Naturally occurring canine laminopathy leading to a dilated and fibrosing cardiomyopathy in the Nova Scotia Duck Tolling Retriever

Abstract

Dilated cardiomyopathy (DCM) is characterized by decreased systolic function and dilation of one or both ventricles, often leading to heart failure or sudden death. Two 10-month-old sibling Nova Scotia Duck Tolling Retrievers (NSDTR) died acutely with evidence of dilated cardiomyopathy with myocardial fibrosis. Association analysis using two cases and 35 controls identified three candidate regions homozygous in the two cases. Whole genome sequencing identified a frameshift deletion in the LMNA gene (NC_049228.1:g.41688530del, NP_001274080:p.(Asp576ThrfsTer124)). Three retrospectively identified NSDTRs with sudden death before 2 years of age and severe myocardial fibrosis were also homozygous for the deletion. One 5 year old with sudden death and myocardial fibrosis was heterozygous for the deletion. This variant was not identified in 722 dogs of other breeds, nor was it identified to be homozygous in 784 NSDTR. LMNA codes for lamin A/C proteins, which are type V intermediate filaments that provide structural support to the nuclear membrane. In humans, LMNA variants can cause DCM with sudden death as well as diseases of striated muscles, lipodystrophy, neuropathies, and accelerated aging disorders. This frameshift deletion is predicted to affect processing of prelamin A into lamin A. Pedigree analysis in the NSDTR and functional evaluation of heterozygotes is consistent with a predominantly recessive mode of inheritance and possibly low penetrance in heterozygotes in contrast to people, where most pathogenic LMNA variants are dominantly inherited.

Figure 1

Pedigree of affected dogs. Affected dogs are outlined in red. Squares represent male dogs and circles represent female dogs. The asterisk indicates the proband case. Numbers below the symbols indicate lifespan of dogs when available. Genotype data for the LMNA variant is shown inside each symbol for the dogs with DNA available. A M indicates the mutant allele (NC_049228.1:g.41688530del) and a + indicates the normal allele. The three litters that produced affected puppies are indicated with letters A, B and C. The three litters have inbreeding loops. Age at death is noted under the pedigree symbols in months (m) or years (y).

Figure 2

Manhattan plot. The − log¹⁰P value for SNV that were homozygous in two affected littermates are plotted by chromosome. Four suggestive regions were identified on CHR 3, 7, 19 and 24 based on Bonferroni significance (1.4 × 10⁻⁵) shown by the red line.

Figure 3

LMNA protein alignment of mutant dog, wildtype dog and human. The deletion results in a frameshift starting at codon 576 (red box). The predicted mutant protein contains 34 amino acids beyond the normal carboxy-terminus (red underline). The CaaX motif, CSIM, (blue box) is required for initial processing of prelamin to mature lamin A. The last 13 amino acids of prelamin are cleaved between a tyrosine and leucine residue (blue dotted box) to complete the processing of mature lamin A. Lamin C is cleaved between amino acids 566 and 567 (red dashed line) for processing and is predicted to occur normally.

Figure 4

Severe myocardial Fibrosis. Sections are stained with Masson’s trichrome. Panel (a) is from a normal dog for comparison. Panels (b–d) are from three cases homozygous for the LMNA variant who died suddenly. Panel (e) showed the quantitative difference of the ratio of fibrosis/tissue between cases (M/M) and controls (+/+). The black calibration bar in panel (d) is 90 µm in length and the P value was < 0.0001.