Assessment of PDK4 and TTN gene variants in 48 Doberman Pinschers with dilated cardiomyopathy

Kathryn M. Meurs 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Joshua A. Stern 2Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Darcy Adin 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Bruce W. Keene 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Theresa C. De Francesco 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Sandra P. Tou 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Abstract

OBJECTIVE

To evaluate the frequency of variants in the pyruvate kinase dehydrogenase 4 (PDK4) and titin (TTN) genes in a group of Doberman Pinschers with dilated cardiomyopathy (DCM) and to determine whether there were unique clinical attributes to each variant.

ANIMALS

48 Doberman Pinschers with DCM.

PROCEDURES

Doberman Pinschers with recently diagnosed DCM were identified, and genomic DNA from each was genotyped with a PCR assay for detection of PDK4 and TTN genetic variants. Dogs were grouped on the basis of whether they had the TTN variant alone, PDK4 variant alone, both variants, or neither variant. Descriptive statistics were compiled for dog age, body weight, and left ventricular dimensions and fractional shortening and for the presence of ventricular and supraventricular arrhythmias and heart failure. Results were compared across groups.

RESULTS

Of the 48 dogs, 28 had the TTN variant alone, 10 had both variants, 6 had neither variant, and 4 had the PDK4 variant alone. The mean age was younger for dogs with the PDK4 variant alone, compared with other dogs. However, the number of dogs with the PDK4 variant alone was very small, and there was an overlap in age across groups. No other meaningful differences were detected across groups, and independent genotype-phenotype relationships were not identified.

CONCLUSIONS AND CLINICAL RELEVANCE

Although findings indicated that the TTN variant was most common, 6 dogs had neither variant, and this fact supported the concept of ≥ 1 other genetic contributor to DCM in Doberman Pinschers. Future studies are warranted to evaluate genotype-phenotype relationships in Doberman Pinschers with DCM.

Abstract

OBJECTIVE

To evaluate the frequency of variants in the pyruvate kinase dehydrogenase 4 (PDK4) and titin (TTN) genes in a group of Doberman Pinschers with dilated cardiomyopathy (DCM) and to determine whether there were unique clinical attributes to each variant.

ANIMALS

48 Doberman Pinschers with DCM.

PROCEDURES

Doberman Pinschers with recently diagnosed DCM were identified, and genomic DNA from each was genotyped with a PCR assay for detection of PDK4 and TTN genetic variants. Dogs were grouped on the basis of whether they had the TTN variant alone, PDK4 variant alone, both variants, or neither variant. Descriptive statistics were compiled for dog age, body weight, and left ventricular dimensions and fractional shortening and for the presence of ventricular and supraventricular arrhythmias and heart failure. Results were compared across groups.

RESULTS

Of the 48 dogs, 28 had the TTN variant alone, 10 had both variants, 6 had neither variant, and 4 had the PDK4 variant alone. The mean age was younger for dogs with the PDK4 variant alone, compared with other dogs. However, the number of dogs with the PDK4 variant alone was very small, and there was an overlap in age across groups. No other meaningful differences were detected across groups, and independent genotype-phenotype relationships were not identified.

CONCLUSIONS AND CLINICAL RELEVANCE

Although findings indicated that the TTN variant was most common, 6 dogs had neither variant, and this fact supported the concept of ≥ 1 other genetic contributor to DCM in Doberman Pinschers. Future studies are warranted to evaluate genotype-phenotype relationships in Doberman Pinschers with DCM.

Dilated cardiomyopathy is a familial, adult-onset disease in Doberman Pinschers that is inherited as an autosomal dominant trait, at least in some families of dogs.1 A genetic variant associated with DCM has been identified in both the PDK4 gene2 (a gene involved in mitochondrial energy production) and the TTN gene3 (a sarcomeric gene involved in cardiac contraction). However, some Doberman Pinschers with DCM do not have either of these genetic mutations, suggesting that there may be another contributing genetic cause. One additional genetic locus, on canine chromosome 5, has been associated with DCM in a European cohort of Doberman Pinschers; however, no genetic mutation has been discovered in this locus.4

In people, genetic heterogeneity (the development of a clinical phenotype associated with > 1 genetic mechanism) has been observed, and multiple genetic mutations in different genes have been associated with familial DCM.5,6 Similarly, the discovery of 2 associated variants in the Doberman Pinscher breed confirms that DCM in dogs is also a disease of genetic heterogeneity.2,3 However, the underlying importance of each variant in a population of affected Doberman Pinschers is unknown. The objectives of the study presented here were to evaluate the frequency of variants in the PDK4 and TTN genes in a group of Doberman Pinschers with DCM and to determine whether there were unique clinical attributes to each variant.

Materials and Methods

Animals

Doberman Pinschers with DCM newly diagnosed on the basis of findings from an echocardiographic examination by a board-certified veterinary cardiologist or a cardiology resident under the supervision of a board-certified cardiologist at the North Carolina State University College of Veterinary Medicine, University of California-Davis School of Veterinary Medicine, or Veterinary Specialty Hospital of the Carolinas between March 1, 2018, and August 30, 2019, were eligible for inclusion in assessment of PDK4 and TTN gene variants. Diagnosis of DCM had to have included right parasternal short-axis view echocardiographic findings of an LVDd > 49 mm (reference range,7 35 to 46 mm), LVDs > 40 mm (reference range,7 26 to 37 mm), or FS < 20% (reference range,7 21% to 38%), alone or in combination.8 The study was conducted in accordance with the guidelines of the North Carolina State University Institutional Animal Care and Use Committee (17-168-0), and written owner consent was obtained for all dogs enrolled.

Data and sample collection

Each dog enrolled in the study underwent lead II ECG to determine whether ventricular ectopy or atrial fibrillation was present and underwent thoracic radiography to assess whether radiographic evidence of congestive heart failure was present. Buccal swab samples and EDTA-anticoagulated blood samples were collected from each dog for DNA analysis for the presence of mutations in the PDK4 and TTN genes. Age and body weight of each dog were recorded, and echocardiographic measurements of LVDd, LVDs, and FS on right parasternal short-axis view were obtained from echocardiography reports in the medical records.

DNA analysis

For each dog, genomic DNA was extracted from an EDTA-anticoagulated blood sample or a buccal swab sample. End-point genotypinga was performed for PDK4 and TTN, with 1 μL (5 to 20 ng/μL) of genomic DNA used for each procedure. The PCR assay cycling parameters were preincubation at 95°C (203°F) for 5 minutes, 45 cycles of a 2-step PCR assay at 95°C for 10 seconds each, then primer annealing at 60°C (140°F) for 40 seconds.

Statistical analysis

Descriptive statistics for age, body weight, FS, LVDd, and LVDs were compiled for dogs grouped on the basis of whether their genotype had the PDK4 variant, TTN variant, both variants, or neither variant. A 1-way ANOVA was used to compare results across the 4 genotype groups, and a Tukey multiple comparison test was used for post hoc analysis. The Student t test was used to assess differences in results for dogs grouped on the basis of whether they were heterozygous versus homozygous for a variant. For differences between groups, a value of P < 0.05 was considered significant. Statistical analysis was performed with available software.b

Results

Animals grouped by genotype

Forty-eight Doberman Pinschers with DCM were included in the study. Of these 48 dogs, 28 (58%) had the TTN variant alone (18 were heterozygous and 10 were homozygous), 10 (21%) had both variants (for PDK4, 6 were heterozygous and 4 were homozygous; for TTN, 6 were heterozygous and 4 were homozygous), 6 (13%) had neither variant, and 4 (8%) had the PDK4 variant alone (2 were heterozygous and 2 were homozygous; Table 1). Because the group of dogs with the PDK4 variant alone contained only 4 dogs, that group was not further analyzed for differences in results for dogs heterozygous versus homozygous for the variant.

Sex

Thirty-two of the dogs were males (11 sexually intact and 21 castrated) and 16 were females (6 sexually intact and 10 spayed; Table 1). There was no difference between genotype groups on the basis of sex.

Table 1—

Descriptive data for 48 client-owned Doberman Pinschers with DCM diagnosed between March 1, 2018, and August 30, 2019, grouped on the basis of whether their genotype contained the TTN variant or PDK4 variant, alone or in combination.

  Age (mo)Males (n = 32)Females (n = 16)
GenotypeNo. (%) of dogsMean ± SDMedian (range)Sexually intactCastratedSexually intactSpayed
TTN variant alone28 (58)71 ± 3168 (22–144)81244
TTN and PDK4 variants10 (21)93 ± 2096 (60–120)1513
No mutation in TTN or PDK46 (13)97 ± 1990 (84–132)1212
PDK4 variant alone4 (8)38 ± 1745 (12–48)2101

Age

Overall, the mean age was 75 months (range, 12 to 144 months). The mean age was significantly (P = 0.002) younger for dogs with the PDK4 variant alone (38 months) versus dogs with both variants (93 months) or neither variant (97 months). In addition, the mean age was significantly (P = 0.02) younger for dogs with the PDK4 variant alone (38 months) versus dogs with the TTN variant alone (71 months). In 1 dog with the PDK4 variant alone, DCM had been diagnosed when the dog was only 12 months old; this dog was the youngest in the study and contributed to the lower mean age for dogs that had the PDK4 variant alone. When age was considered for dogs with the TTN variant alone further grouped on the basis of whether they were heterozygous versus homozygous for the variant, no significant (P = 0.54) difference in age was detected.

Body weight

Mean body weight for all 48 dogs was 38 kg (83.6 lb). Body weight did not differ significantly among the 4 genotype groups (P = 0.81) or between dogs with the TTN variant alone further grouped on the basis of whether they were heterozygous versus homozygous for the variant (P = 0.07).

Echocardiography

Echocardiographic data were obtained from the dog's medical records. Mean FS for all dogs was 15% (range, 4% to 23%). There was no significant (P = 0.76) difference in the mean FS among the 4 genotype groups (TTN variant alone [16%], TTN and PDK4 variants [14%], no mutation in TTN or PDK4 [15%], or PDK4 variant alone [14%]; Table 2). The overall mean LVDd and LVDs were 58 mm (range, 48 to 78 mm) and 49 mm (range, 35 to 67 mm), respectively, and no significant difference was detected in the mean LVDd (P = 0.54) or mean LVDs (P = 0.61) among the 4 genotype groups. Similarly, in dogs with the TTN variant alone, the mean LVDd, LVDs, and FS did not significantly (P = 0.58, 0.37, and 0.50, respectively) differ for dogs homozygous (56 mm, 47 mm, and 17%, respectively) versus heterozygous (58 mm, 50 mm, and 15%, respectively) for the variant.

Table 2—

Echocardiographic measurements of LVDd, LVDs, and FS for the dogs in Table 1 grouped by genotype.

 LVDd (mm)LVDs (mm)FS (%)
GenotypeMean ± SDMedian (range)Mean ± SDMedian (range)Mean ± SDMedian (range)
TTN variant alone57 ± 956 (47–78)48 ± 945 (35–67)16 ± 717 (4–27)
TTN and PDK4 variants58 ± 860 (48–70)50 ± 953 (38–65)14 ± 513 (7–23)
No mutation in TTN or PDK461 ± 162 (51–71)53 ± 755 (40–60)15 ± 514 (7–22)
PDK4 variant alone55 ± 155 (51–58)47 ± 648 (39–52)14 ± 612 (10–23)

ECG

Twelve dogs had supraventricular arrhythmias (eg, atrial fibrillation or supraventricular premature beats) on ECG. Of these 12 dogs, 8 had the TTN variant alone (4 were heterozygous and 4 were homozygous), 2 had the PDK4 variant alone (both were heterozygous), and 2 had the both the TTN variant (heterozygous) and the PDK4 variant (homozygous).

Thirty-two dogs had ventricular ectopy. Of these 32 dogs, 19 had the TTN variant alone (15 were heterozygous and 4 were homozygous), 2 had the PDK4 variant alone (1 dog was heterozygous and 1 was homozygous), and 7 had both variants (3 were heterozygous for both variants, 2 were homozygous for both variants, and 2 were heterozygous for the PDK4 variant and homozygous for the TTN variant). The remaining 4 dogs with ventricular ectopy had neither variant. There was no significant difference between the groups.

Radiography

Twenty-four dogs had radiographic evidence of congestive heart failure. Affected dogs included 15 with the TTN variant alone (11 were heterozygous and 4 were homozygous), 1 with the PDK4 variant alone (heterozygous), 5 with both variants (1 was homozygous for both, 2 were heterozygous for both, 1 was homozygous for the PDK4 variant and heterozygous for the TTN variant, and 1 was heterozygous for the PDK4 variant and homozygous for the TTN variant), and 3 with neither variant. There was no significant difference between groups.

Discussion

The objective of the present study was to evaluate the frequency of the PDK4 and TTN variants in a group of Doberman Pinschers with DCM and to determine whether there were unique clinical attributes to each variant. In the dogs of the present study, the isolated TTN variant alone was most common, followed by the presence of both variants, neither variant, and the PDK4 variant alone, in that order.

We were interested to explore whether Doberman Pinschers with different PDK4 and TTN genotypes had different clinical phenotypes, and our findings were consistent with the genetic heterogeneity observed in humans with multiple genes or genetic mechanisms resulting in DCM. In humans, > 50 different genes, including those that encode for proteins that impact the sarcomere (including TTN as reported3 in dogs), nuclear envelope, cytoskeleton, sarcoplasmic reticulum, and cardiac channels, have been associated with the development of familial DCM.5,6,9 For instance, in humans, variants in the lamin A/C gene are associated with high genetic penetrance and a form of DCM with conduction disturbances, ventricular and supraventricular arrhythmias, and sudden cardiac death.5 Additionally, pathogenic variants in the TTN gene in humans have been associated with ventricular arrhythmias, fibrosis, and reverse remodeling.5 However, the relationship between the specific genotypes and the resulting phenotypic characteristics (genotype-phenotype relationship) is poorly understood with the exception of these examples in humans.10 Likewise, in the small group of dogs of the present study, we were not able to identify specific clinical phenotypic differences among the various genotype groups (dogs with the TTN variant alone, PDK4 variant alone, both the TTN and PDK4 variants, or neither the TTN nor PDK4 variant). Although we observed that the mean age was younger for dogs with the PDK4 variant alone, compared with the other groups, the number of dogs with only the PDK4 variant was very small, and there was an overlap in age across groups.

The present study had several limitations. The number of dogs evaluated was small, and the group sizes were uneven. Only 4 dogs with the PDK4 variant alone were identified. This finding could have indicated the lesser importance of this variant (vs the TTN variant) in the development of DCM, or it simply could have been a consequence of the longer duration of breeder awareness of the PDK4 variant. The PDK4 variant was first identified in 2012, and Doberman Pinscher breeders may have been applying strategic breeding practices over the past several years to reduce the prevalence of the variant in the breed's population.2 An important question that the present study could not answer was whether different variants are associated with differences in severity of DCM. This question would be best addressed by a prospective study in which investigators could assess genetic penetrance (ie, proportion of dogs with the variant that developed the disease) combined with age of onset and disease progression of DCM in Doberman Pinschers. Future studies that evaluate such aspects of genotype-phenotype relationships for DCM in Doberman Pinschers are needed. Additionally, given the known relationship between familial DCM and the Doberman Pinscher breed, none of the dogs in the present study were thoroughly evaluated for other possible causes of DCM, including associations with diet, infectious agents, or other potential causes. It was unknown whether the coexistence of such other potential causes of DCM impacted phenotypic expression of TTN or PDK4 variant genotypes evaluated in the present study.

Although findings indicated that the TTN variant was most common, 6 dogs had neither variant, and this fact supported the concept of ≥ 1 other genetic contributor to DCM in Doberman Pinschers. Future studies are warranted to evaluate genotype-phenotype relationships in Doberman Pinschers with DCM.

Acknowledgments

North Carolina State University holds an invention disclosure on the use of genetic testing for management of DCM in Doberman Pinschers, and Dr. Meurs is listed as one of the inventors on this disclosure. Views presented here are those of the authors, independent of the policies and invention disclosure held by North Carolina State University.

The authors thank Dr. Michael Cocharo and the Veterinary Specialty Hospital of the Carolinas for assistance in identifying eligible dogs.

ABBREVIATIONS

DCM

Dilated cardiomyopathy

FS

Fractional shortening

LVDd

Left ventricular diameter in diastole

LVDs

Left ventricular diameter in systole

PDK4

Pyruvate kinase dehydrogenase 4

TTN

Titin

Footnotes

a.

LightCycler, 480 Real-Time PCR system, Roche Molecular Systems Inc, Pleasanton, Calif.

b.

Prism, version 7.0, GraphPad Software, La Jolla, Calif.

References

  • 1. 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;21:10161020.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Meurs KM, Lahmers S, Keene BW, et al. 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. Hum Genet 2012;131:13191325.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Meurs KM, Friedenberg SG, Kolb J, et al. A missense variant in the titin gene in Doberman Pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death. Hum Genet 2019;138:515524.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Mausberg TB, Wess G, Simak J, et al. A locus on chromosome 5 is associated with dilated cardiomyopathy in Doberman Pinschers. PLoS One 2011;6:e20042.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Paldino A, De Angelis G, Merlo M, et al. Genetics of dilated cardiomyopathy: clinical implications. Curr Cardiol Rep 2018;20:83.

  • 6. Wilcox JE, Hershberger RE. Genetic cardiomyopathies. Curr Opin Cardiol 2018;33:354362.

  • 7. Belanger MC. Echocardiography. In: Ettinger SJ, Feldman EC, Cote E, eds. Textbook of veterinary internal medicine. 8th ed. St Louis: Elsevier, 2017;393410.

    • Search Google Scholar
    • Export Citation
  • 8. Wess G, Schulze A, Butz V, et al. Prevalence of dilated cardiomyopathy in Doberman Pinschers in various age groups. J Vet Intern Med 2010;24:533538.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Reichart D, Magnussen C, Zeller T, et al. Dilated cardiomyopathy: from epidemiologic to genetic phenotypes: a translational review of current literature. J Intern Med 2019;286:362372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Zhang Y, Maccosham A. Mind the gap: genetic variation and personalized therapies for cardiomyopathies. Lifestyle Genom 2018;11:7779.

  • 1. 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;21:10161020.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Meurs KM, Lahmers S, Keene BW, et al. 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. Hum Genet 2012;131:13191325.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Meurs KM, Friedenberg SG, Kolb J, et al. A missense variant in the titin gene in Doberman Pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death. Hum Genet 2019;138:515524.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Mausberg TB, Wess G, Simak J, et al. A locus on chromosome 5 is associated with dilated cardiomyopathy in Doberman Pinschers. PLoS One 2011;6:e20042.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Paldino A, De Angelis G, Merlo M, et al. Genetics of dilated cardiomyopathy: clinical implications. Curr Cardiol Rep 2018;20:83.

  • 6. Wilcox JE, Hershberger RE. Genetic cardiomyopathies. Curr Opin Cardiol 2018;33:354362.

  • 7. Belanger MC. Echocardiography. In: Ettinger SJ, Feldman EC, Cote E, eds. Textbook of veterinary internal medicine. 8th ed. St Louis: Elsevier, 2017;393410.

    • Search Google Scholar
    • Export Citation
  • 8. Wess G, Schulze A, Butz V, et al. Prevalence of dilated cardiomyopathy in Doberman Pinschers in various age groups. J Vet Intern Med 2010;24:533538.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Reichart D, Magnussen C, Zeller T, et al. Dilated cardiomyopathy: from epidemiologic to genetic phenotypes: a translational review of current literature. J Intern Med 2019;286:362372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Zhang Y, Maccosham A. Mind the gap: genetic variation and personalized therapies for cardiomyopathies. Lifestyle Genom 2018;11:7779.

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