applied Biosystems). Eleven polymorphic microsatellites were developed for ACTC (2 markers), CAV1 (1), CSRP3 (1), LMNA (2), MYH7 (1), TNNI3 (3) and VCL (1) (Table 3). The markers, mostly CA-repeats, showed multiple allele sizes (2–6 alleles/marker) in a group of 16 Newfoundland dogs (Table 3). To describe the informativeness of our microsatellite markers, the polymorphism information content (PIC) was obtained based on the genotypes of unrelated founders of a family of Newfoundland dogs (Table 3). According to [41], 2 of the 11 newly designed microsatellites were considered highly informative (PIC>0.50), 7 reasonable informative (0.25<PIC<0.50) and 2 slightly informative (PIC<0.25) in the Newfoundland founder dogs. Besides the 11 polymorphic microsatellites, 2 other markers were found to be monomorphic in the group of Newfoundland dogs, but might be polymorphic in other breeds. This was a MYH7 CA-repeat (position 11,730 of [Genbank: AAEX010141100]) and a TNNI3 CA-repeat (position 17,739 of [Genbank: AAEX01053915]. An already available microsatellite for TPM1 [31] was shown to be highly informative in our group (Table 3).
Table 3

Polymorphic microsatellite markers for canine DCM candidate genes1

Gene

Repeat

Primers (5'-3')

Forward;

Reverse

Ta (°C)

Detected alleles

Informativeness2

Origin (bp ... of contig AAEX 010...)

Distance to gene3

    

bp

#

PIC

#chr

  

ACTC

15CA

actccgaagaaggaagtcaac4

gttcccatctatgagggctat

57.0

234–238

2

0.17

10

bp 37,720/..13479

69.2 kb downstr. Stop

 

20CA

ggaacaaggtgctgttagacc5

cacattccaccgagtaggc7

59.0

338–356

5

0.42

24

bp 5,945/..13478

intragenic (intron 4)

CAV1

13CA

ccacagagctagaaagctacg4

tgttgcaaacaccctatgat

54.5

240–242

2

0.28

24

bp 39,315/...48546

8.2 kb downstr. Stop

CSRP3

15CA

catgtcctgcaagttaatggt4

ggatttctattctgggtttcc

53.0

237–245

3

0.41

24

bp 38,367/..17412

2.8 kb upstr. Start

LMNA

16CA

gggtggtagatgagcatttc6

gaagagaacaagtgggcaag

54.5

204–212

4

0.36

12

bp 5,128/..12734

9.3 kb downstr. Stop

 

18GAAA

ggaagatgagactgttagaatgc5

caggccatgattacttttcc7

57.0

321–344

6

0.67

24

bp 26,754/..12735

22.6 kb downstr. Stop

MYH7

21CA

gatatcctgggattaaagactgg5

ctattttgccctcttcatgg7

58.0

351–363

4

0.37

24

bp 1,418/..41098

36.6 kb downstr. Stop

TNNI3

20CAa

tcaaacagggaaacctgaac6

gattattcagctcccagaacc7

57.0

297–301

3

0.38

24

bp 597/..53929

119.3 kb upstr. Start

 

20CAb

ttccagttgattgtttctctgc5 gcggtttagcactgcattc7

59.0

302–306

2

0.08

24

bp 13,248/..53916

110.2 kb downstr. Stop

 

17GA

tccaacctcagggtactgg5 catgccatggagctatgc7

59.0

304–312

3

0.37

24

bp 48,910/..53930

179.8 kb upstr. Start

TPM1

19CA8

actgtgtccagagtgcagcta4

gattgctagactggc

60.0

467–483

4

0.67

12

bp 88,113/..08742

6.5 kb downstr. Stop

VCL

15GAAA9

caatttcttttccaatcacattag 10

gccattttgcattctcttcaa

54.0

150–170

6

0.69

24

bp 12,680/..16406

88.6 kb downstr. Stop

1 Microsatellites for DES and SGCD were demonstrated in, respectively, [28] and [30]; 2 The informativeness of each SNP was described by its polymorphism information content (PIC), based on the number of genotyped chromosomes (#chr) listed; 3 Based on genomic build 1.1; 4 F-primer fluorescently labelled with 6FAM; 5 Three-primer protocol used; 6 F-primer fluorescently labelled with HEX; 7 extra tail on R-primer: GTGTCTT---- (5'-3') to promote addition of an Adenosine residue at the 3'-end of the complementary DNA strand; 8 Microsatellite demonstrated in [31]; 9 Personal communication P.Stabej; 10 F-primer fluorescently labelled with TET.

The distance between the microsatellite and the corresponding gene was derived from the dog genome build 1.1 [42] 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 [28] 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 [30]. 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 [31] 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.

Conclusion

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.

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Small Animal Teaching Hospital, University of Liverpool
(2)
Centre for Integrated Genomic Medical Research, Division of Epidemiology and Health Sciences, The University of Manchester
(3)
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University
(4)
American University of the Caribbean, Department of Molecular Cell Biology

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